Altered expression of integrins in RSV-transformed chick epiphyseal chondrocytes.

Chondrocytes have been shown to express both in vivo and in vitro a number of integrins of the beta1-, beta3- and beta5-subfamilies (Biorheology 37 (2000) 109). Normal and v-Src-transformed chick epiphyseal chondrocytes (CEC) display different adhesion properties. While normal CEC with time in culture tends to increase their adhesion to the substrate by organizing focal adhesions and actin stress fibers, v-Src-transformed chondrocytes display a refractile morphology and disorganization of actin cytoskeleton. We wondered whether the reduced adhesion and spreading of v-Src-transformed chondrocytes could be ascribed to changes in integrin expression and/or function. Integrin expression by normal CEC is studied and compared to v-Src-transformed chick chondrocytes, using monoclonal and polyclonal antibodies to integrins alpha- and beta-chains. We show the presence of alpha1-, alpha3-, alphav-, alpha6-, beta1- and beta3-chains on CEC, with very low levels of alpha2- and alpha5-chains. Alphav chain associates with multiple beta subunits in normal and transformed chondrocytes. With the exception of alpha1- and alpha2-chains, the levels of the integrin chains analyzed are higher in transformed chondrocytes as compared with normal chondrocytes. In spite of the increased levels of integrin expression, transformed chondrocytes exhibit loss of focal adhesion and actin stress fibers and low adhesion activity on several extracellular matrix constituents. These observations raise the possibility that, in addition to its effects on global pattern of integrin expression, v-Src can influence integrin function in chondrocytes.

alpha1(I) collagen gene expression in quail epiphyseal chondrocytes.

We examined alpha1(I) collagen expression by using primary cultures of quail epiphyseal chondrocytes which exhibit high levels of synthesis of cartilage-specific collagens and do not undergo phenotypic modulation when replated onto collagens I and II or fibronectin. These cells also synthesize proalpha1(I) collagen chain, however, alpha1(I) mRNA fails to be detected by Northern blot and RNase protection analysis. Nuclear transcription rate with a 5-end specific probe is detected in suspension quail chondrocytes and RT-PCR analysis shows the presence of low levels of alpha1(I) mRNA in these cells. The lack of correspondence between procollagen mRNA levels and the rate of collagen synthesis is consistent with previous reports describing the regulation of this transcript in chondrocytes and in collagen I-producing cells.

Tissue transglutaminase expression in quail epiphyseal chondrocytes.

Tissue transglutaminase (tTGase) is a GTP-binding Ca(2+)-dependent enzyme which catalyses the post-translational modification via epsilon(gamma-glutamyl)lysine bridges. The physiological role of tTGase is not fully understood. It has been shown that in cartilage the expression of tTGase correlates with terminal differentiation of chondrocytes. Recent evidence suggests that the GTP-binding activity of tTGase may play a role in the control of cell cycle progression thus explaining some of the suggested roles for the enzyme.tTGase activity is present in primary cultures of epiphyseal chondrocytes and increases transiently upon retinoic acid (RA) treatment. Increase in enzyme activity occurs upon RA addition and is accompanied by a parallel increase in protein and mRNA levels. Stimulation of tTGase expression by RA correlates with suppression of cell growth and occurs independently of cell adhesion and cell differentiation.tTGase expression is not observed in MC2, a permanent chondrocyte cell line derived from retrovirus infected chondrocytes. RA treatment fails to activate tTGase expression in MC2 cells and to completely suppress cell proliferation. Our findings lend support to the idea that tTGase might play a role in non-dividing cultured chondrocytes.

The role of cell adhesion in retinoic acid-induced modulation of chondrocyte phenotype.

Retinoic acid (RA) treatment of a suspension of quail chondrocytes inhibits the expression of cartilage collagens and induces cell adhesion along with fibronectin expression. We asked whether the RA-induced modulation of the chondrocyte phenotype was dependent on cell adhesion. Prevention of cell adhesion blocks cell growth and many of the effects associated with RA, such as collagen II inhibition, collagen I activation and fibronectin induction. The activity of the bone/tendon promoter of the alpha 2(I) collagen gene was determined by measuring the transient expression of COL1A2-CAT, a chimaeric gene bearing 3500 bp from upstream of the transcription start site of the human alpha 2(I) gene fused to the chloramphenicol acetyltransferase (CAT) gene. This promoter is activated only in permissive conditions for cell adhesion. The attachment activities of chondrocytes on protein substrates was studied by an in vitro cell adhesion assay. Untreated cells or cells maintained in suspension while undergoing RA treatment do not attach when replated on protein substrates. Chondrocytes treated with RA in permissive conditions for cell adhesion rapidly attach and spread instead on collagen-coated wells. Altogether the results suggest that cell adhesion plays a major role in RA-induced modulation of the chondrocyte phenotype.

