RESUMEN
The presence of gap junctions between osteoblastic cells has been previously reported. For this study we used the rat osteosarcoma cell line UMR 106, which expresses the osteoblastic phenotype, as a model to characterize further the nature, physiology, and regulation of gap junctions. Northern blot analysis identified a 3.0-kilobase RNA species corresponding to the gap junction protein connexin 43. The presence of two other connexin RNA species (26 and 32) could not be detected by this method in these cells. The identified connexin RNA was amplified by reverse transcription coupled to polymerase chain reaction; the sequence of the amplified product appears identical to the sequence of a cloned rat heart connexin 43 gene. After treatment with PTH, forskolin, and 8-Br-cAMP (a cAMP analog), the levels of connexin 43 RNA in UMR 106 cells increased. Further evidence for the role of PTH and cAMP in the physiology of gap junctions in these cells was obtained with Lucifer yellow dye transfer experiments. Gap-junctional intercellular communication increased in response to PTH and forskolin (an inducer of adenylate cyclase activity). Expression of connexin 43 RNA increased severalfold in response to PTH in a concentration- and time-dependent fashion. Connexin 43 RNA and its PTH-mediated stimulation were also observed in several other osteoblastic cell lines. The roles of PTH and forskolin in regulating the physiological state of gap junctions were confirmed in primary cultures of rat calvaria osteoblasts.
Asunto(s)
Comunicación Celular/efectos de los fármacos , Proteínas de la Membrana/biosíntesis , Proteínas de Neoplasias/biosíntesis , Osteoblastos/efectos de los fármacos , Hormona Paratiroidea/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Neoplasias Óseas/patología , Huesos/química , Colforsina/farmacología , Conexinas , AMP Cíclico/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Miocardio/química , Proteínas de Neoplasias/genética , Osteoblastos/metabolismo , Osteosarcoma/patología , Ratas , Cráneo/citología , Células Tumorales CultivadasRESUMEN
Accumulating evidence indicates that gap junctions, primarily composed of connexin 43 (Cx43), are distributed extensively throughout bone. We have previously reported that in osteoblastic cells parathyroid hormone (PTH) increases both the steady-state levels of transcripts for Cx43 and gap-junctional intercellular communication in a process involving cyclic adenosine monophosphate (cAMP). We now present data showing that the mechanism of stimulation of Cx43 gene expression by PTH involves an increased rate of Cx43 gene transcription without affecting Cx43 transcript stability in UMR 106 osteoblastic cells. Activation of the protein kinase C pathway is not involved in this process. Inhibiting translation consistently decreases the PTH-mediated stimulation of Cx43 gene expression at all the times we tested (1-3 h). However, this effect is only partial, demonstrating that de novo protein synthesis is required for full stimulation. PTH increases the steady-state levels of Cx43 mRNA in several osteoblastic cell lines, albeit to different levels. We were unable to detect PTH stimulation in ROS 17/2.8 osteoblastic cells, suggesting that the effect of PTH on Cx43 gene expression may depend on the developmental state of the cell along the osteoblastic differentiation pathway. In the MC3T3-E1 preosteoblastic cell line, we find that PTH increases Cx43 gene expression in proliferating and maturing osteoblastic cells, but not in nondividing, differentiated osteoblasts, where the basal level of Cx43 gene expression is elevated. Unlike PTH, the osteotropic hormones 1,25-dihydroxyvitamin D3 and 17beta-estradiol do not appear to affect Cx43 gene expression in UMR 106 osteoblastic cells.
Asunto(s)
Conexina 43/efectos de los fármacos , Conexina 43/genética , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Hormona Paratiroidea/farmacología , Células 3T3 , Animales , Calcitriol/farmacología , Carcinógenos/farmacología , Estrógenos/farmacología , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica , Genes/efectos de los fármacos , Genes/genética , Ratones , Osteoblastos/citología , Osteosarcoma , Fenotipo , Proteínas/efectos de los fármacos , Proteínas/genética , Proteínas/metabolismo , ARN/efectos de los fármacos , ARN/metabolismo , Ratas , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Células Tumorales CultivadasRESUMEN
We have investigated signaling (cAMP) and anabolic responses (mineralization of extracellular matrix [ECM]) to parathyroid hormone (PTH) in long-term (30 days) cultures of MC3T3-E1 cells, a murine model of osteoblast differentiation. Expression of PTH/PTH-related peptide receptor (PTH1R) mRNA is detected early and remains relatively constant for 2 weeks with somewhat higher levels observed during the second half of the culture period. In contrast to the relatively stable PTH1R mRNA expression, the cAMP response to PTH varies markedly with no response at day 5 and a marked response (80-fold versus control) by day 10. Responsiveness to PTH remains elevated with fluctuations of 30- to 80-fold stimulation throughout the remainder of the culture period. The timing and duration of PTH treatment to achieve in vitro mineralization of ECM was evaluated. When continuous PTH treatment was initiated before day 20, mineralization decreased. If continuous PTH treatment began on or after day 20, mineralization was unaffected. However, if treatment began on day 20 and then stopped on day 25, mineralization on day 30 was increased 5-fold. This mineralization response to intermittent PTH was confirmed in primary cultures of murine and human osteoblastic cells. These data provide a potential basis for understanding the differential responses to PTH (anabolic versus catabolic) and indicate the developmental temporal variance of anabolic and catabolic responses. Since cAMP signaling was relatively unchanged during this interval (day 10-30) and stimulation of adenylate cyclase only partially mimicked the PTH effect on increased mineralization, other signaling pathways are likely to be involved in order to determine the specific anabolic response to short-term PTH treatment during the differentiation process.
