RESUMEN
BACKGROUND AND OBJECTIVE: While it has long been known that amelogenin is essential for the proper development of enamel, its role has generally been seen as structural in nature. However, our new data implicate this protein in the regulation of cell signaling pathways in periodontal ligament cells and osteoblasts. In this article we report the successful purification of a recombinant mouse amelogenin protein and demonstrate that it has signaling activity in isolated mouse calvarial cells and human periodontal ligament cells. MATERIAL AND METHODS: To determine the regulatory function of canonical Wnt signaling by amelogenin, we used TOPGAL transgenic mice. These mice express a beta-galactosidase transgene under the control of a LEF/TCF and beta-catenin-inducible promoter. To investigate in greater detail the molecular mechanisms involved in the beta-catenin signaling pathway, isolated osteoblasts and periodontal ligament cells were exposed to full-length recombinant mouse amelogenin and were evaluated for phenotypic changes and beta-catenin signaling using a TOPFLASH construct and the LacZ reporter gene. RESULTS: In these in vitro models, we showed that amelogenin can activate beta-catenin signaling. CONCLUSION: Using the TOPGAL transgenic mouse we showed that amelogenin expression in vivo is localized mainly around the root, the periodontal ligament and the alveolar bone.
Asunto(s)
Amelogenina/fisiología , Osteoblastos/metabolismo , Ligamento Periodontal/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proceso Alveolar/metabolismo , Amelogenina/biosíntesis , Amelogenina/genética , Animales , Células Cultivadas , Expresión Génica , Genes Reporteros , Humanos , Ratones , Ratones Transgénicos , Ligamento Periodontal/citología , Proteínas Recombinantes/farmacología , Factores de Transcripción TCF/metabolismo , Raíz del Diente/metabolismo , Transfección , beta Catenina/biosíntesis , beta-Galactosidasa/biosíntesisRESUMEN
The etiology of midface retrusion remains largely unclear. We hypothesized that the cranial base synchondroses play a key role in the development of the craniofacial skeleton in the Sandhoff mouse model. We observed that developmental abnormalities of the cranial base synchondroses involving proliferative chondrocytes are important in craniofacial growth and development. Neonatal restitution of beta-hexosaminidase in mutant mice by gene therapy successfully ameliorated the attendant skeletal defects and restored craniofacial morphology in vivo, suggesting this as a critical temporal window in craniofacial development. Analysis of our data implicates parathyroid-related peptide (PTHrP) and cyclo-oxygenase-2 (COX-2) as possible factors underlying the development of the aforementioned skeletal defects. Hence, timely restitution of a genetic deficiency or, alternatively, the restoration of PTHrP or cyclo-oxygenase activity by the administration of PTH and/or non-steroidal anti-inflammatory drugs or COX-2 selective inhibitors to affected individuals may prove beneficial in the management of midface retrusion.
Asunto(s)
Huesos Faciales/anomalías , Desarrollo Maxilofacial/fisiología , Enfermedad de Sandhoff/genética , Base del Cráneo/crecimiento & desarrollo , beta-N-Acetilhexosaminidasas/fisiología , Animales , Cefalometría , Condrocitos/patología , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Terapia Genética , Placa de Crecimiento/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteína Relacionada con la Hormona Paratiroidea/deficiencia , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Enfermedad de Sandhoff/terapia , beta-N-Acetilhexosaminidasas/deficiencia , beta-N-Acetilhexosaminidasas/genéticaRESUMEN
Multiple inositol polyphosphate phosphatase (Minpp1) metabolizes inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) and inositol hexakisphosphate (InsP(6)) with high affinity in vitro. However, Minpp1 is compartmentalized in the endoplasmic reticulum (ER) lumen, where access of enzyme to these predominantly cytosolic substrates in vivo has not previously been demonstrated. To gain insight into the physiological activity of Minpp1, Minpp1-deficient mice were generated by homologous recombination. Tissue extracts from Minpp1-deficient mice lacked detectable Minpp1 mRNA expression and Minpp1 enzyme activity. Unexpectedly, Minpp1-deficient mice were viable, fertile, and without obvious defects. Although Minpp1 expression is upregulated during chondrocyte hypertrophy, normal chondrocyte differentiation and bone development were observed in Minpp1-deficient mice. Biochemical analyses demonstrate that InsP(5) and InsP(6) are in vivo substrates for ER-based Minpp1, as levels of these polyphosphates in Minpp1-deficient embryonic fibroblasts were 30 to 45% higher than in wild-type cells. This increase was reversed by reintroducing exogenous Minpp1 into the ER. Thus, ER-based Minpp1 plays a significant role in the maintenance of steady-state levels of InsP(5) and InsP(6). These polyphosphates could be reduced below their natural levels by aberrant expression in the cytosol of a truncated Minpp1 lacking its ER-targeting N terminus. This was accompanied by slowed cellular proliferation, indicating that maintenance of cellular InsP(5) and InsP(6) is essential to normal cell growth. Yet, depletion of cellular inositol polyphosphates during erythropoiesis emerges as an additional physiological activity of Minpp1; loss of this enzyme activity in erythrocytes from Minpp1-deficient mice was accompanied by upregulation of a novel, substitutive inositol polyphosphate phosphatase.
