RESUMEN
Articular chondrocytes derived from osteoarthritic tissues (OA HAC) show a severely reduced chondrogenic commitment. This impairment undermines their use for tissue-engineered cartilage repair, which relies on cell proliferation and growth to meet therapeutic needs, but also on efficient cell plasticity to recover the chondrogenic phenotype. Reversine (Rev), a 2,6-disubstituted purine inhibitor of spindle-assembly checkpoints, was described to convert differentiated mesenchymal cells to their undifferentiated precursors. We hypothesized that Rev exposure could divert OA HAC to more plastic cells, re-boosting their subsequent commitment. HAC were enzymatically released from OA cartilage specimens, expanded for 2 weeks and treated with 5 µm Rev in dimethylsulphoxide (DMSO) or with DMSO alone for 6 days. Cell growth was assessed using the AlamarBlueTM assay. Cytoskeletal structure, endoproliferation and caspase-3-immunopositivity were assayed by epifluorescence microscopy. The OA HAC chondrogenic performance was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate dehydrogenase, Sox9, Aggrecan (Agg), type II collagen (Col2), Ki67, cyclinD1, transforming growth factor-ß1 (TGF-ß1), -2 and -3, interleukin-1ß (IL-1ß) and -6 , SMAD3 and -7, and vascular endothelial growth factor. Rev-treated OA HAC recovered polygonal morphology and reduced Ki67 expression and proliferation. Cell-cycle impairment accounted for altered cytoskeletal organization, endoproliferation and apoptosis, whereas a compensatory mechanism sustained the increased cyclinD1 transcript levels. Sox9, Agg and TGFs were overexpressed, but not Col2. IL transcripts were massively downregulated. These events were dose-related and transient. Overall, in spite of a higher Rev-induced transcriptional activity for extracellular matrix components and in spite of a Rev-treated cell phenotype closer to that of the three-dimensional native articular chondrocyte, Rev effects seem unleashed from a full regained chondrogenic potential.
Asunto(s)
Forma de la Célula/efectos de los fármacos , Condrocitos/citología , Morfolinas/farmacología , Purinas/farmacología , Anciano , Anciano de 80 o más Años , Cartílago Articular , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Dimetilsulfóxido/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Osteoartritis/patología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismoRESUMEN
OBJECTIVES: DNA aneuploidy has been reported to be a predictor of poor prognosis in both premalignant and malignant lesions. In oral lichen planus (OLP), this hypothesis remains to be proved. This study aimed to determine the rate of occurrence of DNA aneuploidy in patients with OLP by high-resolution DNA flow cytometry. METHODS: Patients with OLP were consecutively enrolled. Tissue samples were subdivided for formalin fixation and routine histological assessment and for immediate storage at -20°C for later DNA ploidy analysis, which was performed by DAPI staining of the extracted nuclei and excitation with a UV lamp. The DNA aneuploid sublines were characterized by the DNA Index. RESULTS: A DNA aneuploid status was observed in two of 77 patients with OLP (2.6%). When considering the clinical aspect of the OLP lesions, both DNA aneuploid cases had a reticular clinical aspect. CONCLUSIONS: DNA aneuploidy is an uncommon event in OLP and less frequent compared to other non-dysplastic and non-OLP oral potentially malignant disorders. The extremely low rate of DNA aneuploidy could represent an occasional finding or reflect the low rate of malignant transformation observed in patients with OLP even if the real prognostic value of DNA ploidy analysis in patients with OLP remains to be confirmed.