Asunto(s)
Osteoblastos/metabolismo , Hormona Paratiroidea/farmacología , Fosfatasa Alcalina/metabolismo , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Humanos , Ratones , Minerales/metabolismo , Osteoblastos/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Hormona Paratiroidea/genética , Receptores de Hormona Paratiroidea/metabolismo , Factores de TiempoRESUMEN
Mesenchymal stem cells (MSCs) residing in bone marrow (BM) are the progenitors for osteoblasts and for several other cell types. In humans, the age-related decrease in bone mass could reflect decreased osteoblasts secondary to an age-related loss of osteoprogenitors. To test this hypothesis, BM cells were isolated from vertebral bodies of thoracic and lumbar spine (T1-L5) from 41 donors (16 women and 25 men) of various ages (3-70 years old) after death from traumatic injury. Primary cultures were grown in alpha modified essential medium with fetal bovine serum for 13 days until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP+) were considered to have osteogenic potential. BM nucleated cells were plated (0.5, 1, 2.5, 5, or 10 x 106 cells/10-cm dish) and grown in dexamethasone (Dex), which promotes osteoblastic differentiation. The optimal plating efficiency using BM-derived cells from donors of various ages was 5 x 106 cells/10-cm dish. BM-derived cells were also grown in the absence of Dex at this plating density. At the optimal plating density, in the presence of Dex, the number of CFU-F/ALP+ present in the BM of the younger donors (3-36 years old) was 66.2 +/- 9.6 per 106 cells (mean +/- SEM), but only 14.7 +/- 2.6 per 106 cells in the older donors (41-70 years old). With longer-term culture (4-5 weeks) of these BM cells in medium containing 10 mM beta-glycerophosphate and 100 microg/ml ascorbic acid, the extracellular matrix mineralized, a result consistent with mature osteoblastic function. These results demonstrate that the number of MSCs with osteogenic potential (CFU-F/ALP+) decreases early during aging in humans and may be responsible for the age-related reduction in osteoblast number. Our results are particularly important in that the vertebrae are a site of high turnover osteoporosis and, possibly, the earliest site of bone loss in age-related osteoporosis.
Asunto(s)
Células de la Médula Ósea/citología , Mesodermo/citología , Osteogénesis/fisiología , Células Madre/citología , Células del Estroma/citología , Adolescente , Adulto , Factores de Edad , Anciano , Fosfatasa Alcalina/metabolismo , Células de la Médula Ósea/enzimología , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Niño , Preescolar , Ensayo de Unidades Formadoras de Colonias , Dexametasona/farmacología , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Osteoblastic cells in long-term culture undergo a phenotypic maturation process leading to extracellular matrix (ECM) production and bone nodule (BN) formation. Cell-to-cell communication via gap junctions (GJC) can be detected between osteoblastic cells within 24 h of plating. We evaluated, in long-term cultures of osteoblastic cells, the effect of inhibiting GJC on the phenotypic maturation process and the expression of specific genes associated with this process. MC3T3-E1 cells were plated, and, after 24 h (day 0), cells were exposed to 18-alpha-glycyrrhetinic acid (AGA), a nontoxic reversible inhibitor of GJC. GJC, alkaline phosphatase (AP) activity, BN formation, and the relative level of transcripts encoding osteocalcin (OC), bone sialoprotein (bSP), osteopontin (OP), collagen alpha1 type I (alpha1ICol), and elongation factor-1a (EF1a) were evaluated on day 0 and every 4-7 days thereafter through day 30. GJC was assessed by fluorescent dye transfer. Gene expression was analyzed by northern blot and semiquantitative reverse transcription-polymerase chain reaction. GJC was detectable at day 0 and increased with time in culture. AGA (100 micromol/L) strongly inhibited GJC at all timepoints tested. Moreover, AGA-exposed cells showed a dose-dependent decrease in AP activity and a delay in the appearance of BN. This delayed phenotypic expression coincided with an inhibitory effect on the expression of the osteoblast-specific genes OC and bSP. Expression of alpha1ICol mRNA was also affected, but to a lesser extent, whereas OP and EF1a were not affected. Similar results were obtained with oleamide, an additional reversible inhibitor of GJC. In contrast, cells exposed to either vehicle or 100 micromol/L glycyrrhizic acid (a noninhibitory glycoside of 18-beta-glycyrrhetinic acid) were indistinguishable from untreated cells for all parameters evaluated. We conclude that GJC inhibition interferes with the maturation process of osteoblastic cells in culture, possibly by affecting signals regulating the expression of genes involved in the maturation/differentiation of the osteoblastic phenotype.