Asunto(s)
Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/fisiología , Células 3T3 , Animales , Northern Blotting , Diferenciación Celular , División Celular , Células Cultivadas , Condrocitos/citología , Cromatografía Líquida de Alta Presión , Citosol/metabolismo , Embrión de Mamíferos/metabolismo , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Hibridación in Situ , Fosfatos de Inositol/metabolismo , Ratones , Ratones Transgénicos , Modelos Genéticos , Fenotipo , Monoéster Fosfórico Hidrolasas/biosíntesis , Ácido Fítico/metabolismo , ARN Mensajero/metabolismo , Recombinación Genética , Factores de Tiempo , Regulación hacia ArribaRESUMEN
To investigate the role of the transcription factor nuclear factor kappaB (NFkappaB) in tumor metastasis, we generated a murine lung alveolar carcinoma cell line (Line 1) defective in NFkappaB-signaling by retroviral delivery of a dominant negative inhibitor of NFkappaB. The NFkappaB signal blockade resulted in the down-regulation of prometastatic matrix metalloproteinase 9, a urokinase-like plasminogen activator, and heparanase and reciprocal up-regulation of antimetastatic tissue inhibitors of matrix metalloproteinases 1 and 2 and plasminogen activator inhibitor 2. NFkappaB signal blockade did not affect tumor cell proliferation in vitro or in vivo but prevented intravasation of tumor cells in an in vivo chick chorioallantoic membrane model of metastasis as well as spontaneous metastasis in a murine model. These findings suggest that NFkappaB plays a central and specific role in the regulation of tumor metastasis and may be an important therapeutic target for development of antimetastatic cancer treatments.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , FN-kappa B/fisiología , Metástasis de la Neoplasia/genética , Adenocarcinoma Bronquioloalveolar/enzimología , Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma Bronquioloalveolar/secundario , Alantoides/irrigación sanguínea , Animales , División Celular/fisiología , Embrión de Pollo , Corion/irrigación sanguínea , Regulación hacia Abajo , Glucuronidasa/biosíntesis , Glucuronidasa/genética , Humanos , Proteínas I-kappa B/genética , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Metaloproteinasa 9 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos BALB C , FN-kappa B/antagonistas & inhibidores , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-2/biosíntesis , Inhibidor Tisular de Metaloproteinasa-2/genética , Transfección , Regulación hacia Arriba , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
A true collagenase (EC 3.4.24.3) which had been discovered previously in bone culture fluids and extracts of whole bone has now been localized to the cellular component of bone. The cellular enzyme bears the same characteristics as that of bone collagenases described earlier. Moreover, it is directly extractable in relatively large quantities. Attempts to demonstrate the presence of a bone cell procollagease were unsuccessful. It was also observed that the cells secrete significant amounts of collagenase in vitro. With increasing incubation time the extracellular collagenase levels rise and the intracellular collagenase levels drop.