Asunto(s)
Aneuploidia , ADN/análisis , Liquen Plano Oral/genética , Liquen Plano Oral/patología , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mucosa Bucal/química , Mucosa Bucal/patología , Estudios ProspectivosRESUMEN
OBJECTIVES: Oral fields of visually normal and non-dysplastic mucosa (ODFs) may represent the precursors of oral potentially malignant lesions (OPMLs). Aim of the study was to provide new evidence for the concept of the "field carcinogenesis" model by comparing the ODF and OPML genomic aberration profiles obtained by high resolution DNA flow cytometry (hr DNA-FCM) and array-Comparative Genomic Hybridization (a-CGH). A second aim was to investigate if specific CGH aberrations were associated with DNA aneuploidy. METHODS: Nineteen patients with single OPMLs were recruited for the study. In parallel with obtaining samples of OPML tissue from 11 leukoplakias without dysplasia (nd-OPMLs) and 8 with dysplasia (d-OPMLs), we also obtained samples from distant ODFs. DNA aneuploid nuclei detected by hr DNA-FCM were physically separated, based on DNA content, from the DNA diploid components with a DNA-FCM-Sorter. These relatively pure subpopulations of epithelial nuclei were then submitted to DNA extraction and a-CGH for a genome-wide analysis of DNA copy number aberrations (CNAs). RESULTS: The frequencies of DNA aneuploidy (DI ≠ 1) among ODFs and OPMLs were respectively 5.3% and 32%. The DI aneuploid values of ODFs and nd-OPMLs were all near-diploid (DI ≠ 1 and DI ≤ 1.4), while for d-OPMLs were high-aneuploid (DI > 1.4) in 40% of the cases. CNA averages were 1.9 in ODFs and 6.5 in OPMLs. The gain of the chromosomal region 20q13.33-qter was observed in 37% of both ODFs and corresponding OPMLs. Additional common regions included 7p22.2-pter, 11p15.5-pter and 16p13.3-pter where gains were observed. Furthermore, gains of 20q13.31-q13.33 and of 5p13.33-pter and loss of 9p21.3 were detected at high frequency (respectively, at 62.5%, 50% and 50%) only in d-OPMLs. In particular, loss at 9p21.3, gain at 5p13.33-pter and gain of 20q13.31-q13.33 were associated with DNA aneuploidy (p = 0.00004; p = 0.0005; p = 0.01). CONCLUSIONS: ODFs and OPMLs showed common CNAs in specific chromosomal regions suggesting that they may represent early events of the natural history of oral carcinogenesis according to the field effect cancerization and may contribute to the ODF-OPML transition. In addition, loss at 9p21.3 and gains at 5p13.33-pter and 20q13.31-q13.33 may contribute to DNA aneuploidization.
Asunto(s)
Aberraciones Cromosómicas , Genoma Humano/genética , Mucosa Bucal/patología , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Adulto , Anciano , Aneuploidia , Cromosomas Humanos/genética , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN/genética , ADN de Neoplasias/genética , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana EdadRESUMEN
We developed a novel injectable carrageenan/fibrin/hyaluronic acid-based hydrogel with in situ gelling properties to be seeded with chondrogenic cells and used for cartilage tissue engineering applications. We first analysed the distribution within the hydrogel construct and the phenotype of human articular chondrocytes (HACs) cultured for 3 weeks in vitro. We observed a statistically significant increase in the cell number during the first 2 weeks and maintenance of cell viability throughout the cell culture, together with the deposition/formation of a cartilage-specific extracellular matrix (ECM). Taking advantage of a new in vivo model that allows the integration between newly formed and preexisting cartilage in immunodeficient mice to be investigated, we showed that injectable hydrogel seeded with human articular chondrocytes was able to regenerate and repair an experimentally made lesion in bovine articular cartilage, thus demonstrating the potential of this novel cell delivery system for cartilage tissue engineering.