Asunto(s)
Comunicación Celular , Uniones Comunicantes/fisiología , Osteoblastos/citología , Células 3T3 , Fosfatasa Alcalina/metabolismo , Animales , Secuencia de Bases , Comunicación Celular/efectos de los fármacos , Cartilla de ADN , Uniones Comunicantes/efectos de los fármacos , Ácido Glicirretínico/farmacología , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Osteoblastos/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Previously we showed that physiological levels of parathyroid hormone (PTH) can increase the mineralization of extracellular matrix (ECM) by osteoblast-like cells in vitro. In this study, we assess the role of gap-junctional intercellular communication (GJC) in the PTH-enhanced mineralization of ECM in MC3T3-E1 cells, a murine culture model of osteoblastic differentiation. Messenger RNA and protein for connexin 43 (Cx43), the major component of MC3T3-E1 gap junctions, and GJC increased as the cells progressed toward a mature phenotype. Immunocytochemistry showed accumulation of Cx43 at the area of close contact between cells. The timing of the PTH treatment that increased matrix mineralization in these cells coincided with the highest expression of Cx43 and GJC. Administration of 18-alpha-glycyrrhetinic acid (AGA) promptly blocked GJC in cultures of MC3T3-E1 cells in a dose-dependent and reversible manner at all times tested during the culture period. Treatment with AGA, but not with an inactive analog, reversed the PTH-induced ECM mineralization. These data suggest that GJC mediates anabolic actions of PTH related to osteoblast-mediated mineralization.
Asunto(s)
Calcificación Fisiológica/efectos de los fármacos , Comunicación Celular/fisiología , Uniones Comunicantes/fisiología , Osteoblastos/metabolismo , Hormona Paratiroidea/farmacología , Animales , Comunicación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Conexina 43/metabolismo , Ratones , Osteoblastos/citología , Osteoblastos/efectos de los fármacosRESUMEN
Bone formation and remodeling require continuous generation of osteoprogenitor cells from bone marrow stromal cells (MSC), which generate and respond to a variety of growth factors with putative roles in hematopoiesis and mesenchymal differentiation. In this study we examine the interaction of two such factors on the maturation of skeletal components. We previously reported that these factors, hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D(3) (vitD(3)), act together to increase alkaline phosphatase in chondroblasts. We now describe the cooperative effect of these agents on MSC isolated and cultured from human vertebral bone marrow. MSC (passages 3-9) isolated from bone marrow cells of human vertebrae (T1-L5) from 22-36-year-old normal donors were first expanded in vitro and then plated in the presence or absence of 10 ng/mL HGF and/or 10 nmol/L vitD(3), for 7-18 days. HGF treatment increased cell proliferation 2.5-fold, with no effect on alkaline phosphatase activity. Whereas vitD(3) treatment inhibited cell growth by 50%, alkaline phosphatase activity was stimulated eightfold, although no mineralization was observed. HGF together with vitD(3) increased cell proliferation 1.5-fold and alkaline phosphatase activity 13-fold over untreated control. Moreover, mineralization was detected only with this combination. Our findings provide evidence that HGF in concert with vitamin D may promote growth and differentiation of human MSC into osteogenic cells.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Colecalciferol/farmacología , Factor de Crecimiento de Hepatocito/farmacología , Osteoblastos/efectos de los fármacos , Adulto , Fosfatasa Alcalina/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/enzimología , Recuento de Células/métodos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Humanos , Masculino , Osteoblastos/citología , Osteoblastos/enzimología , Columna Vertebral/citología , Columna Vertebral/efectos de los fármacos , Columna Vertebral/enzimología , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/enzimologíaRESUMEN
It is frequently important to identify the types of cells that are infected with human immunodeficiency virus type-1 (HIV-1) in sections of formalin-fixed paraffin-embedded (FFPE) brain tissue. Currently, both immunocytochemical and in situ hybridization methods are used for this purpose. Combined in situ hybridization and immunocytochemistry results in simultaneous detection of HIV-1 nucleic acids and proteins and allow comparison of transcriptional and translational events of cells infected with HIV-1 in the same section. In addition, this technique allows morphologic and immunologic identification of the cells within which in situ hybridization occurs and confirmation of the identity of the cells that are not hybridized. Procedures are described for use with FFPE brain tissue.
