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1.
Cancer Res ; 54(1): 28-32, 1994 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-8261453

RESUMO

Interrelationships between nuclear architecture and gene expression were examined by comparing the representation of nuclear matrix proteins in ROS 17/2.8 rat and MG-63 human osteosarcoma cells with those in normal diploid osteoblasts. The tumor-derived cells coexpress genes which are expressed in a sequential and mutually exclusive manner during the progressive stages of osteoblast differentiation. In osteosarcoma cells two-dimensional electrophoretic analysis indicates a composite representation of nuclear matrix proteins characteristic of both the proliferative and postproliferative periods of osteoblast phenotype development. In addition, nuclear matrix proteins unique to the tumor cells and the absence of nuclear matrix proteins found only in normal diploid osteoblasts are observed. Tumor-specific nuclear matrix proteins include those expressed in a proliferation-dependent and independent manner. There is a parallel relationship between nuclear matrix proteins and the expression of cell growth and tissue-specific genes during osteoblast differentiation and in osteosarcoma cells where the developmental sequence of gene expression has been abrogated. Nuclear matrix proteins therefore provide markers reflecting defined periods of bone cell differentiation and phenotypic characteristics of an osteosarcoma.


Assuntos
Biomarcadores Tumorais/análise , Proteínas Nucleares/análise , Osteoblastos/química , Osteossarcoma/química , Animais , Antígenos Nucleares , Diferenciação Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Osteoblastos/citologia , Osteossarcoma/genética , Osteossarcoma/patologia , Fenótipo , Gravidez , Ratos , Ratos Sprague-Dawley , Células Tumorais Cultivadas
2.
J Bone Miner Res ; 13(2): 155-67, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9495508

RESUMO

The molecular mechanisms that couple osteoblast structure and gene expression are emerging from recent studies on the bone extracellular matrix, integrins, the cytoskeleton, and the nucleoskeleton (nuclear matrix). These proteins form a dynamic structural network, the tissue matrix, that physically links the genes with the substructure of the cell and its substrate. The molecular analog of cell structure is the geometry of the promoter. The degree of supercoiling and bending of promoter DNA can regulate transcriptional activity. Nuclear matrix proteins may render a change in cytoskeletal organization into a bend or twist in the promoter of target genes. We review the role of nuclear matrix proteins in the regulation of gene expression with special emphasis on osseous tissue. Nuclear matrix proteins bind to the osteocalcin and type I collagen promoters in osteoblasts. One such protein is Cbfa1, a recently described transcriptional activator of osteoblast differentiation. Although their mechanisms of action are unknown, some nuclear matrix proteins may act as "architectural" transcription factors, regulating gene expression by bending the promoter and altering the interactions between other trans-acting proteins. The osteoblast nuclear matrix is comprised of cell- and phenotype-specific proteins including proteins common to all cells. Nuclear matrix proteins specific to the osteoblast developmental stage and proteins that distinguish osteosarcoma from the osteoblast have been identified. Recent studies indicating that nuclear matrix proteins mediate bone cell response to parathyroid hormone and vitamin D are discussed.


Assuntos
Proteínas de Neoplasias , Proteínas Nucleares/metabolismo , Osteoblastos/metabolismo , Antígenos Nucleares , Diferenciação Celular/genética , Tamanho Celular/genética , Colágeno/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core , DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Proteínas Nucleares/genética , Osteoblastos/ultraestrutura , Osteocalcina/metabolismo , Hormônio Paratireóideo/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Transcrição Gênica
3.
J Bone Miner Res ; 16(6): 975-84, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11393794

RESUMO

With the discoveries of different death mechanisms, an emerging definition of apoptosis is the process of cell death associated with caspase activation or caspase-mediated cell death. This definition accepts that caspases represent the final common mechanistic pathway in apoptosis. Apoptosis may be triggered either by activation events that target mitochondria or endoplasmic reticulum or by activation of cell surface "death receptors," for example, those in the tumor necrosis factor (TNF) superfamily. In the postnatal and adult skeleton, apoptosis is integral to physiological bone turnover, repair, and regeneration. The balance of osteoblast proliferation, differentiation, and apoptosis determines the size of the osteoblast population at any given time. Although apoptosis has been recorded in many studies of bone, the selective mechanisms invoked in the different models studied rarely have been identified. This review offers a broad overview of the current general concepts and controversies in apoptosis research and then considers specific examples of osteoblast apoptosis pertinent to skeletal development and to the regulation of bone turnover. In reviewing selected work on interdigital apoptosis in the developing skeleton, we discuss the putative roles of the bone morphogenetic proteins (BMPs), Msx2, RAR-gamma, and death inducer obliterator 1 (DIO-1). In reviewing factors regulating apoptosis in the postnatal skeleton, we discuss roles of cytokines, growth factors, members of the TNF pathway, and the extracellular matrix (ECM). Finally, the paradoxical effects of parathyroid hormone (PTH) on osteoblast apoptosis in vivo are considered in the perspective of a recent hypothesis speculating that this may be a key mechanism to explain the anabolic effects of the hormone. An improved understanding of the apoptotic pathways and their functional outcomes in bone turnover and fracture healing may facilitate development of more targeted therapeutics to control bone balance in patients with osteoporosis and other skeletal diseases.