Asunto(s)
Cartílago Articular/citología , Condrocitos/citología , Hidrogeles , Regeneración , Anciano , Animales , Secuencia de Bases , Cartílago Articular/fisiología , Bovinos , Células Cultivadas , Cartilla de ADN , Matriz Extracelular , Femenino , Humanos , Inmunohistoquímica , Masculino , Ratones , Persona de Mediana Edad , Modelos Animales , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ingeniería de TejidosRESUMEN
Proliferation and apoptosis are two fundamental processes that occur during limb development, and in particular in joint formation. To study the role of hedgehog proteins in limbs, we have misexpressed Sonic Hedgehog specifically in chondrocytes. We found that the appendicular skeleton was severely misshapen while pelvic and shoulder girdles developed normally. In particular, we detected fusion of the elbow/knee joint, no definite carpal/tarsal, metacarpal/metatarsal bones and absence of distinct phalanges, fused in a continuous cartilaginous rod. Molecular markers of joints, such as Gdf5 and sFrp2 were absent at presumptive joint sites and Tenascin C, a molecule associated with joint formation and expressed in permanent cartilage, was expressed in a wider region in transgenic animals as compared to the wild type. The ratio of proliferating to non-proliferating chondrocytes was about two times higher in transgenic developing cartilage as compared to the wild type. Accordingly, the proapoptotic gene Bax was barely detectable in the growth plate of transgenic mice and Tunel assay showed the absence of apoptosis in presumptive joints at E15.5. Taken together, these results suggest that misexpression of Sonic Hedgehog causes apoptosis and proliferation defects leading to the lack of joint cavity and fusion of selected limb skeletal elements.
Asunto(s)
Apoptosis/fisiología , Condrocitos/citología , Condrocitos/fisiología , Proteínas Hedgehog/metabolismo , Articulaciones/embriología , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Cartílago/anomalías , Cartílago/embriología , Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Factor 5 de Diferenciación de Crecimiento , Proteínas Hedgehog/genética , Articulaciones/anomalías , Articulaciones/citología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Transducción de Señal , Tenascina/genética , Tenascina/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
BACKGROUND: Loss of TP53 function through gene mutation is a critical event in the development and progression of many tumour types including colorectal cancer (CRC). In vitro studies have found considerable heterogeneity amongst different TP53 mutants in terms of their transactivating abilities. The aim of this work was to evaluate whether TP53 mutations classified as functionally inactive (< or=20% of wildtype transactivation ability) had different prognostic and predictive values in CRC compared with mutations that retained significant activity. MATERIALS AND METHODS: TP53 mutations within a large, international database of CRC (n = 3583) were classified according to functional status for transactivation. RESULTS: Inactive TP53 mutations were found in 29% of all CRCs and were more frequent in rectal (32%) than proximal colon (22%) tumours (P < 0.001). Higher frequencies of inactive TP53 mutations were also seen in advanced stage tumours (P = 0.0003) and in tumours with the poor prognostic features of vascular (P = 0.006) and lymphatic invasion (P = 0.002). Inactive TP53 mutations were associated with significantly worse outcome only in patients with Dukes' stage D tumours (RR = 1.71, 95%CI 1.25-2.33, P < 0.001). Patients with Dukes' C stage tumours appeared to gain a survival benefit from 5-fluorouracil-based chemotherapy regardless of TP53 functional status for transactivation ability. CONCLUSIONS: Mutations that inactivate the transactivational ability of TP53 are more frequent in advanced CRC and are associated with worse prognosis in this stage of disease.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Colorrectales/genética , Mutación , Proteína p53 Supresora de Tumor/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patología , Anciano , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Análisis Mutacional de ADN , Exones , Femenino , Estudios de Seguimiento , Humanos , Agencias Internacionales , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Estadificación de Neoplasias , Tasa de SupervivenciaRESUMEN
During vertebrate embryogenesis, bones of the vertebral column, pelvis, and upper and lower limbs, are formed on an initial cartilaginous model. This process, called endochondral ossification, is characterized by a precise series of events such as aggregation and differentiation of mesenchymal cells, and proliferation, hypertrophy and death of chondrocytes. Bone formation initiates in the collar surrounding the hypertrophic cartilage core that is eventually invaded by blood vessels and replaced by bone tissue and bone marrow. Over the last years we have extensively investigated cellular and molecular events leading to cartilage and bone formation. This has been partially accomplished by using a cell culture model developed in our laboratory. In several cases observations have been confirmed or directly made in the developing embryonic bone of normal and genetically modified chick and mouse embryos. In this article we will review our work in this field.