Asunto(s)
Huesos/enzimología , Colagenasa Microbiana/metabolismo , Animales , Huesos/citología , Calcio/farmacología , Clostridium/enzimología , Ácido Edético/farmacología , Precursores Enzimáticos , Calor , Masculino , Peso Molecular , RatasRESUMEN
Transforming growth factor beta and fibroblast growth factor are mitogens for chick growth plate chondrocytes. TGF-beta stimulated a 3.5-fold increase, and FGF a 13.5-fold increase in the rate of thymidine incorporation after a 24 h exposure. TGF-beta and FGF were synergistic in chondrocytes, causing a 73-fold stimulation in thymidine incorporation compared with control. This synergistic response was not dependent upon the simultaneous presence of both mitogens. Sequential exposure of chondrocytes to TGF-beta and FGF in either order reproduced in large part the synergistic interaction observed when both growth factors were present simultaneously. The time required for induction of the subsequent synergistic response was brief and, in the case of TGF-beta, corresponded to the time required for [125I]TGF-beta receptor binding. EGF and PDGF were not mitogenic for chondrocytes, and neither of these factors enhanced the response of the cells to either TGF-beta or FGF. Finally, TGF-beta and FGF did not, either alone or in combination, elevate intracellular cAMP levels. These results emphasize the importance of examining growth factor effects in the context of other growth regulators. Furthermore, this specific and dramatic synergistic stimulation of thymidine incorporation may provide a useful tool in elucidating the mitogenic mechanism of the individual growth factors.
Asunto(s)
ADN/biosíntesis , Factores de Crecimiento de Fibroblastos/administración & dosificación , Placa de Crecimiento/efectos de los fármacos , Factor de Crecimiento Transformador beta/administración & dosificación , Animales , Pollos , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Técnicas In Vitro , CinéticaRESUMEN
Type X collagen is produced by hypertrophic chondrocytes and serves as a highly specific marker for chondrocyte maturation. This study was designed to compare the expression of type II and type X collagen in growth plate sections and in distinct populations of chondrocytes in culture by in situ hybridization. Growth plate sections were treated with type II and type X collagen cDNA probes. Type II collagen mRNA was present throughout the growth plate but greatest in the lower proliferating and upper hypertrophic regions. In contrast, type X collagen was expressed only in the hypertrophic region. Northern analysis confirmed the specificity of the probe for type X collagen mRNA. Chick growth plate chondrocytes were separated by countercurrent centrifugal elutriation into five distinct populations and plated in serum-containing medium. These cultures were examined at varying times after plating for the expression of type II and type X collagen mRNA. At 3 h, type II collagen was present in the majority of the cells in all fractions, and approximately 15-20% of the cells expressed type X collagen mRNA. The cells expressing type X were from the hypertrophic region. At 24 h, however, nearly all cells in culture expressed type X mRNA, and there was a decrease in expression of type II collagen mRNA. Similar results were obtained in cultures in the absence of serum, and SDS-PAGE analysis of collagen synthesis confirmed the expression of type X collagen in all populations of fractionated cells at 24 h at the protein level. Type X collagen is an important marker through which cellular matruation can be evaluated in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Colágeno/biosíntesis , Placa de Crecimiento/metabolismo , Animales , Autorradiografía , Northern Blotting , Fraccionamiento Celular , Células Cultivadas , Pollos , Colágeno/genética , ADN Complementario/genética , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Placa de Crecimiento/citología , Hibridación in Situ , Sondas de Oligonucleótidos , ARN Mensajero/metabolismoRESUMEN
Prostaglandins are locally produced in a number of tissues in response to a variety of stimuli, including local growth factors and systemic hormones. The present investigation characterizes prostaglandin effects on growth plate chondrocytes. Since cyclic adenosine monophosphate (cAMP) may act as a prostaglandin-stimulated second messenger, the effects of prostaglandins A1, D2, E1, E2, F2 alpha, and I2 (10(-10)-10(-6) M) on cAMP levels and thymidine incorporation were evaluated. The stimulation of cAMP and thymidine incorporation by the various prostaglandin metabolites were dose dependent and highly correlated (r = 0.99, p less than 0.001). The magnitude of the effect varied but was maximal at 10(-6) M for each of the