Assuntos
Apoptose/fisiologia , Remodelação Óssea/fisiologia , Osteoblastos/patologia , Animais , Caspases/metabolismo , Matriz Extracelular/fisiologia , Humanos , Osteoblastos/metabolismo , Hormônio Paratireóideo/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
4.
J Bone Miner Res ; 16(1): 10-23, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11149472

RESUMO

Collagen expression is coupled to cell structure in connective tissue. We propose that nuclear matrix architectural transcription factors link cell shape with collagen promoter geometry and activity. We previously indicated that nuclear matrix proteins (NP/NMP4) interact with the rat type I collagen alpha1(I) polypeptide chain (COL1A1) promoter at two poly(dT) sequences (sites A and B) and bend the DNA. Here, our objective was to determine whether NP/NMP4-COL1A1 binding influences promoter activity and to clone NP/NMP4. Promoter-reporter constructs containing 3.5 kilobases (kb) of COL1A1 5' flanking sequence were fused to a reporter gene. Mutation of site A or site B increased promoter activity in rat UMR-106 osteoblast-like cells. Several full-length complementary DNAs (cDNAs) were isolated from an expression library using site B as a probe. These clones expressed proteins with molecular weights and COLIA1 binding activity similar to NP/NMP4. Antibodies to these proteins disrupted native NP/NMP4-COL1A1 binding activity. Overexpression of specific clones in UMR-106 cells repressed COL1A1 promoter activity. The isolated cDNAs encode isoforms of Cys2His2 zinc finger proteins that contain an AT-hook, a motif found in architectural transcription factors. Some of these isoforms recently have been identified as Cas-interacting zinc finger proteins (CIZ) that localize to fibroblast focal adhesions and enhance metalloproteinase gene expression. We observed NP/NMP4/CIZ expression in osteocytes, osteoblasts, and chondrocytes in rat bone. We conclude that NP/NMP4/CIZ is a novel family of nuclear matrix transcription factors that may be part of a general mechanical pathway that couples cell structure and function during extracellular matrix remodeling.


Assuntos
Colágeno/genética , Regulação da Expressão Gênica , Proteínas Associadas à Matriz Nuclear , Matriz Nuclear/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteoblastos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Antígenos Nucleares , Desenvolvimento Ósseo/genética , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Linhagem Celular , Clonagem Molecular , Colágeno/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Mutação/genética , Proteínas Nucleares/química , Proteínas Nucleares/imunologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Elementos de Resposta/genética , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/imunologia , Dedos de Zinco/genética
5.
Endocrinology ; 129(6): 2993-3000, 1991 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1954883

RESUMO

We showed recently that the initial peak cytosolic ionized calcium ([Ca2+]i) response to PTH (2-min exposure) is preserved relative to the cAMP response in osteoblast-like rat osteosarcoma cells (ROS 17/2.8) desensitized by 72-h exposure to PTH. We attempted in the present studies to determine the mechanisms for preservation of the [Ca2+]i response and to explore the effects of longer PTH rechallenges. The [Ca2+]i response to a 20-min perifusion with rat PTH [rPTH-(1-34)] was monitored by aequorin luminescence in both naive and PTH-desensitized ROS 17/2.8 cells. The responses of both naive and desensitized cells consisted of two phases: an initial peak, followed by an intermediate plateau that was sustained in the presence of PTH. We observed in the naive cell populations synchronous oscillations in [Ca2+]i concentration during this second phase (amplitude, 10-60 nM; frequency, 1-3/100 sec). These oscillations were maintained through extracellular calcium (EC Ca2+) entry; the initial peak was the result of Ca2+ release from intracellular stores. In desensitized cells, these two phases could not be clearly separated with respect to Ca2+ source, but, as we showed before, exhibited an enhanced dependence on EC Ca2+ entry for the response to PTH. Nevertheless, in the desensitized cells, the sustained [Ca2+]i response was diminished in magnitude and showed little oscillatory behavior. Brief exposure to neomycin sulfate, an inhibitor of phosphoinositide turnover, attenuated the PTH-induced [Ca2+]i rise in both naive and desensitized cells. Protein kinase-C activity did not appear to be required for either phase of the PTH-induced [Ca2+]i response. Exposure to cholera toxin attenuated the [Ca2+]i response to hormone in both naive and desensitized cells, more markedly in the latter. Cholera toxin treatment dramatically increased basal cAMP levels in both cell preparations; PTH-stimulated cAMP production was unchanged in naive cells, but increased nearly 4-fold in desensitized cells. We propose that the preserved PTH-induced peak [Ca2+]i rise in desensitized cells results primarily from the diminished regulation of EC Ca2+ entry by the cAMP response limb. The attenuated sustained oscillatory behavior observed in desensitized cells upon rechallenge with hormone may be the result of reduced phosphoinositide turnover and reduced Ca2+-stimulated Ca2+ release. Thus, the [Ca2+]i response to PTH in osteoblast-like cells is complex and modulable and seems to provide a number of ways to regulate intracellular metabolism under various conditions. We speculate that this plasticity of the [Ca2+]i response to PTH is related to the pleiotropic actions of the hormone on cells of the osteoblast lineage.