Asunto(s)
Condrogénesis/genética , Regulación del Desarrollo de la Expresión Génica , Osteogénesis/genética , Animales , Células de la Médula Ósea/metabolismo , Huesos/embriología , Comunicación Celular , Diferenciación Celular , Células Cultivadas , Embrión de Pollo , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/fisiología , Humanos , Ratones , Modelos Biológicos , Neovascularización Fisiológica/genética , Osteoblastos/metabolismoRESUMEN
Recently we have isolated and characterized a cDNA coding for a novel developmentally regulated chick embryo protein, cartilage associated protein (CASP). Here we describe the isolation and characterization of the cDNAs coding for the mouse CASP. Comparison of the mammalian putative protein sequence with the chick sequence shows a very high identity overall (51%); in particular the chick protein is homologous to the half amino terminus of the mouse protein. Furthermore, the comparison of the CASP cDNA sequence with sequences of the genebank database confirms our hypothesis that the CASP genes belong to a novel family that also includes genes encoding for some nuclear antigens. In all mouse tissues examined three CASP mRNAs species are detected, whereas in chick tissues a single mRNA is present. Immunohistochemistry studies show that the protein is expressed in all mouse embryonic cartilages. The mouse cartilage associated protein gene (Crtap) was assigned to chromosome 9F3-F4 by fluorescence in situ hybridization.
Asunto(s)
Cartílago Articular/metabolismo , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartílago Articular/embriología , Embrión de Pollo , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Desarrollo Embrionario y Fetal , Proteínas de la Matriz Extracelular , Expresión Génica , Ratones , Chaperonas Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
We have recently isolated and characterized cDNA clones coding for a novel developmentally regulated avian and mouse embryo protein, CASP for Cartilage Associated Protein. Here we describe the isolation and characterization of the gene coding for the human CASP. The comparison of the putative human and mouse protein sequences with the chick sequence revealed an overall high identity (89% and 51%, respectively). Homology search with known DNA and protein sequences showed that CASPs are related to two mammalian nuclear proteins. Here we demonstrate definitively that CASPs are distinct from these nuclear proteins. However, sequence comparison analyses suggest that all of these proteins belong to a new family. In all human tissues examined two CASP mRNA species were detected, whereas a single mRNA and three mRNAs were found in chick and mouse, respectively. The human CASP gene (CRTAP) was assigned to chromosome 3p22 by fluorescence in situ hybridization.
Asunto(s)
Cromosomas Humanos Par 3/genética , Mapeo Físico de Cromosoma , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Proteínas de la Matriz Extracelular , Perfilación de la Expresión Génica , Glicosilación , Humanos , Hibridación Fluorescente in Situ , Chaperonas Moleculares , Datos de Secuencia Molecular , Peso Molecular , Filogenia , Señales de Clasificación de Proteína/genética , Proteínas/química , ARN Mensajero/análisis , ARN Mensajero/genética , Alineación de Secuencia , Terminología como AsuntoRESUMEN
We have investigated the expression and localization of fibronectin, laminin, and their receptors, and we used an in vitro chick chondrocyte differentiation model to define a time hierarchy for their appearance in early chondro-genesis and to determine their role in the cell condensation process. By serum fibronectin depletion/reconstitution, or GRGDSP peptide competition experiments, we show that fibronectin contributes to the initial cell-cell interactions that occur during condensation. In later stages, a down-regulation of both fibronectin and of its alpha5beta1 integrin receptor occur, as demonstrated by mRNA and protein kinetics. Immunolocalisation studies suggest that the reduction of fibronectin in discrete areas is involved in local activation of the cell differentiation program. Furthermore, we show that laminin is expressed during the in vitro cell condensation process in areas that are negative for fibronectin staining. The types of laminin as well as the timing of expression have been determined by northern blot and RT-PCR analyses. The highest levels of expression are coincident with maximal cell aggregation. The alpha3beta1 laminin receptor, highly expressed in dedifferentiated cells, follows later on the ligand trend. During in vitro chondrogenesis, a down-regulation in the B isoform, and an up-regulation of the A isoform, of the alpha subunit of the alpha6beta1 laminin receptor occurs. Immunolocalisation studies suggest that laminin is involved in the definition of differentiating areas as opposed to non differentiating areas of the condensed region, i.e. the periphery, which eventually gives rise to the perichondrium.