prostaglandins. Prostaglandins of the E series (E1 and E2) were the most potent, causing significant effects at 10(-10) M and with maximal 12- and 13-fold increases in DNA synthesis after a 24 h exposure. Prostaglandins D2 and A1 maximally stimulated thymidine incorporation by 4.7- and 3.1-fold but caused significant increases only at 10(-8) M. Prostaglandins F2 alpha and I2 were the least stimulatory, producing small but significant increases in thymidine incorporation at 10(-6) M (30 and 100% stimulations). A causal relationship between cAMP and thymidine incorporation was further verified by the ability of dibutyryl-cAMP to increase DNA synthesis. Long-term chondrocyte cultures treated continuously with PGE2 demonstrated an increase in cell number, confirming the proliferative effect. Indomethacin did not alter the potent dose-dependent stimulations of chondrocyte DNA synthesis by TGF-beta 1, basic FGF, or PTH, indicating that these known mitogens act independently of prostaglandin metabolism. PGE2 was further examined for its effects of matrix synthesis. PGE2 inhibited collagen synthesis with a maximal 42% decrease but did not alter noncollagen protein synthesis. In contrast, PGE2 maximally increased sulfate incorporation by 35% and caused a small dose-dependent inhibition in alkaline phosphatase activity. Thus, prostaglandins alter DNA and matrix synthesis in growth plate chondrocytes and may have an important role in chondrocyte metabolism in the growth plate, fracture callus, and other areas of endochondral ossification.
Asunto(s)
Matriz Ósea/efectos de los fármacos , ADN/biosíntesis , Placa de Crecimiento/efectos de los fármacos , Indometacina/farmacología , Prostaglandinas/fisiología , Fosfatasa Alcalina/metabolismo , Animales , Matriz Ósea/crecimiento & desarrollo , Células Cultivadas , Pollos , AMP Cíclico/análisis , Dinoprostona/análisis , Factor 2 de Crecimiento de Fibroblastos/farmacología , Placa de Crecimiento/citología , Placa de Crecimiento/crecimiento & desarrollo , Hormona Paratiroidea/farmacología , Biosíntesis de Proteínas , Proteoglicanos/biosíntesis , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
While parathyroid hormone-related protein (PTHrP) has been characterized as an important negative regulator of chondrocyte maturation in the growth plate, the autocrine or paracrine factors that stimulate chondrocyte maturation are not well characterized. Cephalic sternal chondrocytes were isolated from 13-day embryos, and the role of bone morphogenetic protein-6 (BMP-6) as a positive regulator of chondrocyte maturation was examined in monolayer cultures. Progressive maturation, which was accelerated in the presence of ascorbate, occurred in the cultures. During maturation, the cultures expressed high levels of BMP-6 mRNA which preceded the induction of type X collagen mRNA. Treatment of the cultures with PTHrP (10(-7) M) at the time of plating completely abolished BMP-6 and type X collagen mRNA expression. Removal of PTHrP after 6 days was followed by the rapid (within 24 h) expression of BMP-6 and type X collagen mRNA, with BMP-6 again preceding type X collagen expression. The addition of exogenous BMP-6 (100 ng/ml) to the cultures accelerated the maturation process both in the presence and absence of ascorbate and resulted in the highest levels of type X collagen. When exogenous BMP-6 was added to PTHrP containing cultures, maturation occurred with the expression of high levels of type X collagen, despite the presence of PTHrP in the cultures. Furthermore, BMP-6 did not stimulate expression of its own mRNA in the PTHrP treated cultures, but it did stimulate the expression of Indian hedgehog (Ihh) mRNA. These latter findings suggest that while PTHrP directly inhibits BMP-6, it indirectly regulates Ihh expression through BMP-6. Other phenotypic changes associated with chondrocyte differentiation were also stimulated by BMP-6, including increased alkaline phosphatase activity and decreased proliferation. The results suggest that BMP-6 is an autocrine factor that initiates chondrocyte maturation and that PTHrP may prevent maturation by inhibiting the expression of BMP-6.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Condrocitos/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Ácido Ascórbico/farmacología , Proteína Morfogenética Ósea 6 , Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Condrocitos/citología , Condrocitos/metabolismo , Colágeno/genética , Expresión Génica/efectos de los fármacos , Modelos Biológicos , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Timidina/metabolismoRESUMEN