Assuntos
Cálcio/metabolismo , Osteoblastos/metabolismo , Hormônio Paratireóideo/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Toxina da Cólera/farmacologia , Tolerância a Medicamentos , Neomicina/farmacologia , Osteoblastos/efeitos dos fármacos , Osteossarcoma , Periodicidade , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologia
6.
Endocrinology ; 128(2): 1021-8, 1991 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1846574

RESUMO

We have investigated the effects of PTH-induced desensitization on second messenger interactions in the rat osteosarcoma cell line ROS 17/2.8. Adenylate cyclase activation was assessed by accumulation of immunoassayable cAMP, and cytosolic calcium ion ([Ca2+]i) concentrations were measured in adherent perifused cells loaded with the Ca2(+)-sensitive bioluminescent protein aequorin. Preexposure to rat PTH-(1-34) [rPTH-(1-34); 10(-8) M for 48 h, then 10(-7) M for 24 h] dramatically reduced (by 85%) the cAMP response to fresh challenge [2 min; 10(-9)-10(-7) M rPTH-(1-34)], but the peak PTH-induced rise of [Ca2+]i was not diminished significantly (0-20%). Nevertheless, we did observe other changes in the PTH-induced [Ca2+]i response. Exposure of treated cells to (Bu)2cAMP nearly abolished the [Ca2+]i response to PTH (greater than 80% reduction), but had much less effect on the PTH-stimulated [Ca2+]i increment of the naive cells (less than 35% reduction). Treated cells also had a blunted [Ca2+]i response to PTH in the presence of low extracellular calcium (greater than 60% reduction), but in the naive cells, low extracellular Ca2+ did not significantly diminish the peak PTH-induced [Ca2+]i rise, although low extracellular Ca2+ dramatically reduced the area under this [Ca2+]i transient (greater than 50%). Low extracellular Ca2+ had no influence on the peak [Ca2+]i responses of treated cells to bradykinin or prostaglandin F2 alpha. Although the peak PTH-stimulated [Ca2+]i rise of treated cells in normal Ca2+ medium was not significantly attenuated, the time to half-maximum [Ca2+]i concentration was significantly increased (greater than 100%), and the area under the [Ca2+]i transient was diminished. These alterations in the [Ca2+]i response of treated cells were not observed upon challenge with bradykinin or prostaglandin F2 alpha. Thus, 1) the cAMP and [Ca2+]i responses of ROS 17/2.8 cells to rPTH-(1-34) are not obligatorily coupled; 2) the response of naive cells to PTH includes both the release of Ca2+ from intracellular stores and the entry of extracellular Ca2+; and 3) pretreatment of these cells with rPTH-(1-34) augments the dependence on Ca2+ entry during hormone rechallenge. We propose that the preserved PTH-stimulated [Ca2+]i rise in treated cells results partly from loss of cAMP-mediated inhibition of extracellular Ca2+ entry.


Assuntos
Cálcio/metabolismo , AMP Cíclico/metabolismo , Citosol/metabolismo , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Sistemas do Segundo Mensageiro , Animais , Bucladesina/farmacologia , Linhagem Celular , Íons , Osteoblastos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ratos , Teriparatida
7.
Crit Rev Eukaryot Gene Expr ; 11(4): 279-97, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-12067068