Asunto(s)
Condrocitos/citología , Condrocitos/fisiología , Fibronectinas/genética , Integrinas/genética , Laminina/genética , Animales , Especificidad de Anticuerpos , Adhesión Celular/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Embrión de Pollo , Condrocitos/química , Regulación hacia Abajo/fisiología , Proteínas de la Matriz Extracelular/análisis , Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/análisis , Fibronectinas/inmunología , Regulación del Desarrollo de la Expresión Génica/fisiología , Integrinas/análisis , Integrinas/inmunología , Laminina/análisis , Laminina/inmunología , Esbozos de los Miembros/citología , Oligopéptidos/farmacología , ARN Mensajero/análisis , Receptores de Laminina/análisis , Tibia/citologíaRESUMEN
A subtracted cDNA library was generated to identify cDNAs specific for chondrocyte mRNAs preferentially expressed at the hypertrophic stage with respect to early differentiation stages. The characterization of a cDNA isolated from this library that hybridizes with a 1.8 kb mRNA is described here. This mRNA is expressed at extremely low levels in dedifferentiated chondrocytes cultured in adherent conditions, at very low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes cultured in suspension conditions. In the developing chick embryo this mRNA is detectable in RNAs extracted from several other tissues besides cartilage. The described cDNA contains a complete open reading frame coding for a polypeptide of about 33 kDa. Homology searches with known cDNA and protein sequences have revealed that the chicken protein is related to the amino-terminal half of two mammalian nuclear antigens. By immunohistochemistry with specific rabbit antisera a strong signal was detected in the cartilage extracellular matrix of selected regions of the developing skeleton. Because of this localization of the antigen we named this protein cartilage associated protein (hereafter referred to as CASP).
Asunto(s)
Cartílago/embriología , Cartílago/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Embrión de Pollo , Clonación Molecular , Matriz Extracelular/metabolismo , Biblioteca de Genes , Inmunohistoquímica , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Proteínas/inmunología , ARN Mensajero/metabolismo , Proteínas Recombinantes/inmunología , Distribución TisularAsunto(s)
Ácido Ascórbico/farmacología , Proteínas Aviares , Proteínas Portadoras/biosíntesis , Cartílago/metabolismo , Citocinas , 2,2'-Dipiridil/farmacología , Animales , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cartílago/citología , Diferenciación Celular , Células Cultivadas , Embrión de Pollo , Matriz Extracelular/metabolismo , Expresión Génica , Glicerofosfatos/farmacología , Péptidos y Proteínas de Señalización Intracelular , Midkina , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , ARN MensajeroRESUMEN
The runt locus in Drosophila encodes a nuclear protein involved in embryo segmentation, sex determination/X dosage compensation, and neurogenesis. runt homologues have been identified in higher vertebrates. The encoded proteins share a domain of 128 amino acids called the runt domain. It has been reported that this domain mediates DNA binding and heterodimerization. Here, we analyze runtB expression during chondrocyte differentiation "in vitro" and "in vivo." We have first isolated, from a chondrocyte library, a cDNA clone coding for a runtB chicken homologue and containing a complete open reading frame. The predicted protein product is 84% identical to the mouse PEBP2alphaB2 isoform. By RT-PCR analysis we have also cloned the chicken cDNA fragment coding for delta alphaB2, the exon sequence included in the B1 isoform mRNA. On Northern blot analysis of cultured chondrocytes, runtB mRNA levels increase dramatically with the transition from stage 0 (dedifferentiated) to stages I and II (hypertrophic chondrocytes). Moreover, runt polypeptides were demonstrated in chondrocytes both in vivo and in vitro. These results suggest that runt plays a role in chondrogenic differentiation.