Myogenesis in Xenopus laevis.

The amphibian embryo provides a convenient experimental system with which to study myogenesis. The earliest steps in the formation of axial and cardiac muscle are accessible for investigation using both embryological and molecular approaches. We review the origins of skeletal and cardiac muscle in the Xenopus embryo, the molecular markers available to detect muscle differentiation, and the use of embryo explants to investigate the regulation of myogenesis.

Alpha 2(I) collagen gene expression is up-regulated in quail chondrocytes pretreated with retinoic acid.

alpha 2(I) collagen gene expression is induced in quail embryo chondrocytes pretreated with retinoic acid (RA). The initial appearance of alpha 2(I) mRNA occurs around day 3 of culture in RA-free medium and rapidly progresses over the next 4 days. In transient transfection assays, expression of COL1A2-CAT, a chimeric gene bearing 3500 bp upstream the bone/tendon transcription start site from the human alpha 2(I) gene fused to the CAT gene, is stimulated severalfold in RA-treated chondrocytes. In contrast, enzyme activity is very low in untreated chondrocytes, suggesting that the sequences required for RA-induced transcription of the alpha 2(I) gene are present in this plasmid. Analysis of alpha 2(I) promoter sequences performed with deletion mutants gives overlapping results in collagen type I-producing fibroblasts and chondrocytes withdrawn from RA treatment. These experiments suggest that RA-induced transcription of the alpha 2(I) collagen gene in chondrocytes is regulated by the binding of transcription factors to the same regulatory sequences that control transcription in fibroblasts.

Expression of type X collagen is transiently stimulated in redifferentiating chondrocytes pretreated with retinoic acid.

Growth of quail chondrocytes in the presence of retinoic acid (RA) results in the suppression of the differentiated phenotype. RA-treated chondrocytes recover their differentiated phenotype if they are cultured for an additional 15 days in the absence of RA. A few days after removal from RA, treated chondrocytes acquire the polygonal morphology characteristic of chondrocytes growing as attached cells; they also gradually resume collagen II expression and synthesize cultures. The levels of collagen X mRNA decrease during the second week of culture in the absence of RA. Finally, at the end of 15 days, the absolute levels of collagen II and collagen X mRNAs are very similar in control and recovering chondrocytes.

The podosomes of Rous sarcoma virus transformed chondrocytes show a peculiar ultrastructural organization.

The ultrastructure of F-actin-containing punctate adhesion structures (podosomes) and of their rosette-like clusters has been studied by transmission electron microscopy in Rous sarcoma virus transformed chick embryo chondrocytes. Peculiar "glove finger" invaginations were found to take origin from the ventral membrane at sites of close contact; they were directed toward the center of the cell perpendicularly from the substratum. These new structures may be the sites where the local release of proteases takes place at the side of cell-to-substratum adhesion in podosome-bearing cells. The cytoplasmic face of glove finger invaginations and of the plasma membrane at cell-to-cell contact is lined by thick accumulations of microfilamentous material.

A continuous line of chicken embryo cells derived from a chondrocyte culture infected with RSV.

A continuous line of chicken embryo cells was derived from a culture of chondrocytes infected with Rous sarcoma virus (RSV). These cells, designated as NA101, have reduced serum requirements and are able to grow in a semisolid medium. NA101 cells show the same phenotype as freshly RSV infected chondrocyte cultures, i.e. synthesis of fibronectin and type I collagen, elongated bipolar shape and absence of tumorigenicity following grafting onto the chorioallantoic membrane of embryonated duck eggs; thus they appear to be distinct from transformed fibroblasts. Neither NA101 cells nor freshly infected chondrocyte cultures synthesize type II or type X collagen, which are the differentiation markers of normal chicken chondrocytes in culture.

Avian myelocytomatosis virus immortalizes differentiated quail chondrocytes.