RESUMO

The functional role of the osteoblast nuclear matrix has been a matter of supposition. Its presumed function as an architectural agent of transcription derives primarily from the low solubility of nuclear matrix proteins and their typical localization into discrete subnuclear domains. In addressing how the nuclear matrix regulates skeletal genes, the authors compare Nmp4, Cbfal, and YY1 for the purpose of profiling osteoblast nuclear matrix transcription factors. All three proteins contribute to the transcription of ECM genes and partition into the osteoblast nuclear matrix via a nuclear matrix targeting domain. The authors propose that osteoblast nuclear matrix transcription factors involved in ECM regulation generally have the capacity to alter DNA geometry and reciprocally respond to DNA as an allosteric ligand. This may allow these proteins to adapt to the local nuclear architecture and generate the pattern of regulation specified by that architecture via unmasking of the appropriate transactivation domains. Osteoblast nuclear matrix transcription factors may also act as transcriptional adaptor molecules by supporting the formation of higher order protein complexes along target gene promoters. The genes encoding all three proteins considered here have trinucleotide repeat domains, although the significance of this is unclear. There is no canonical nuclear matrix binding motif, but finger-like structures may be suited for anchoring proteins to discrete subnuclear domains. Finally, the ability to leave the osteoblast nuclear matrix may be as important to the function of some nuclear matrix transcription factors as their association with this subcompartment.


Assuntos
Osso e Ossos/fisiologia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Proteínas Associadas à Matriz Nuclear , Matriz Nuclear/fisiologia , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genética , Animais , Antígenos Nucleares , Sequência de Bases , Sequência Consenso , Fatores de Ligação de DNA Eritroide Específicos , Humanos , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Ratos , Fator de Transcrição YY1
8.
Bone ; 22(4): 317-24, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9556130

RESUMO

The parathyroid hormone (PTH) signaling pathways that effect changes in osteoblast gene expression also alter the organization of the cytoskeletal proteins. PTH regulates the expression of nucleoskeletal proteins, such as nuclear mitotic apparatus protein (NuMA) and topoisomerase II-alpha. NuMA is a structural component of the interphase nucleus and organizes the microtubules of the mitotic spindle during mitogenesis. We propose that PTH-induced alterations in osteoblast cytoarchitecture are accompanied by changes in osteoblast nuclear structure that contribute to changes in gene expression. We used immunofluorescence and confocal microscopy to determine the effect of PTH on the expression and nuclear distribution of NuMA in the rat osteosarcoma cell line, ROS 17/2.8. Cells were treated with PTH or vehicle, then fixed and stained with NuMA antibody. Optical sections of interphase naive cells revealed a diffuse distribution of NuMA, interspersed with speckles, in the central nuclear planes but not in nucleoli. During the metaphase and anaphase, NuMA localized at the mitotic spindle apparatus. The percentage of NuMA-immunopositive ROS 17/2.8 cells decreased with increasing confluence, but serum starvation did not attenuate NuMA expression. Cell density-dependent changes in cytoskeletal organization were observed in these cells. PTH treatment induced changes in cytoskeletal organization and increased the percentage of NuMA-immunopositive ROS 17/2.8 cells. These data suggest that PTH effects changes in osteoblast nuclear architecture by regulating NuMA, and that these alterations may be coupled to cytoskeletal organization.


Assuntos
Proteínas Nucleares/biossíntese , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Animais , Autoantígenos/biossíntese , Autoantígenos/genética , Proteínas de Ciclo Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Microscopia Confocal , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Ratos , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Células Tumorais Cultivadas
9.
Bone ; 26(3): 227-34, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10709994

RESUMO

Bone cells undergo changes in cell structure during phenotypic development. Parathyroid hormone (PTH) induces a change in osteoblast shape, a determinant of collagen expression. We hypothesize that alterations in bone cell shape reflect and direct gene expression as governed, in part, by nuclear organization. In this study, we determined whether the expression of nuclear matrix proteins that mediate nuclear architecture, NuMA, topoisomerase II (topo II)-alpha, and -beta, were altered during osteoblast development and response to PTH in vivo. NuMA forms an interphase nuclear scaffold in some cells, the absence of which may accommodate alterations in nuclear organization necessary for specific functions. Topo II enzymes are expressed in bone cells; the alpha-isoform is specific to proliferating cells. We used immunohistochemistry and flow cytometry to determine whether NuMA is expressed in the primary spongiosa of the rat metaphyseal femur and whether expression of NuMA, topo II-alpha, and II-beta changes during osteoblast development or with PTH treatment. NuMA and topo II-beta were expressed in marrow cells, osteoblasts, osteocytes, and chondrocytes. These proteins were not detected in osteoclasts in vivo, but were observed in cultured cells. Bone marrow cells expressed topo II-alpha. All three proteins were expressed in cultures of rat osteoblast-like UMR-106 cells. PTH treatment downregulated the number of topo II-alpha-immunopositive cells, correlated with a decrease in S-phase cells, in both bone tissue and cell culture. We conclude that, in vivo, nuclear matrix composition is altered during bone cell development and that anabolic doses of PTH attenuate the proliferative capacity of osteogenic cells, in part, by targeting topo II-alpha expression.