Asunto(s)
Cartílago/citología , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartílago/metabolismo , Diferenciación Celular , Células Cultivadas , Embrión de Pollo , Clonación Molecular , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/biosíntesis , Proteínas de Drosophila , Exones/genética , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Tibia , Factores de TranscripciónRESUMEN
Data obtained while investigating growth plate chondrocyte differentiation during endochondral bone formation both in vivo and in vitro indicate that initial chondrogenesis depends on positional signaling mediated by selected homeobox-containing genes and soluble mediators. Continuation of the process strongly relies on interactions of the differentiating cells with the microenvironment, that is, other cells and extracellular matrix. Production of and response to different hormones and growth factors are observed at all times and autocrine and paracrine cell stimulations are key elements of the process. Particularly relevant is the role of the TGF-beta superfamily, and more specifically of the BMP subfamily. Other factors include retinoids, FGFs, GH, and IGFs, and perhaps transferrin. The influence of local microenvironment might also offer an acceptable settlement to the debate about whether hypertrophic chondrocytes convert to bone cells and live, or remain chondrocytes and die. We suggest that the ultimate fate of hypertrophic chondrocytes may be different at different microanatomical sites.
Asunto(s)
Cartílago/citología , Animales , Diferenciación Celular/fisiologíaRESUMEN
Mesenchymal cell condensation in chick limb bud occurs at embryonic stage 22 and is the starting event of chondrogenesis. Several mechanisms have been proposed to have an active role in the induction of this process. Among them the establishment of cell-cell contacts represents a key event. Here we have investigated the modulation of N-CAM and N-cadherin gene expression in an in vitro culture system which allows chondrocyte differentiation to proceed from condensation of prechondrogenic cells to hypertrophic chondrocytes and eventually to osteoblast-like cells. Both Northern and Western blots demonstrated that they were developmentally regulated in differentiating chondrocytes. Both cell adhesion proteins were detectable in prechondrogenic cells, increased during cell aggregation, became undetectable in hypertrophic chondrocytes, and resulted in reexpression during their maturation to osteoblast-like cells. The timing of appearance of N-cadherin and N-CAM suggests that N-cadherin initiates the in vitro cell condensation thereafter stabilized by N-CAM. In agreement with the above findings, the immunolocalization of these molecules in the cell aggregates revealed that N-CAM and N-cadherin appear, after 12 h of suspension culture, on the surface of all cells at the membrane regions participating in cell-cell contacts. At 72 h N-CAM became restricted to cells at the aggregate periphery, while N-cadherin was detected both in type II collagen-negative and -positive regions. At this time of culture, electron microscopy shows a number of cell-cell contacts at the perifery of the cell aggregates, while only a few of them were observed in the aggregate interior. The expression of N-CAM and type II collagen by chondrocytes was mutually exclusive and a sorting out between differentiating and nondifferentiating cells occurred.
