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  • 1
    Electronic Resource
    Electronic Resource
    Abnormalities of phosphoprotein gene expression in three osteopetrotic rat mutations: Elevated mrna transcripts, protein synthesis, and accumulation in bone of mutant animals (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 158 (1994), S. 110-120 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Osteoclast abnormalities that characterize osteopetrosis, a disorder of bone resorption, may derive from aberrant signals from the osteoblast or the bone matrix. In the present studies, both synthesis and the bone matrix content of the major bone phosphoprotein component, osteopontin, were found to be elevated in three osteopetrotic rat mutations (ia, op, and tl). In whole bone, a twofold increase in the content of the characteristic amino acid O-phosphoserine for osteopontin occurred in op and tl mutant long bone, but a smaller (15%) and more variable increase was observed in ia mutant rat long bone. Extraction of the bone matrix components and partial purification by reverse phase chromatography showed a twofold increase in a phosphoprotein fraction relative to other noncollagenous components. Amino acid analysis and staining characteristics of SDS-PAGE fractionated proteins indicated this to be osteopontin. Organ cultures of calvarial bone from 4 day ia osteopetrotic mutant and normal rats in the presence of 3H-proline showed increased synthesis of this 60 kD protein, which was stimulated by vitamin D. Preparation of total cellular RNA from bone of 2- and 6-weekold mutants and normal rats supported increased synthesis of osteopontin as reflected by hybridization with osteopontin cDNA probe, showing significantly higher levels of mRNA transcripts in ia (3-5 fold), tl (1.4-2 fold), and op (6-25 fold) mutant bone compared to normal littermates. The changes in osteopontin mRNA levels in mutant bone were also examined in relation to other growth and phenotype-expressed genes. The findings of increased accumulation of osteopontin in osteopetrotic bone and increased synthesis by osteoblasts are interesting in light of the previously reported decrease in bone osteocalcin content (Endocrinology, 126:966, 1990), confirmed here by decreased osteocalcin mRNA transcripts. Such aberrations in the composition of skeletal extracellular matrix could be a reflection of or a contributing factor to the osteoclast abnormalities of some of these osteopetrotic disorders. © 1994 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041580114
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  • 2
    Electronic Resource
    Electronic Resource
    Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 143 (1990), S. 213-221 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Rat calvaria osteoblasts derived from 21-day-old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al: Biochem. Biophis. Res. Commun. 148:1129, 1987) and is similar to that observed in cultures of subcultivated 16-day-old embryonic chick calvaria-derived osteoblasts (COB) (Gerstenfeld, et. al: Dev. Biol. 122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of β-glycerol-phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200-fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/maturation in vitro.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041430203
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  • 3
    Electronic Resource
    Electronic Resource
    Transcriptionally active nuclei isolated from intact bone reflect modified levels of gene expression in skeletal development and pathology (1994)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 55 (1994), S. 182-189 
    Details
    ISSN: 0730-2312
    Keywords: rat bone transcription ; rat bone transcription factors ; osteopetrotic bone transcription ; osteocalcin transcription ; collagen transcription ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Transcriptional regulation of gene expression in vivo in bone, associated with normal development or skeletal disorders, to date, has not been studied. We report the successful isolation of nuclei that are transcriptionally active from normal and osteopetrotic rat bone. Transcription rates of cell growth and bone-related genes (including histone H4, c-fos, c-jun, TGFβ1, β2 macroglobulin, collagen, fibronectin, osteocalcin, osteopontin, and tartrate resistent acid phosphatase) change as a function of calvarial development from birth to 6 weeks and are selectively modified in osteopetrotic animals. Additionally, nuclei isolated from intact bone yield promoter binding factors. Bone nuclei, which transcribe faithfully and contain the normal complement of nuclear protein factors, offer a powerful approach for investigating in vivo gene regulation in skeletal development and pathology. © 1994 Wiley-Liss, Inc.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240550205
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  • 4
    Electronic Resource
    Electronic Resource
    Variations in vitamin D receptor transcription factor complexes associated with the osteocalcin gene vitamin D responsive element in osteoblasts and osteosarcoma cells (1994)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 55 (1994), S. 218-229 