Assuntos
Osso e Ossos/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Isoenzimas/metabolismo , Proteínas Nucleares/metabolismo , Hormônio Paratireóideo/farmacologia , Animais , Antígenos de Neoplasias , Antígenos Nucleares , Osso e Ossos/enzimologia , Osso e Ossos/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular , Células Cultivadas , Proteínas de Ligação a DNA , Masculino , Microscopia de Fluorescência , Ratos , Ratos Sprague-Dawley
10.
Bone ; 30(6): 931-6, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12052465

RESUMO

Nmp4 proteins are transcription factors that contribute to the expression of type I collagen and many of the matrix metalloproteinase genes. Numerous Nmp4 isoforms have been identified. These proteins, all derived from a single gene, have from five to eight Cys(2)His(2) zinc fingers, the arrangement of which directs specific isoforms to nuclear matrix subdomains. Nmp4 isoforms also have an SH3 binding domain, typical of cytoplasmic docking proteins. Although recent evidence indicates that Nmp4 proteins also reside in the osteoblast cytoplasm, whether they localize to specific organelles or structures is not well defined. The intracellular localization of a protein is a determinant of its function and provides insights into its mechanism of action. As a first step toward determining the functional relationship between the cytoplasmic and nuclear Nmp4 compartments, we mapped their location in the osteoblast cytoplasm. Immunocytochemical analysis of osteoblasts demonstrated that Nmp4 antibodies labeled the mitochondria, colocalized with Golgi protein 58K, and lightly stained the cytoplasm. Western analysis using Nmp4 antibodies revealed a complex profile of protein bands in the nuclear, mitochondrial, and cytosolic fractions. Several of these proteins were specific to defined intracellular domains. Consistent with the western analyses, reverse transcription-polymerase chain reaction (RT-PCR) analysis detected previously uncharacterized Nmp4 isoforms. These data necessarily enlarge the known Nmp4 family from nuclear matrix transcription factors to a more widely extended class of intracellular proteins.


Assuntos
Líquido Intracelular/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Osteoblastos/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3/química , Células 3T3/metabolismo , Animais , Animais Recém-Nascidos , Citoplasma/química , Citoplasma/metabolismo , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Líquido Intracelular/química , Masculino , Camundongos , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas Associadas à Matriz Nuclear/biossíntese , Osteoblastos/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/biossíntese
11.
Bone ; 27(2): 209-18, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10913913

RESUMO

Osteoblast differentiation and function can be studied in situ in the metaphysis of growing long bones. Proliferation and apoptosis dominate in the primary spongiosa subjacent to the growth plate, and differentiation and function dominate in the proximal metaphysis. Apoptosis of osteocytes dominates at the termination of the trabeculae in diaphyseal marrow. As parathyroid hormone regulates all phases of osteoblast development, we studied the in vivo regulation by human parathyroid hormone (1-34) (PTH) of apoptosis in bone cells of the distal metaphysis of young male rats. Rats were given PTH at 80 microg/kg per day, once daily, for 1-28 days. Bone cells were defined for flow cytometry as PTH1-receptor-positive (PTH1R(+)) and growth factor-receptor-positive (GFR(+)) cells. Apoptotic cells stained positive for either TdT-mediated dUTP-X nick end labeling (TUNEL) or annexin V (annV(+)) were detected by either flow cytometry or immunohistochemistry. Apoptosis was also assessed at the tissue level by RNAse protection and caspase enzyme activity assays. PTH increased apoptotic osteoblasts in the proliferating zone and apoptotic osteocytes in the terminal trabecular zone, by 40%-60% within 2-6 days of PTH treatment, but values became equivalent to controls after 21-28 days of treatment. This transient increase was confirmed in PTH1R(+), GFR(+) bone cells isolated by flow cytometry. There was no detectable change in the steady-state mRNA levels of selected apoptotic genes. Starting at 3 days, at the tissue level, PTH inhibited activity of caspases, which recognize the DEVD peptide substrate (caspases 2, 3, and/or 7), but not those caspases recognizing LEHD or YVAD peptide sequences. We speculate that the localized and tissue level effects of PTH on apoptosis can be explained on the basis of its anabolic effect on bone. The transient increase in apoptosis in the proliferating zone and terminal trabecular zone may be the result of the increased activation frequency and bone turnover seen with daily PTH treatment. As once-daily PTH increases the number of differentiated osteoblasts, and as these and hematopoietic marrow cells dominate metaphyseal tissue, inhibition of caspase activity may contribute to their prolonged survival, enabling extension of trabecular bone into the diaphyseal marrow to increase bone mass.