Asunto(s)
Cadherinas/metabolismo , Cartílago/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Animales , Secuencia de Bases , Cadherinas/genética , Cartílago/citología , Cartílago/embriología , Adhesión Celular , Moléculas de Adhesión Celular Neuronal/genética , Agregación Celular , Comunicación Celular , Diferenciación Celular , Embrión de Pollo , Colágeno/metabolismo , Cartilla de ADN/genética , Sondas de ADN/genética , Técnica del Anticuerpo Fluorescente , Expresión Génica , Microscopía Electrónica , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
It has been proposed that tensin, in association with several other proteins, mediates the micro-filament-integrin link. Here we describe the isolation of clones spanning about 5 kb from the 3' end of tensin mRNA from cultured chick embryo chondrocyte and embryonic heart cDNA libraries. Tensin expression was investigated in cultured chick embryo cells. It was observed that tensin expression is dependent upon substrate adhesion and it is turned off after 7 days of suspension culture. This process is reversible. Tensin expression is also regulated during cartilage cell differentiation in vivo; at Hamburger and Hamilton stage 39-40, non-hypertrophic tibial chondrocytes express both RNA and protein while hypertrophic chondrocytes do not. In the culture system the expression of vimentin, a major component of intermediate filaments, showed an opposite behaviour since the suspension culture enhances the accumulation of both vimentin and its mRNAs. Therefore in chick embryo cultured chondrocytes and in vivo, during cartilage development, cell shape changes and/or integrin-extracellular matrix protein interactions may be involved in the regulation of these two genes coding for cytoskeletal proteins.
Asunto(s)
Cartílago/metabolismo , Proteínas de Microfilamentos/biosíntesis , Vimentina/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Cartílago/citología , Cartílago/embriología , Adhesión Celular , Diferenciación Celular , Células Cultivadas , Embrión de Pollo , Clonación Molecular , ADN Complementario/química , Regulación hacia Abajo , Hibridación in Situ , Datos de Secuencia Molecular , ARN Mensajero/química , TensinasRESUMEN
Differentiation of hypertrophic chondrocytes toward an osteoblast-like phenotype occurs in vitro when cells are transferred to anchorage-dependent culture conditions in the presence of ascorbic acid (Descalzi Cancedda, F., C. Gentili, P. Manduca, and R. Cancedda. 1992. J. Cell Biol. 117:427-435). This process is enhanced by retinoic acid addition to the culture medium. Here we compare the growth of hypertrophic chondrocytes undergoing this differentiation process to the growth of hypertrophic chondrocytes maintained in suspension culture as such. The proliferation rate is significantly higher in the adherent hypertrophic chondrocytes differentiating to osteoblast-like cells. In cultures supplemented with retinoic acid the proliferation rate is further increased. In both cases cells stop proliferating when mineralization of the extracellular matrix begins. We also report on the ultrastructural organization of the osteoblast-like cell cultures and we show virtual identity with cultures of osteoblasts grown from bone chips. Cells are embedded in a dense meshwork of type I collagen fibers and mineral is observed in the extracellular matrix associated with collagen fibrils. Differentiating hypertrophic chondrocytes secrete large amounts of an 82-kD glycoprotein. The protein has been purified from conditioned medium and identified as ovotransferrin. It is transiently expressed during the in vitro differentiation of hypertrophic chondrocytes into osteoblast-like cells. In cultured hypertrophic chondrocytes treated with 500 nM retinoic acid, ovotransferrin is maximally expressed 3 d after retinoic acid addition, when the cartilage-bone-specific collagen shift occurs, and decays between the 5th and the 10th day, when cells have fully acquired the osteoblast-like phenotype. Similar results were obtained when retinoic acid was added to the culture at the 50 nM "physiological" concentration. Cells expressing ovotransferrin also coexpress ovotransferrin receptors. This suggests an autocrine mechanism in the control of chondrocyte differentiation to osteoblast-like cells.