Endochondral ossification in fracture healing of rats at 4, 8, 11, 14, and 21 days was analyzed using immunological and molecular probes for markers of the chondrocyte and osteoblast phenotype. These markers were osteocalcin, type I and type II collagen, including the probes homologous to the alternatively spliced forms of alpha 1 type II collagen, type IIA and type IIB. Histologic examination was performed on serial sections of the same tissue blocks to correlate cellular morphology with the immunohistochemical and in situ hybridization findings. At the junction of the cartilaginous and osseous tissue, an overlap of phenotype and morphology was noted. At the 8-day time point, the cells with chondrocyte morphology expressed intracellular message for osteocalcin and type I collagen. Immunohistochemical analysis of these cells also demonstrated intracellular osteocalcin. However, high levels of the type IIA collagen mRNA, which has previously been associated with less differentiated mesenchymal precursor cells, were expressed in both chondrocytes and osteoblasts. At the later time point (21 days) there was a substantial decrease in the number of cells displaying shared phenotypic characteristics. In situ hybridization and immunohistochemistry have permitted identification of an overlapping or shared phenotype in osteoblasts and chondroblasts in fracture callus. The findings raise important questions regarding the possible plasticity of mesenchymal cell phenotypes within the dynamic environment of fracture healing. Additional examination of these issues will further define factors involved in origin, differentiation, and maturation of bone and cartilage cells.
Asunto(s)
Callo Óseo/citología , Cartílago/metabolismo , Curación de Fractura/fisiología , Osteoblastos/metabolismo , Animales , Calcificación Fisiológica/genética , Calcificación Fisiológica/fisiología , Cartílago/citología , Diferenciación Celular/genética , Colágeno/genética , Colágeno/metabolismo , ADN Complementario/metabolismo , Modelos Animales de Enfermedad , Femenino , Curación de Fractura/genética , Fracturas Óseas/metabolismo , Inmunohistoquímica , Hibridación in Situ , Sondas de Oligonucleótidos , Osteoblastos/citología , Osteocalcina/genética , Osteocalcina/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Tibia/lesionesRESUMEN
Isolated avian growth plate chondrocytes convert the acetoxymethyl ester (AM) form of Fura-2 quickly and efficiently to the Ca2(+)-sensitive pentacarboxylic acid (FA) form. Control experiments indicate that the Kd for intracellular Fura-2/FA is very close to that of extracellular Fura-2/FA at the same ionic strength and pH and that the Fura-2/FA fluorescence from indicator converted by intracellular organelles is quite small. Correcting for the effects of extracellular Fura-2/FA and partial hydrolysis products has improved the accuracy of determination of intracellular [Ca2+] over earlier measurements in chondrocytes. Cytosolic [Ca2+] in isolated growth plate chondrocytes (containing cells from each maturational stage) is found to require approximately 9 hours to recover from the isolation process. After this recovery period, cytosolic [Ca2+] in these cells converges to approximately 70 nM regardless of the [Ca2+] of the recovery medium, suggesting regulation of cytosolic [Ca2+] to a set point. Chondrocytes that are separated into maturationally distinct fractions using countercurrent centrifugal elutriation show an increase in cytosolic [Ca2+] with cellular maturation. The least mature resting cells have a [Ca2+] near 57 nM, while the most mature hypertrophic cells are around 95 nM.
Asunto(s)
Calcio/análisis , Citosol/química , Placa de Crecimiento/citología , Animales , Calcificación Fisiológica/fisiología , Diferenciación Celular , Fraccionamiento Celular , Separación Celular , Pollos , Fura-2/análogos & derivados , Fura-2/metabolismo , Fura-2/farmacología , Placa de Crecimiento/química , Placa de Crecimiento/fisiología , Orgánulos/metabolismoRESUMEN
Parathyroid hormone (PTH) is one of the most potent stimulators of growth plate chondrocyte mitogenesis that has been reported. However, study of the second messenger signaling mechanisms involved in the transduction of the hormone's effects on these cells is incomplete. Our data indicate that in addition to stimulating cyclic adenosine-3'5'-monophosphate metabolism, PTH also activates the phosphoinositide cascade, the pathway responsible for the generation of inositol-1,4,5-trisphosphate dependent Ca2+ signals. Our conclusion that PTH activates the phosphoinositide cascade is based on data that demonstrate: (1) the Ca2+ transients evoked by the hormone are dependent on intracellular Ca2+ stores; (2) the hormone stimulates the release of radiolabeled inositol from GPC plasma membranes; and (3) the hormone stimulates a greater than 8-fold increase in cytosolic inositol-1,4,5-trisphosphate pool size.