Assuntos
Apoptose/efeitos dos fármacos , Fêmur/citologia , Osteócitos/citologia , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Fatores Etários , Animais , Anexina A5/análise , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Diáfises/citologia , Citometria de Fluxo , Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Osteócitos/química , Osteócitos/enzimologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/análise , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptor IGF Tipo 1/análise , Receptores de Superfície Celular/análise , Receptores de Fatores de Crescimento de Fibroblastos/análise , Receptores de Hormônios Paratireóideos/análise , Receptores do Fator de Crescimento Derivado de Plaquetas/análise , Fator de Crescimento Transformador beta/análise , Receptor fas/genética
12.
Arch Oral Biol ; 40(11): 1047-51, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8670023

RESUMO

Tooth eruption activates a localized resorption and formation of alveolar bone and these activities depend upon the adjacent parts, coronal and basal, respectively, of the dental follicle-enamel epithelium. In this study the nuclear matrix-intermediate filament (NM-IF) proteins of these tissues were isolated in order to continue investigations into the molecular mechanisms underlying eruption. Dental follicles were removed from the third and fourth premolar of dogs at 13, 16 and 20 weeks (pre-, early, and mid-to-late eruption of these teeth) and NM-IF proteins were extracted from the coronal and basal halves. Most of the NM-IF protein profiles of these coronal and basal parts on one-dimensional, sodium dodecyl sulphate-polyacrylamide gel electrophoresis were remarkably constant, indicating an essentially uniform cellular composition. However, differences between these tissues were observed and some of these changed during eruption. Based on recent observations that nuclear matrix changes reflect and may even mediate cell-specific changes in gene expression, these findings suggest that changes in nuclear matrix proteins may be related to the molecular basis for some aspects of differential gene expression in the coronal and basal regions of the dental follicle and account for the ability of these tissues to activate bone resorption and formation during tooth eruption.


Assuntos
Esmalte Dentário/ultraestrutura , Saco Dentário/ultraestrutura , Proteínas de Filamentos Intermediários/análise , Proteínas Nucleares/análise , Erupção Dentária , Processo Alveolar/patologia , Animais , Dente Pré-Molar , Reabsorção Óssea/patologia , Cães , Eletroforese em Gel de Poliacrilamida , Epitélio/ultraestrutura , Expressão Gênica , Proteínas de Filamentos Intermediários/genética , Matriz Nuclear/química , Matriz Nuclear/genética , Matriz Nuclear/ultraestrutura , Proteínas Nucleares/genética , Dodecilsulfato de Sódio , Erupção Dentária/genética
13.
J Bone Joint Surg Br ; 82(8): 1174-6, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11132282

RESUMO

We describe ten patients with Turner's syndrome (karyotype 45, XO) who had leg lengthening for short stature. A high incidence of postoperative complications was encountered and many patients required intramedullary fixation as a salvage procedure. We discuss the reasons for this and highlight the differences between our findings and those of a similar series recently reported. In view of the considerable difficulties encountered, we do not recommend leg lengthening in Turner's syndrome.


Assuntos
Estatura , Alongamento Ósseo/efeitos adversos , Alongamento Ósseo/métodos , Desigualdade de Membros Inferiores/etiologia , Desigualdade de Membros Inferiores/cirurgia , Síndrome de Turner/complicações , Adolescente , Adulto , Alongamento Ósseo/instrumentação , Feminino , Fraturas do Fêmur/diagnóstico por imagem , Fraturas do Fêmur/etiologia , Fraturas do Fêmur/cirurgia , Fixação Intramedular de Fraturas , Consolidação da Fratura , Humanos , Cariotipagem , Desigualdade de Membros Inferiores/diagnóstico por imagem , Fenótipo , Radiografia , Resultado do Tratamento , Síndrome de Turner/genética
14.
J Exp Biol ; 141: 187-95, 1989 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2926318

RESUMO

When cephalopod eggs were incubated in artificial sea water it was found that they sometimes resulted in hatchlings with defects of the statocyst suprastructure, leading to the severe behavioural defect of uncontrolled swimming. Experiments in defined media (seven basic salts mixed in deionized water) with seven species of cephalopods demonstrated clearly that there is 100% normal development of the aragonite statoliths when strontium levels were 8 mg l-1. Conversely, statoliths did not develop when strontium was absent. In cuttlefish, the growth of the cuttlebone was also affected adversely when strontium was absent. In mariculture production tanks, supplementing commercial artificial sea water with strontium to normal levels of 8 mg l-1 almost eliminated the occurrence of abnormal hatchlings. Circumstantial evidence indicates that there is a critical window in development during which strontium is required for normal development. The role of strontium in biomineralization during embryogenesis is unknown, but it appears to be important in the Mollusca.