    Details
    ISSN: 0730-2312
    Keywords: osteosarcoma cells ; osteocalcin gene ; osteoblasts ; vitamin D response element (VDRE) ; transcription factor complexes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Vitamin D responsive transcription of the bone-specific osteocalcin gene differs markedly in osteosarcoma cells and normal diploid osteoblasts. In osteoblasts the osteocalcin gene is transcribed, and upregulated by Vitamin D, only in post-proliferative cells, but in osteosarcoma cells expression is constitutive. This distinction in transcriptional regulation of the osteocalcin gene correlates with striking differences in the relative representation of two principal Vitamin D-dependent protein/DNA complexes designated V1 and V2 at the Vitamin D responsive element in the osteocalcin promoter. Formation of both complexes is Vitamin D dependent and they contain the Vitamin D receptor as well as an RXR related protein. Pore size exclusion and sedimentation velocity analyses suggest that the V1 and V2 complexes represent oligomeric protein assemblies (respectively, tetramers and trimers), and reflect primarily DNA-directed association of the monomeric protein components at the osteocalcin Vitamin D responsive element. UV crosslinking and methylation interference analyses of the V1 and V2 complexes at the osteocalcin Vitamin D responsive element indicate differences in protein/DNA recognition. For example, the V1 complex interacts with both steroid half-elements, whereas the V2 complex appears to recognize the proximal half-element. Our findings suggest variations in protein/protein and protein/DNA interactions of the VDR and RXR related complexes V1 and V2 at the osteocalcin Vitamin D responsive element that reflect unique properties of the osteosarcoma and normal diploid osteoblast phenotype. © 1994 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240550209
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  • 5
    Electronic Resource
    Electronic Resource
    Sequence-specific DNA binding activities of nuclear matrix proteins of mammalian lens epithelial cells (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 58 (1995), S. 1-5 
    Details
    ISSN: 0730-2312
    Keywords: DNA-binding proteins ; lens epithelial cell ; nuclear matrix ; Oct-1 ; SP-1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: This study examines matrix and nonmatrix nuclear proteins of the rabbit lens epithelial cells. The nuclear matrix proteins were isolated by modified Penman technique, which requires presence of detergents and nucleases, whereas nonmatrix nuclear proteins were obtained by high salt extraction. The data from these experiments revealed presence of DNA binding activities for SP-1 and OCT-1 proteins in both matrix and non-matrix compartments of rabbit lens epithelial cells. Comparison of the relative abundance of SP-1 and OCT-1 binding activities in nuclear matrix and nonmatrix fraction suggest the distribution between these two compartment is cell type specific and possibly related to the control of cell growth. © Wiley-Liss, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240580102
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  • 6
    Electronic Resource
    Electronic Resource
    Multiple types of mRNA-cytoskeleton interactions (1990)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 44 (1990), S. 177-187 
    Details
    ISSN: 0730-2312
    Keywords: translated mRNAs ; cytoskeleton ; HeLa cells ; signal peptide-histone fusion mRNA ; histone mRNA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Nearly all actively translated mRNAs are associated with the cytoskeleton in HeLa cells and the nature of this association is poorly understood. To gain insight into this association, we have examined and compared the cytoskeleton-mRNA interactions of a signal peptide-histone fusion mRNA (membrane-bound polysomal mRNA) to those of endogenous histone mRNA (nonmembrane-bound polysomal mRNA). We report here the detection of a cytoskeleton attachment site within the signal peptide-histone fusion mRNP/mRNA nucleotide sequence that is not present in wild-type histone mRNA or in HLA-B7 and chorionic gonadotropin-α membrane-bound polysomal mRNAs. These results support the possibility that there are multiple mechanisms for the attachment of specific classes of mRNAs to the cytoskeleton.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240440306
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  • 7
    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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  • 8
    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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  • 9
    Electronic Resource
    Electronic Resource
    Inhibition of induced endochondral bone development in caffeine-treated rats (1993)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 52 (1993), S. 171-182 
    Details
    ISSN: 0730-2312