Asunto(s)
Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Hormona Paratiroidea/farmacología , Fosfatidilinositoles/metabolismo , Animales , División Celular/efectos de los fármacos , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , Pollos , Placa de Crecimiento/química , Inositol 1,4,5-Trifosfato/análisis , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatidilinositoles/análisisRESUMEN
Cells isolated from fetal rat calvaria were cultured in a [14C]glycine-labeled collagen gel. This method of culture places the cells in an environment similar to that in the intact tissue and provides a means for assaying the release of collagenase from the cells. Degradation of the collagen matrix begins within the first 3 h of cell culture, the earliest time point at which samples were taken, and continues for at least 12 h. When cells were prepared from calvaria incubated for 30 min with parathyroid hormone (PTH), there was a marked decrease in the collagenase content of the freshly isolated cells, indicating that secretion of collagenase had been enhanced by PTH. Upon subsequent culture of control and PTH-pretreated cells, there was an increase in lysis of collagen gels surrounding the hormone-treated cells within 8 h. The increased collagenolysis was prevented by exposure of the cells to puromycin before and during culture, suggesting that collagenase synthesis also was stimulated by PTH.
Asunto(s)
Huesos/enzimología , Colagenasa Microbiana/metabolismo , Hormona Paratiroidea/farmacología , Animales , Huesos/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Feto , Puromicina/farmacología , RatasRESUMEN
Isolated bone cells in culture contain an enzyme capable of hydrolyzing the phosphate ester of phosphotyrosine. This enzyme, which we have termed phosphotyrosine phosphatase, has not previously been reported in bone. Some of its characteristics include: 1) maximum activity near physiological pH, 2) a Km for substrate of 52 microM, 3) marked inhibition by the phosphate analog vanadate ion, 4) activity correlation with bone cell alkaline phosphatase, and 5) regulation by bone target hormones. Data obtained with vanadate ion support the contention that this enzyme may play a role in the regulation of bone cell growth.
Asunto(s)
Huesos/enzimología , Calcitriol/farmacología , Hormona Paratiroidea/farmacología , Fosfoproteínas Fosfatasas/análisis , Tirosina/análogos & derivados , Fosfatasa Alcalina/análisis , Animales , Huesos/efectos de los fármacos , División Celular/efectos de los fármacos , Embrión de Pollo , Concentración de Iones de Hidrógeno , Cinética , Masculino , Fosfoproteínas Fosfatasas/aislamiento & purificación , Fosfotirosina , Proteínas Tirosina Fosfatasas , Ratas , Tirosina/metabolismoRESUMEN
Transforming growth factor-beta (TGF beta) is capable of regulating the proliferation and phenotypic expression of growth plate chondrocytes in culture. Chondrocytes were isolated from the growth plates from the long bones of 3- to 5-week-old chicks. Conditioned medium was harvested from short term monolayer cultures for the assay of TGF beta production by these cells. A receptor competition assay using [125I]TGF beta was used to quantitate the amount of TGF beta in the conditioned medium. Acid-activated conditioned medium contained 5.0 +/- 0.4 ng/ml TGF beta, while conditioned medium that had not been exposed to acid had undetectable levels of the peptide by this assay. The initial cell plating density was inversely related to the amount of TGF beta produced on a per cell basis. Growth plate chondrocytes separated by countercurrent centrifugal elutriation into maturationally distinct subpopulations had different rates of TGF beta production; hypertropic chondrocytes produced significantly more TGF beta (4.5 ng/10(6) cells) than the smallest chondrocytes isolated (2.3 ng/10(6) cells). A variety of other growth mediators were tested for their ability to influence TGF beta production by chondrocytes, and it was found that only basic fibroblast growth factor could significantly influence TGF beta production, producing a 6-fold increase in TGF beta recovered in the conditioned medium. The production of TGF beta by growth plate chondrocytes implicates it as an important autocrine or paracrine regulator in the process of endochondral calcification.