Asunto(s)
Encéfalo/patología , Encéfalo/virología , Infecciones por VIH/patología , Infecciones por VIH/virología , Inmunohistoquímica , Hibridación in Situ , VIH-1/aislamiento & purificación , HumanosRESUMEN
Osteoblasts and adipocytes are thought to differentiate from a common stromal progenitor cell. These two phenotypically mature cell types show a high degree of plasticity, which can be observed when cells are grown under specific culture conditions. Gap junctions are abundant among osteoblastic cells in vivo and in vitro, whereas they are down-regulated during adipogenesis. Gap junctional communication (GJC) modulates the expression of genes associated with the mature osteoblastic phenotype. Inhibition of GJC utilizing 18-alpha-glycyrrhetinic acid (AGRA) blocks the maturation of pre-osteoblastic cells in vitro. Moreover, cytoplasmic lipid droplets are detectable at the end of the culture period, suggesting that GJC inhibition may favor an adipocytic phenotype. We used several human osteoblastic cell lines, as well as bone-derived primary osteoblastic cells, to show that confluent cultures of human osteoblastic cells grown under osteogenic conditions developed an adipocytic phenotype after 3 days of complete inhibition of GJC using AGRA or oleamide, two dissimilar nontoxic reversible inhibitors. Development of an adipogenic phenotype was confirmed by the accumulation of triglyceride droplets and the increase in mRNA expression of the adipocytic markers peroxisome proliferator-activated receptor gamma2 and lipoprotein lipase. Glycyrrhizic acid, a noninhibitory AGRA analog, or alpha-bromopalmitate, a nondegradable fatty acid, had no effect. Modulation of skeletal GJC may represent a new pharmacological target by which inhibition of marrow adipogenesis can take place with the parallel enhancement of osteoblastogenesis, thus providing a novel therapeutic approach to the treatment of human age-related osteopenic diseases and postmenopausal osteoporosis.
Asunto(s)
Adipocitos/citología , Uniones Comunicantes/fisiología , Osteoblastos/citología , Células 3T3 , Administración Tópica , Adolescente , Anciano , Animales , Antiinflamatorios/farmacología , Northern Blotting , Diferenciación Celular , Línea Celular , Células Cultivadas , Citoplasma/metabolismo , Regulación hacia Abajo , Femenino , Regulación del Desarrollo de la Expresión Génica , Ácido Glicirretínico/farmacología , Ácido Glicirrínico/farmacología , Humanos , Lipoproteína Lipasa/metabolismo , Masculino , Ratones , Músculo Esquelético/metabolismo , Ácidos Oléicos/farmacología , Osteosarcoma/metabolismo , Palmitatos/farmacología , Fenotipo , ARN/metabolismo , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Columna Vertebral/citología , Factores de Tiempo , Factores de Transcripción/farmacología , Triglicéridos/metabolismoRESUMEN
Gap-junctional communication and expression of gap junction-forming proteins were investigated in normal human prostate epithelial cells and in several malignant prostate cell lines. In comparison with normal cells, gap-junctional communication in malignant cells, as assayed by the transfer of 443-Da fluorescent tracer Lucifer yellow, was either reduced or not detected. Malignant cells expressed mRNA transcripts for connexin (Cx) 43, whereas normal cells expressed mRNA transcripts for Cx32 and Cx40. In both normal and malignant cells, gap-junctional communication was enhanced twofold to fivefold by treatment with forskolin, an agent known to increase intracellular levels of cAMP. Immunocytochemical staining with a Cx43-specific antibody revealed that in malignant cells this enhancement correlated with the number of gap junctions and occurred without any qualitative or quantitative alteration in Cx43 mRNA or protein. Moreover, western blot analyses showed that both control and forskolin-treated malignant cells expressed only one form of Cx43. Our data suggest that gap-junctional communication in both normal and malignant prostate cells may be regulated by hormones that work via a cAMP-dependent signal transduction pathway. Thus, both normal and malignant cells offer a new experimental model system in which interactions between a hormonal form of cellular communication and intercellular communication mediated via gap junctions can be studied.