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  • 1
    Electronic Resource
    Electronic Resource
    Aberrant gene expression in cultured mammalian bone cells demonstrates an osteoblast defect in osteopetrosis (1994)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 55 (1994), S. 366-372 
    Details
    ISSN: 0730-2312
    Keywords: osteoclast ; gene regulation ; rat ; skeleton ; osteopontin ; osteocalcin ; mineralization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Osteopetrosis is a skeletal condition in which a generalized radioopacity of bone is caused by reduced resorption of bone by osteoclasts. However, it has recently been shown that during skeletal development in several osteopetrotic rat mutations specific aberrations occur in gene expression reflecting the activity of the bone forming cells, osteoblasts, and the development of tissue organization. To evaluate their pathogenetic significance, progressive osteoblast differentiation was studied in vitro. Primary cultures of normal osteoblasts undergo a sequential expression a cell growth and tissue-related genes associated with development of skeletal tissue. We report that osteoblast cultures can be established from one of these mutants, toothless; that these cells in vitro exhibit similar aberrations in gene expression during cell proliferation and extracellular matrix formation and mineralization observed in vivo; and that an accelerated maturation sequence by mutant osteoblasts mimics the characteristic skeletal sclerosis of this disease. These data are the first direct evidence for an intrinsic osteoblast defect in osteopetrosis and establish an in vitro model for the study of heritable skeletal disorders. © 1994 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240550314
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    Identification and characterization of two proximal elements in the rat osteocalcin gene promoter that may confer species-specific regulation (1993)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 53 (1993), S. 240-250 
    Details
    ISSN: 0730-2312
    Keywords: osteocalcin ; CCAAT ; transcription ; phosphatase ; steroid-like half-elements ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The rat osteocalcin gene encodes a 6-kD osteoblast-specific protein that is expressed postproliferatively. The developmental and steroid hormone responsive expression of the osteocalcin gene is transcriptionally regulated by a promoter with multiple basal and enhancer elements that exhibit activity controlled by a series of physiological mediators (e.g., 1,25(OH)2D3, glucocorticoids). In this study, we established the contribution of the rat osteocalcin (OC) box domain ( -99 to -76), a proximal basal element with a CCAAT motif as a central core, to transcriptional activity of the rat osteocalcin gene with in vivo co-transfection assays. By this same assay, however, the highly homologous (22 of 24 nt) human OC box element was unable to compete for transcription factor binding with the rat OC promoter. In vitro protein/DNA interaction studies confirm the presence of two protein binding sites in the OC box region, one of which overlaps the CCAAT motif and, at least in part, accounts for species-specific expression. Competition analysis established that the single nucleotide substitution of adenine for thymine, which converts the core motif of the rat OC box (CCAAT) to the core motif of the human OC box (CCAAA), accounts for observed species differences in transcription factor interactions. The CCAAT-specific protein/DNA interactions are heat stable and insensitive to phosphatase treatment. A second protein/DNA interaction located upstream of the CCAAT motif includes two steroid-like half-elements. These interactions are heat labile and sensitive to phosphatase treatment in contrast to the CCAAT-specific interactions. The human OC promoter contains only a single steroid-like half-element, while two steroid half-elements with an 11 nucleotide spacer are present in the rat OC promoter. These observed variations in sequence organization and transactivation factor binding in analogous proximal basal regulatory regions of the OC gene promoter may provide a basis for species-restricted variations in responsiveness to physiological mediators of OC gene expression at the transcriptional level.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240530309
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  • 3
    Electronic Resource
    Electronic Resource
    Effect of gallium nitrate in vitro and in normal rats (1993)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 52 (1993), S. 330-336 
    Details
    ISSN: 0730-2312
    Keywords: gallium nitrate ; bone mineral density ; osteocalcin ; collagen ; tartrate-resistant acid phosphatase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Gallium nitrate (GN) is an inhibitor of bone resorption and thereby may result in a change in coupled bone formation. In the present investigation the effects of GN on bone formation were studied in the rat osteosarcoma (ROS) 17/2.8 cell line and normal diploid rat osteoblasts (ROB) in vitro and the femur of rats treated in vivo, measuring mRNA levels for two osteoblast parameters, type I collagen, a marker of matrix formation, and osteocalcin, a bone specific protein and also histone H4, a marker of cell proliferation. GN, at 50 μM for 3 h, increased type I collagen mRNA levels by 132% in ROS 17/2.8 cells and by 122% in proliferating ROB cells. Osteocalcin (OC) mRNA levels were decreased by 61% in ROS 17/2.8 cells and by 97% in differentiated ROB cells. These changes occurred in the absence of any effects on cell proliferation. Seventy-day-old female rats were then treated with GN, 0.5 mg/kg/day, for 3 weeks. As previously reported, GN decreased serum calcium levels, but had no effect on lumbar or femoral bone density. In contrast to the in vitro effects, GN had no effect on type I collagen steady-state mRNA levels in the femur; however, it decreased OC steady-state mRNA levels in the femur by 58%. These results suggest that GN has similar in vitro effects in transformed and normal osteoblasts, while the collagen-stimulatory effects observed in vitro cannot be extrapolated to in vivo models. The consistent inhibition of osteocalcin in vitro and in vivo suggests a more specific target for GN that may relate to its effects in inhibiting bone resorption in normal rats.
    Additional Material: 7 Tab.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240520309
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  • 4
    Electronic Resource
    Electronic Resource
    Bone tissue-specific transcription of the osteocalcin gene: Role of an activator osteoblast-specific complex and suppressor hox proteins that bind the OC box (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 310-324 
    Details
    ISSN: 0730-2312
    Keywords: osteocalcin ; transcriptional regulation ; homeodomain protein ; Msx ; bone-specific ; OC box ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bone-specific expression of the osteocalcin gene is transcriptionally controlled. Deletion analysis of osteocalcin promoter sequences by transient transfection of osseous (ROS 17/2.8) and nonosseous (R2 fibroblast) cells revealed that the most proximal 108 nucleotides are sufficient to confer tissue-specific expression. By gel mobility shift assays with wild-type and mutated oligonucleotides and nuclear extracts from several different cell lines we identified a novel transcription factor complex which exhibits sequence-specific interactions with the primary transcriptional element, the OC box (nt -99 to -76). This OC box binding protein (OCBP) is present only in osteoblast-like cells. Methylation interference demonstrated association of the factor with OC box sequences overlapping the Msx homeodomain consensus binding site. By assaying several mutations of the OC box, both in gel shift and transient transfection studies using ROS 17/2.8, we show the following. First, binding of OCBP correlates with osteocalcin promoter activity in ROS 17/2.8 cells. Increased binding leads to a 2-3-fold increase in transcription, while decreased binding results in transcription 30-40% of control. Second, homeodomain protein binding suppresses transcription. However, Msx expression is critical for full development of the bone phenotype as determined by antisense studies. Last, we show that one of the mutations of the OC box permits expression of osteocalcin in non-osseous cell lines. In summary, we demonstrate association of at least two classes of tissue-restricted transcription factors with the OC box element, the OCBP and Msx proteins, supporting the concept that these sequences contribute to defining tissue specificity. © 1996 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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