Asunto(s)
Placa de Crecimiento/metabolismo , Factores de Crecimiento Transformadores/biosíntesis , Animales , Unión Competitiva , Recuento de Células , Línea Celular , Células Cultivadas , Pollos , Factores de Crecimiento de Fibroblastos/farmacología , Fibroblastos/metabolismo , Sustancias de Crecimiento/farmacología , Concentración de Iones de HidrógenoRESUMEN
Transforming growth factor-beta (TGF beta) is a local regulator of cell metabolism and growth. TGF beta increases the synthesis of collagen and enhances the deposition of matrix by almost all cells studied to date. The presence of TGF beta in cartilage suggests an important autocrine function, and the present study was designed to examine its influence on the matrix synthesis of chick epiphyseal chondrocytes. Chondrocytes were plated in serum-free (BSA-supplemented) medium or medium containing 10% fetal bovine serum (FBS), and after 24 h in monolayer culture were treated with TGF beta in identical medium. A 24-h incubation with TGF beta caused a dose-dependent decrease in collagen synthesis (-14%) and increase in noncollagen protein synthesis (+25%), with greater effects in serum-containing medium (-22% and +58%, respectively). Similarly, the stimulation of sulfate incorporation by TGF beta was greater in FBS-containing medium (+140%) than in serum-free medium (+70%). These changes were present by 6 h, were maximal in the 0.3-3.0 ng/ml dose range, and were found to reflect an alteration in extracellular protein synthesis. The enhancement of TGF beta effects by serum was abolished when chondrocytes were plated and exposed to TGF beta in medium containing dialyzed FBS (12-14K membrane). The present study indicates that TGF beta influences the synthesis of matrix components by growth plate chondrocytes. The effects are enhanced by factors present in serum.
Asunto(s)
Cartílago/metabolismo , Matriz Extracelular/metabolismo , Placa de Crecimiento/metabolismo , Péptidos/farmacología , Animales , Sangre , Cartílago/efectos de los fármacos , Células Cultivadas , Pollos , Colágeno/biosíntesis , Medios de Cultivo , Matriz Extracelular/efectos de los fármacos , Placa de Crecimiento/efectos de los fármacos , Sustancias de Crecimiento , Cinética , Biosíntesis de Proteínas , Proteoglicanos/biosíntesis , Sulfatos/metabolismo , Factores de Crecimiento TransformadoresRESUMEN
Cells isolated from embryonic chick intestine in culture convert 25-hydroxyvitamin-D3 to a number of more polar metabolites. Two of these metabolites have been identified with chemical and chromatographic methods as 24,25-dihydroxyvitamin-D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin-D3 (1,25(OH)2D3). The enzymes conform to substrate saturation kinetics. The apparent Km for substrate for the synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 is 70 nM and 29 nM, respectively.
Asunto(s)
Calcitriol/biosíntesis , Dihidroxicolecalciferoles/biosíntesis , Intestinos/embriología , 24,25-Dihidroxivitamina D 3 , Animales , Células Cultivadas , Embrión de Pollo , Cromatografía Líquida de Alta Presión , Mucosa Intestinal/metabolismo , CinéticaRESUMEN
Transforming growth factor-beta (TGF-beta) is a multifunctional regulator of a variety of cellular functions, including proliferation, differentiation, matrix synthesis, and apoptosis. In growth plate chondrocytes, TGF-beta slows the rate of maturation. Because the current paradigm of TGF-beta signaling involves Smad proteins as downstream regulators of target genes, we have characterized their role as mediators of TGF-beta effects on chondrocyte maturation. Both Smad2 and 3 translocated to the nucleus upon TGF-beta1 signaling, but not upon BMP-2 signaling. Cotransfection experiments using the TGF-beta responsive and Smad3 sensitive p3TP-Lux luciferase reporter demonstrated that wild-type Smad3 potentiated, whereas dominant negative Smad3 inhibited TGF-beta1 induced luciferase activity. To confirm the role of Smad2 and 3 as essential mediators of TGF-beta1 effects on chondrocyte maturation, we overexpressed both wild-type and dominant negative Smad2 and 3 in virally infected chondrocyte cultures. Overexpression of both wild-type Smad2 and 3 potentiated the inhibitory effect of TGF-beta on chondrocyte maturation, as determined by colx and alkaline phosphatase activity, whereas dominant negative Smad2 and 3 blocked these effects. Wild-type and dominant negative forms of Smad3 had more pronounced effects than Smad2. Our results define Smad2 and 3 as key mediators of the inhibitory effect of TGF-beta1 signaling on chondrocyte maturation.