Assuntos
Moluscos/embriologia , Atividade Motora/fisiologia , Estrôncio , Animais , Decapodiformes/embriologia , Moluscos/crescimento & desenvolvimento , Moluscos/fisiologia , Necessidades Nutricionais , Octopodiformes/embriologia , Água do Mar , Natação
15.
Br J Sports Med ; 30(3): 264-5, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8889126

RESUMO

Compartment syndrome of the thigh is a rare but serious condition that is normally associated with closed trauma or compressive injury. A case of acute compartment syndrome of the thigh occurred in a 16 year old boy after intensive weight training. There was no evidence of muscle tear or focal haemorrhage during subsequent fasciotomy.


Assuntos
Síndromes Compartimentais/etiologia , Coxa da Perna , Levantamento de Peso/lesões , Doença Aguda , Adolescente , Síndromes Compartimentais/cirurgia , Edema/patologia , Fasciotomia , Hemorragia/patologia , Humanos , Masculino , Músculo Esquelético/patologia , Ruptura , Coxa da Perna/cirurgia
16.
Biol Bull ; 178(3): 231-238, 1990 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29314941

RESUMO

During embryogenesis of the marine opisthobranch gastropod Aplysia californica Cooper, 1863, there is a brief critical time (window) during which strontium is essential for the onset of calcification. The present study was undertaken to elucidate the role of this element in mineralization. Strontium performed no structural function; deformed shells of strontium-deprived animals had normal atomic crystal structure and the element was excluded during calcification. Calcium deposition and fixation was reduced by approximately 80% in the absence of strontium but was not significantly altered in the presence of sub-optimal concentrations of this metal ion despite dramatic deficits in shell and statolith morphology. This suggests that calcium deficiency per se is not responsible for deficits induced by strontium deprivation. The reduced total calcium may be a secondary effect resulting from the complete inhibition of precipitation. Strontium did not modulate total alkaline phosphatase activity or total sulfated mucopolysaccharide synthesis during embryogenesis, and no morphological abnormalities of the organic shell were observed. Although the role of strontium in embryonic calcification of Aplysia californica remains enigmatic, these data suggest that strontium affects a highly discrete regulatory component because these more general indicators of calcification and differentiation are unaffected by its absence.

17.
Calcif Tissue Int ; 75(5): 396-404, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15592796

RESUMO

beta-Catenin plays a dual role in cells: one at cell-cell junctions and one regulating gene transcription together with TCF (T-cell Factor) in the nucleus. Recently, a role for beta-catenin in osteoblast differentiation and gene expression has begun to be elucidated. Herein we investigated the effects of fluid shear stress (FSS) on beta-catenin signaling. FSS is a well-characterized anabolic stimulus for osteoblasts; however, the molecular mechanisms for the effects of this stimulation remain largely unknown. We found that 1 hour of laminar FSS (10 dynes/cm(2)) induced translocation of beta-catenin to the nucleus and activated a TCF-reporter gene. Analysis of upstream signals that may regulate beta-catenin signaling activity revealed two potential mechanisms for increased beta-catenin signaling. First, FSS induced a transient, but significant, increase in the phosphorylation of both glycogen synthase kinase 3beta (GSK-3beta) and Akt. Second, FSS reduced the levels of beta-catenin associated with N-cadherin, suggesting that less sequestration of beta-catenin by cadherins occurs in osteoblasts subjected to FSS. Functional analysts of potential genes regulated by beta-catenin signaling in osteoblasts revealed two novel observations. First, endogenous, nuclear beta-catenin purified from osteoblasts formed a complex with a TCF -binding element in the cyclooxygenase-2 promoter, and, second, overexpression of either a constitutively active beta-catenin molecule or inhibition of GSK-3beta activity increased basal cyclooxygenase-2 levels. Together, these data demonstrate for the first time that FSS modulates the activity of both GSK-3beta and beta-catenin and that these signaling molecules regulate cyclooxygenase-2 expression in osteoblasts.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Osteoblastos/fisiologia , Transdução de Sinais , Transativadores/metabolismo , Células 3T3 , Animais , Animais Recém-Nascidos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/genética , Ensaio de Desvio de Mobilidade Eletroforética , Técnica Indireta de Fluorescência para Anticorpo , Genes Reporter , Quinases da Glicogênio Sintase/metabolismo , Immunoblotting , Camundongos , Mutação , Fosforilação , Testes de Precipitina , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Crânio/citologia , Estresse Mecânico , Fatores de Tempo , Transativadores/genética , beta Catenina
18.
J Cell Biochem ; 69(3): 336-52, 1998 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-9581872

RESUMO

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 alpha1(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.