Quail embryo chondrocytes in culture display two morphological phenotypes: polygonal epithelial-like and floating cells. Both cell populations synthesize cartilage extracellular matrix proteins (type II collagen and specific proteoglycans), whereas type X collagen, which appears to be a marker of later stages of chondrocyte differentiation, is expressed only by the epithelial-like cells. Avian myelocytomatosis virus strain MC29 does not induce morphological transformation in quail embryo chondrocytes but stimulates these cells to proliferate with a progressively reduced doubling time. MC29-infected chondrocytes can be established in culture as a continuous cell line, whereas control (uninfected) cultures only survive a few months. Rapidly dividing MC29-infected chondrocytes still express type II collagen and cartilage proteoglycans but do not synthesize type X collagen.

Cytoskeleton and adhesion patterns of cultured chick embryo chondrocytes during cell spreading and Rous sarcoma virus transformation.

The cytoskeleton and the adhesion complex of chick embryo chondrocytes maintained in vitro have been studied by fluorescence and interference reflection microscopy during the process of cell spreading. The pattern of actin-containing microfilaments and the distribution of vinculin speckles on adhesion plaques have been found to change as a function of the culture time. Newly plated chondrocytes adhere to the substratum mostly around a peripheral ring-like region and show a complex tridimensional array of microfilaments. When chondrocytes flatten, they develop stress fibres and show a diffuse system of vinculin-containing adhesion plaques scattered over the entire ventral side of the cells. Upon infection with Rous sarcoma virus (RSV) chondrocytes display one or more actin-containing ruffles located on the dorsal side similar to the 'actin flowers' earlier described in other cell types. These structures have been found to accumulate vinculin too. In chondrocytes infected with two td-ts mutants of RSV, 'actin flowers' have been found to persist at the restrictive temperature. At this temperature, however, in the majority of cells, stress fibres and adhesion plaques reappear.

The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. Transformation by rous sarcoma virus induces a switch in the collagen type synthesis and enhances fibronectin expression.

Epithelial-like chondrocytes obtained from chick embryo were transformed with Rous sarcoma virus. Cellular transformation was monitored looking at the morphology change, the cell growth, and the expression of plasminogen activator. Analysis on polyacrylamide gel of intracellular and secreted proteins showed: 1) a disappearance of the specific products of differentiated chondrocytes; 2) a switch in the collagen synthesis from the type II, the chondrocyte-specific type, to the type I, characteristic of fibroblasts and other cells of mesenchymal origin; 3) an enhancement of fibronectin synthesis. Analysis of the proteins from chondrocytes infected with Rous-associated virus 1, a virus unable to induce cell transformation in vitro, indicated that the altered expression of the differentiated proteins in Rous sarcoma virus-infected chondrocytes depended upon the action of src gene product.

The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. I. Characterization of the chondrocyte-specific phenotypes.

We have maintained chick embryo chondrocytes in culture for more than 2 months, passaging the floating cells in the absence of ascorbic acid. Throughout the culture period some of the cells attached to the dish, assuming an epithelial-like morphology and subsequently giving rise to new floating cells. The interconversion of the two cell populations was highest in primaries and decreased with the aging of the culture. Cartilage cells synthesized pro-alpha 1 (II) collagen and sulphated proteoglycans in vitro; compared with floaters, the epithelial-like cells secreted relatively large amounts of fibronectin. When ascorbic acid was added to the medium, all cells attached, maintaining their rounded shape; in this condition the pro-alpha, (II) collagen was matured and collagen fibres were detectable outside the cells. Other specific proteins synthesized by the chondrocytes in culture were also identified. One of these, a 64 K collagenase-sensitive protein, was not related to the type II collagen and may represent a new collagen type.

Studies on a temperature sensitive nuclear petite mutant of Saccharomyces cerevisiae: phenotypic reversibility of the mitochondrial functions.

1. We have studied the pleiotropic effect of a single-gene mutation of the pts mutant strain 1511 grown at 23 degrees C and 36 degrees C. 2. Growth of the mutant at the non-permissive temperature results in a decrease of respiration rate to about 50% after one generation and to less than 5% after five generations. The cytochrome spectra analysis revealed that only cytochrome c was present after growth at 36 degrees C. 3. Mitochondrial protein synthesis experiments in vivo demonstrated that the protein synthesizing system was not as rapidly inactivated by high temperature as the respiratory system. 4. The recovery of the respiratory capacity of the cells at 23 degrees C is complete but dependent on the de novo synthesis of a temperature sensitive protein.