Asunto(s)
Condrocitos/citología , Proteínas de Unión al ADN/fisiología , Transactivadores/fisiología , Factor de Crecimiento Transformador beta/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Embrión de Pollo , Condrocitos/efectos de los fármacos , Colágeno/genética , Proteínas de Unión al ADN/genética , Expresión Génica , Luciferasas/genética , ARN Mensajero/análisis , Transducción de Señal , Proteína Smad2 , Proteína smad3 , Transactivadores/genética , TransfecciónRESUMEN
Human prostatic acid phosphatase (hPAP) directly enhances the differentiated characteristics of isolated bone cells in vitro. This enzyme, when added to cell cultures for 24 h in vitro stimulates collagen synthesis and the production of alkaline phosphatase. The effects are dose dependent, with statistically significant effects occurring from 0.1-100 nM hPAP. Concentrations higher than 100 nM do not evoke greater effects. The maximal effect of hPAP occurs between 12 and 24 h of exposure. The cells stimulated to the greatest degree are osteoprogenitor cells and osteoblasts. Fibroblasts isolated from the same tissue show a lesser sensitivity to hPAP. hPAP has no detectable effect on cell proliferation, as measured by radiolabeled thymidine incorporation or total DNA synthesis. None of the observations reported in this work can be attributed to contaminating proteins in the hPAP preparation. hPAP was radiolabeled with 125I and was used for affinity binding and cross-linking studies. Scatchard analysis of specific binding indicated the presence of 1.0 X 10(5) high affinity binding sites/cell, with a Kd of 6.5 nM. Cross-linking studies demonstrated the presence of one 320-kDa binding complex. The pH profile and kinetic determinations of Km and maximum velocity for hPAP were similar to those previously reported, except for the finding of positive cooperativity of the substrate with the enzyme under the conditions of our assay. We believe that the direct stimulation of bone-forming cells by hPAP may contribute to the sclerotic nature of skeletal bone around sites of neoplastic prostatic metastases and that the effect of the enzyme is probably mediated by a plasma membrane receptor.
Asunto(s)
Fosfatasa Ácida/farmacología , Fosfatasa Alcalina/análisis , Huesos/citología , Colágeno/metabolismo , Próstata/enzimología , Fosfatasa Alcalina/metabolismo , Animales , Huesos/enzimología , Huesos/metabolismo , Separación Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Fibroblastos/metabolismo , Concentración de Iones de Hidrógeno , Factor II del Crecimiento Similar a la Insulina/metabolismo , Radioisótopos de Yodo , Masculino , Ratas , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/fisiologíaRESUMEN
The astacin-related metalloproteases Bone Morphogenetic Protein-1 (BMP1) and Tolloid possess multiple functions in the maturation of extracellular matrices containing fibrillar collagens. We are interested in developing an in-vitro model system to study the role of BMP1 and Tolloid in chondrocytes and osteoblasts. Cloning of the cDNAs for chick BMP1 and Tolloid reveals that the two gene products are more than 80% identical to their human and mouse homologs and are similarly derived from the same genetic locus. Anti-BMP1/Tolloid antibodies have been developed, and detect two proteins of 80 and 116kDa. Chick BMP1 and Tolloid message and proteins are found in a variety of embryonic and juvenile tissues, including chondrocytes and osteoblasts. Tolloid message and protein are generally less abundant than BMP1 message; this discrepancy is greatest in growth plate chondrocytes. Tolloid protein is more tightly bound than BMP1 to the extracellular matrix produced by cultured osteoblasts. The Chordin gene is also expressed in chondrocytes and osteoblasts, suggesting that BMP1 and Tolloid influence BMP signaling as well as matrix maturation during skeletogenesis.