Asunto(s)
Conalbúmina/biosíntesis , Matriz Extracelular/metabolismo , Placa de Crecimiento/citología , Osteoblastos/citología , Receptores de Transferrina , Fosfatasa Alcalina/biosíntesis , Secuencia de Aminoácidos , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Células Clonales , Placa de Crecimiento/metabolismo , Datos de Secuencia Molecular , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Receptores de Superficie Celular/biosíntesis , Tretinoina/farmacologíaRESUMEN
The combined nucleotide sequences of several overlapping cDNAs provide the first complete amino acid sequence of type XIV collagen. Independent confirmation of the deduced sequence is provided by amino acid sequencing of several tryptic peptides isolated from purified chicken skin type XIV collagen. Comparative analyses show that the amino-terminal non-triple-helical region of alpha 1(XIV) chains contains sequence motifs that are similar to alpha 1(IX) collagen, fibronectin type III repeats, and von Willebrand's factor A-domains. The results also strongly suggest that the alpha 1(XIV) collagen gene is identical to the gene encoding the matrix component previously named undulin. cDNAs covering the 5' region of alpha 1(XIV) mRNA fall into two classes with distinct sequences in their 5'-untranslated regions. We believe the two alternative sequences result from differential splicing of the primary transcript. Interestingly, one of the untranslated sequences shows a high degree of identity with the cis-regulatory translational control sequence in the 5'-untranslated region of a Drosophila ribosomal protein mRNA. We hypothesize therefore that the sequence in alpha 1(XIV) collagen may play a role in the control of alpha 1(XIV) protein synthesis.
Asunto(s)
Empalme Alternativo , Colágeno/genética , Fibronectinas/genética , Glicoproteínas/genética , ARN Mensajero/genética , Transcripción Genética , Factor de von Willebrand/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Tejido Conectivo/metabolismo , ADN/genética , Drosophila/genética , Biblioteca de Genes , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Estructura Secundaria de Proteína , ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Homología de Secuencia de Aminoácido , Piel/metabolismoRESUMEN
Type IX collagen is a component of cartilage and vitreous humor. Its structure and matrix localization suggest it may serve to mediate interactions between fibrillar collagen, proteoglycan and other matrix components. Consequently, abnormalities in type IX collagen may result in chondrodysplasia. In this paper we describe the preparation and use of two monoclonal antibodies which recognize peptide sequences within the human cartilage alpha 1 (IX) collagen chain. Antibody 23-5D1 is highly sensitive and highly specific. It permits the immunoblot detection of type IX collagen extracted from milligram amounts of normal and chondrodysplastic cartilage; it also identifies the "short" form of the alpha 1 (IX) chain in human vitreous humor. Antibody 37-10H7 is highly specific, but of low sensitivity. It was used to make the new observation that an N-linked oligosaccharide is present in the amino-terminal globular domain of the alpha 1 (IX) chain. We anticipate that these antibodies may be valuable tools in the study of human and other mammalian chondrodysplasias.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Colágeno/inmunología , Epítopos , Secuencia de Aminoácidos , Cartílago/metabolismo , Colágeno/genética , Colágeno/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Cuerpo Vítreo/metabolismoRESUMEN
The collagens represent a highly diverse superfamily of extracellular matrix proteins that can be divided into several distinct families. One of the families, called FACIT (fibril-associated collagens with interrupted triple-helices) family, contains molecules that appear to be associated with cross-striated fibrils composed of members of the fibrillar collagen family. We have determined a portion of the primary structure of a recently discovered member of the FACIT family, chicken alpha 1(XIV) collagen, based on cloning and sequencing cDNAs. A synthetic oligopeptide from within the carboxy-terminal non-triple-helical domain of the alpha 1(XIV) chain has been used for generating specific polyclonal antibodies. The antiserum, PS1, recognizes a 220 kDa polypeptide in immunoblots of extracts of chicken skin, tendons, and cartilage. Sequencing of a tryptic peptide generated from purified, immunoreactive material, gives a sequence identical to that derived from cDNA sequencing, providing strong support for the type XIV-specificity of PS1. We have examined the expression of type XIV collagen in developing chick embryos by immunostaining of sections from 12-day-old embryos with PS1 and by Northern blot analysis of RNA from several tissues from both 12- and 17-day-old embryos. The results show that type XIV collagen is prevalent within relatively dense connective tissues such as dermis, tendons, perichondrium, perimysium, the stroma of lungs and liver, and blood vessels.