    Keywords: caffeine ; bone matrix implants ; delayed ossification ; osteoblasts ; gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have addressed questions raised by the observation in fetal rats of delayed ossification induced by caffeine at maternal doses above 80 mg/kg body weight per day. The effect of caffeine on endochondral bone development and mineralization has been studied in an experimental model system of bone formation which involves implantation of demineralized bone particles (DBP) in subcutaneous pockets of young growing rats. Caffeine's effects on cellular events associated with endochondral ossification were examined directly by quantitating cellular mRNA levels of chondrocyte and osteoblast growth and differentiation markers in DBP implants from caffeine-treated rats harvested at specific stages of development (day 7 through day 15). Oral caffeine administration to rats implanted with DBP resulted in a dose dependent inhibition of the formation of cartilage tissue in the implants. Histologic examination of the implants revealed a decrease in the number of cells which were transformed to chondrocytes compared to control implants. Those cartilaginous areas that did form, however, proceeded through the normal sequelae of calcified cartilage and bone formation. At the 100 mg/kg dose, cellular levels of mRNA for histone, collagen type II, and TGFβ were all reduced by greater than 40% of control implants consistent with the histological findings. Alkaline phosphatase activity in the implants and mRNA levels for proteins reflecting the hypertrophic chondrocyte and bone phenotype, collagen type I and osteocalcin were markedly decreased compared to controls. Lower doses of 50 and 12.5 mg/kg caffeine also resulted in decreased cellular proliferation and transformation to cartilage histologically and reflected by significant inhibition of type II collagen mRNA levels (day 7). The effects of caffeine on gene expression observed in vivo during the period of bone formation (day 11 to day 15) in the DBP model were similar to the inhibited expression of H4, alkaline phosphatase, osteocalcin, and osteopontin found in fetal rat calvarial derived osteoblast cultures following 24 hour exposure of the cultures to 0.4 mM caffeine. Thus the observed delayed mineralization in the fetal skeleton associated with caffeine appears to be related to an inhibition of endochondral bone formation at the early stages of proliferation of undifferentiated mesenchymal cells to cartilage specific cells as well as at later stages of bone formation.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240520209
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  • 10
    Electronic Resource
    Electronic Resource
    Interferon α selectively affects expression of the human myeloid cell nuclear differentiation antigen in late stage cells in the monocytic but not the granulocytic lineage (1994)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 54 (1994), S. 198-206 
    Details
    ISSN: 0730-2312
    Keywords: granulocytes ; monocytes ; human myeloid cell lines ; retinoic acid ; phorbol ester ; mRNA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The human myeloid cell nuclear differentiation antigen (MNDA) is expressed constitutively in cells of the myeloid lineage, appearing in myeloblast cells in some cases of acute myeloid leukemia and consistently being detected in promyelocyte stage cells as well as in all later stage cells including peripheral blood monocytes and granulocytes. The human myeloid leukemia cell lines, HL-60, U937, and THP-1, express similar levels of immunochemically detectable MNDA. Although, the level of MNDA mRNA in primary monocytes is very low it was up-regulated at 6 h following the addition of interferon α. The effect of interferon α on the MNDA mRNA is also observed in the cell lines HL-60, U937, and THP-1. The MNDA mRNA level in primary granulocytes was unaffected by addition of interferon α and other agents including interferon γ, endotoxin, poly (I) · poly (C), and FMLP. The MNDA mRNA level in the myeloid cell lines was also unaffected by the latter four agents. Induction of differentiation in the myeloid cell lines with phorbol ester induces monocyte differentiation which was accompanied by a decrease in MNDA mRNA level. This reduced level of mRNA could then be elevated with subsequent interferon α treatment. The effects of phorbol ester on MNDA mRNA appeared to be associated with induced differentiation since inhibiting cell proliferation did not alter the level of MNDA mRNA and cell cycle variation in MNDA mRNA levels were not observed. The ability of interferon α to up-regulate MNDA mRNA in phorbol ester treated myeloid cell lines is consistent with the observations made in primary monocytes. Granulocyte differentiation induced by retinoic acid treatment of HL-60 cells did not alter the MNDA mRNA level which was also unchanged following subsequent treatment with interferon α. The lack of interferon α effects on retinoic acid treated HL-60 cells is consistent with its inability to influence MNDA mRNA level in primary granulocytes.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240540208
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