Assuntos
Colágeno/genética , Proteínas Associadas à Matriz Nuclear , Proteínas Nucleares/metabolismo , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Animais , Antígenos Nucleares , Sítios de Ligação , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Colágeno/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Peso Molecular , Proteínas Nucleares/química , Osteoblastos/metabolismo , Poli T/metabolismo , Ligação Proteica , Ratos , Fatores de Transcrição/química , Células Tumorais Cultivadas , Raios Ultravioleta
19.
J Cell Biochem ; 79(3): 506-17, 2000 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-10972987

RESUMO

The mechanisms underlying the coupling of type I collagen and matrix metalloproteinase (MMP) expression to cell structure and adhesion are poorly understood. We propose that nuclear matrix architectural transcription factors link cell structure and transcription via their association with nuclear matrix subdomains and by their capacity for altering promoter geometry. NP/NMP4 are nuclear matrix proteins that contain from five to eight Cys(2)His(2) zinc fingers. Some NP/NMP4 isoforms bind to the rat type I collagen alpha1(I) polypeptide chain promoter in the manner of architectural transcription factors and alter basal transcription in osteoblast-like cells (Thunyakitpisal et al. in review). Certain isoforms of NP/NMP4 are identical to CIZ, Cas-interacting zinc finger protein, a nucleocytoplasmic shuttling protein that associates with focal adhesions and regulates MMP expression [Nakamoto et al. (2000): Mol Cell Biol 20:1649-1658]. To better understand the role of subnuclear architecture in collagen and MMP expression, we mapped the osteoblast nuclear distribution of NP/NMP4 proteins and identified the functional motifs necessary for nuclear localization and nuclear matrix targeting. Immunofluorescence microscopy was used to determine the cellular and subnuclear distribution of native NP/NMP4 proteins and green fluorescent protein (GFP)-NP/NMP4 fusion proteins in osteoblast-like cells. All GFP-NP/NMP4 fusion proteins localized to the nucleus, but accumulated in distinct nuclear matrix subdomains. The zinc finger domain was necessary and sufficient for nuclear import and matrix targeting. We conclude that the arrangement of the NP/NMP4 zinc fingers largely determines the subnuclear location of these isoforms.


Assuntos
Colágeno/genética , Proteínas Associadas à Matriz Nuclear , Proteínas Nucleares/química , Osteoblastos/metabolismo , Isoformas de Proteínas/química , Fatores de Transcrição/química , Dedos de Zinco , Motivos de Aminoácidos , Animais , Antígenos Nucleares , Sítios de Ligação , Neoplasias Ósseas/patologia , Linhagem Celular/metabolismo , Núcleo Celular/metabolismo , Humanos , Rim , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Osteossarcoma/patologia , Regiões Promotoras Genéticas , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Splicing de RNA , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas/metabolismo
20.
J Cell Biochem ; 55(1): 4-15, 1994 May.
Artigo em Inglês | MEDLINE | ID: mdl-8083299

RESUMO

During the past several years it has become increasingly evident that the three-dimensional organization of the nucleus plays a critical role in transcriptional control. The principal theme of this prospect will be the contribution of nuclear structure to the regulation of gene expression as functionally related to development and maintenance of the osteoblast phenotype during establishment of bone tissue-like organization. The contributions of nuclear structure as it regulates and is regulated by the progressive developmental expression of cell growth and bone cell related genes will be examined. We will consider signalling mechanisms that integrate the complex and interdependent responsiveness to physiological mediators of osteoblast proliferation and differentiation. The focus will be on the involvement of the nuclear matrix, chromatin structure, and nucleosome organization in transcriptional control of cell growth and bone cell related genes. Findings are presented which are consistent with involvement of nuclear structure in gene regulatory mechanisms which support osteoblast differentiation by addressing four principal questions: 1) Does the representation of nuclear matrix proteins reflect the developmental stage-specific requirements for modifications in transcription during osteoblast differentiation? 2) Are developmental stage-specific transcription factors components of nuclear matrix proteins? 3) Can the nuclear matrix facilitate interrelationships between physiological regulatory signals that control transcription and the integration of activities of multiple promoter regulatory elements? 4) Are alterations in gene expression and cell phenotypic properties in transformed osteoblasts and osteosarcoma cells reflected by modifications in nuclear matrix proteins? There is a striking representation of nuclear matrix proteins unique to cells, tissues as well as developmental stages of differentiation, and tissue organization. Together with selective association of regulatory molecules with the nuclear matrix in a growth and differentiation-specific manner, there is a potential for application of nuclear matrix proteins in tumor diagnosis, assessment of tumor progression, and prognosis of therapies where properties of the transformed state of cells is modified. It is realistic to consider the utilization of nuclear matrix proteins for targeting regions of cell nuclei and specific genomic domains on the basis of developmental phenotypic properties or tissue pathology.


Assuntos
Núcleo Celular/ultraestrutura , Osteoblastos/citologia , Transdução de Sinais/genética , Transcrição Gênica , Animais , Sequência de Bases , Diferenciação Celular/genética , Divisão Celular/genética , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos/genética
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