Modulation of fibronectin gene expression in chondrocytes by viral transformation and substrate attachment

Chicken vertebral chondrocytes, which normally grow in suspension, synthesize large amounts of cartilage extracellular matrix proteins, but little fibronectin. We have analyzed the effects of both substrate attachment and transformation with a temperature-sensitive mutant of Rous sarcoma virus on fibronectin gene expression in these cells. Our experiments show that viral transformation increases fibronectin synthesis to a greater extent than substrate attachment. Furthermore, transformed chondrocytes have lost the ability to decrease fibronectin synthesis in response to suspension culture, suggesting that transformation alters the normal attachment-responsive control of fibronectin gene expression. Finally, infected substrate-attached chondrocytes shifted to the nonpermissive temperature for transformation use fibronectin RNA more efficiently in protein synthesis than cells grown under the other conditions, suggesting for the first time a role for translational control of fibronectin gene expression.     

Transformed human cells release different fibronectin variants than do normal cells

Fibronectin molecules are dimers composed of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor-derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts. Furthermore, we observed that 45-65% of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparin-binding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma.     

Absence of the I-10 protein segment mediates restricted dimerization of the cartilage-specific fibronectin isoform

The cartilage-specific (V + C)(-) fibronectin isoform does not efficiently heterodimerize with other V-region splice variants of fibronectin. To understand better the structural elements that determine this restricted dimerization profile, a series of truncated fibronectin expression constructs with various internal deletions in the V, III-15, or I-10 segments were constructed and co-transfected into COS-7 cells with either the V(+)C(+) or the (V + C)(-) isoform. SDS-PAGE and immunoblot analyses of the resulting conditioned media suggest that the I-10 segment must either be present in both monomeric subunits of fibronectin or absent from both subunits for efficient dimerization to occur. Further studies suggest that the I-10 segment specifically, not simply a balanced number of type I repeats at the carboxyl terminus of each monomeric subunit, plays an important role in determining different fibronectin dimerization patterns. Neither I-11 nor I-12 could be substituted for segment I-10 without significantly reducing the formation of heterodimers. Therefore, absence of segment I-10 explains why (V + C)(-) fibronectin is not found in heterodimeric configurations with other native V-region splice variants in cartilage. The unique dimerization pattern of (V + C)(-) fibronectin does not prevent matrix formation yet is consistent with this isoform having specialized properties in situ that are important for either the structural organization and biomechanical properties of cartilage or the regulation of a chondrocytic phenotype.     

Control of AKR fibroblast phenotype by fibronectin: Regulation of cell-surface fibronectin binding receptor by fibronectin

Results of previous studies show that the expression of fibronectin and its cell-surface fibronectin binding receptor is coregulated in 3-methylchloranthrene transformation of normal AKR-2B cells to form AKR-MCA cells and in N, N,-dimethylformamide (DMF) induction of differentiation of transformed AKR-MCA cells (1990, J. Cell. Physiol., 143:445). In this study, we tested the corgulation hypothesis by transfection experiments using an antisense fibronectin expression vector. We determined the effect of antisense fibronectin RNA expression on untransformed AKR-2B cells, and on the responses of transformed AKR-MCA cells to DMF treatment. Expression of antisense fibronectin RNA in AKR-2B cells down-modulated fibronectin production, reduced adhesion to extracellular fibronectin, and altered cellular morphology Saturation binding and Scatchard analyses using radiolabelled fibronectin revealed a concurrent down-modulation of cell-surface fibronectin binding sites, but the binding affinity of the receptor for the ligand was not affected. Immunoblotting and immunostaining revealed down-modulation of the expression of α5β1 integrins. Expression of antisense fibronectin RNA in AKR-MCA cells down-modulated the ability of DMF to restore normal fibronectin production, cell-surface fibronectin binding receptor, adhesion to extracellular fibronectin, and cellular morphology. These studies show that both fibronectin and its cell-surface fibronectin binding receptor were tightly regulated during transformation and induction of differentiation in these cells, that the ligand and its cell-surface fibronectin binding receptor worked together to bring about phenotypic changes, and that fibronectin production regulated the expression of its cell-surface fibronectin binding receptor. © 1994 Wiley-Liss, Inc.     

Development of osteogenic tissue in diffusion chambers from early precursor cells in bone marrow of adult rats

Diffusion chambers containing bone marrow cells from adult rats were implanted intraperitoneally into rat hosts and cultured in vivo for up to 64 days. Biochemical and histological analyses of the contents of the chambers demonstrate that a connective tissue consisting of bone, cartilage and fibrous tissues is formed by precursor cells present in marrow stroma. The amounts of osteogenic tissue and DNA are directly correlated with time of implantation and with number of cells inoculated. In the chambers there is initial formation of fibrous tissue which is strongly reactive to collagen type III, laminin and fibronectin. In areas of osteogenesis which appear later within this fibrous anlage, expression of collagen type III, laminin and fibronectin decrease and collagen types I and II increase in association with bone and cartilage respectively. Where osteogenesis does not develop, fibrous tissue continues to express collagen type III. The sequential expression of the different extracellular matrix components is similar to that previously observed during osteogenic differentiation in embryonic and adult developmental systems. It is concluded that the formation of fibrous and osteogenic tissues in diffusion chambers by precursor cells present in adult marrow, resembles the normal developmental process.     

Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells

Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered.     

Regulation of cellular morphology by the Rous sarcoma virus src gene: analysis of fusiform mutants.

We have been interested in how Rous sarcoma virus (RSV) influences transformed cell morphology and compared the molecular properties of chicken embryo cells (CEC) infected with mutants of RSV that induce the fusiform transformed cell morphology with those of CEC infected by wild-type RSV, which induces the more normal round transformed cell morphology. We looked for properties shared by all fusiform mutant-infected cells, because these may be responsible for maintaining the fusiform morphology. Five different fusiform mutants, two wild-type RSVs, and one wild-type back revertant of a fusiform mutant were studied. In the fusiform mutant-infected cells, the localization and myristylation of pp60src were determined and the extent of expression of the extracellular matrix protein fibronectin was examined at both the mRNA and protein levels. The phosphorylation of vinculin on tyrosine also was examined in the same CEC. Within all fusiform mutant-transformed CEC, pp60src was dramatically absent from the adhesion plaque sites normally seen in cells transformed with wild-type RSV, and these transformed CEC all expressed more fibronectin mRNA and protein in the extracellular matrix than did the wild-type RSV-transformed CEC. The absence of pp60src from the adhesion plaques was not due to lack of myristylation of the src protein, and tyrosine phosphorylation of vinculin was not related to fibronectin expression. These results suggest that the inverse relationship between pp60src in the adhesion plaques and fibronectin expression in the extracellular matrix may be interconnected phenomena and could be related to the maintenance of the fusiform transformed morphology.     

Molecular and immunologic differences in canine fibronectins from articular cartilage and plasma

Two new monoclonal antibodies (Mabs) which reacted with canine fibronectin were produced and characterized. Data supported the conclusion that the epitope recognized by Mab 1H9A4 is within the first three Type III homology repeats of the Hep 2 domain and that the epitope for Mab 13G3B7 is within the last Type III homology repeat of fibronectin. These antibodies, along with three others, Mabs IST-2, IST-7, and IST-9, produced and characterized in the laboratories of L. Zardi of Genoa, Italy, were used to characterize canine cartilage and plasma fibronectin. In addition, cartilage explants were labeled with [35S]methionine in order to characterize newly synthesized cartilage fibronectin. The following observations were made. (i) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (NaDodSO4-PAGE) of reduced canine plasma fibronectin revealed a characteristic doublet; reduced cartilage fibronectin revealed two major bands and one minor band. The lower molecular weight band was 10 kDa less than the beta subunit of plasma fibronectin. In Western blots, this band stained with Mab 1H9A4 but failed to react with Mab 13G3B7. (ii) Western blots of thermolysin and trypsin digests of cartilage fibronectin revealed cleavage patterns which differed from those obtained from digestions of plasma fibronectin. (iii) The ED-A sequence, detected by Mab IST-9, was present in less than 2% of the cartilage fibronectins. (iv) NaDodSO4-PAGE of purified and reduced 35S-labeled fibronectin revealed two major radioactive bands and one minor radioactive band which comigrated with the fibronectin from the cartilage but not with plasma fibronectin. We concluded that like "cellular" fibronectin, the ratio of alpha-type subunits to beta subunits was greater than 4 to 1 in cartilage fibronectin compared to 1.25 to 1 for plasma fibronectin; however, cartilage fibronectin was not a cellular fibronectin by the criterion of the presence of the ED-A sequence. Another difference between plasma and cartilage fibronectin was the presence in cartilage fibronectin of a subpopulation of subunits on which the last Type III homology repeat could not be detected. Biosynthetic data were consistent with the concept that cartilage fibronectin originates from local synthesis by the chondrocyte.     

Structural comparison of fibronectins from normal and transformed cells

Comparative study of the structures of fibronectins from normal and transformed cells by partial proteolysis as well as by tryptic peptide fingerprinting and analysis of phosphorylation show that: 1) fibronectin molecules from normal and transformed cells probably have very similar primary structures; 2) the phosphorylation of fibronectin is a highly specific and conserved phenomenon; 3) fibronectin from both normal and transformed cells is phosphorylated only on serine residues; 4) although the major sites of phosphorylation in fibronectin are the same in normal and transformed cells, fibronectin from transformed cells appears to be phosphorylated to a much higher extent than that from normal cells.     

The splicing pattern of fibronectin mRNA changes during chondrogenesis resulting in an unusual form of the mRNA in cartilage

Chondrogenesis, the differentiation of mesenchyme into cartilage, results in a change in composition of the extracellular matrix. The cartilage matrix contains several unique components, including type II collagen and chondroitin sulfate proteoglycan; it also contains fibronectin, a glycoprotein that mediates the interaction of cells with their matrix. We show that chick cartilage fibronectin mRNA contains an unusual pattern of alternatively spliced exons. Specifically, it contains exon IIIB but does not contain exon IIIA whereas fibronectin mRNA from mesenchyme contains both exons IIIB and IIIA. Thus the splicing pattern of the fibronectin mRNA must change from B+A+ to B+A- during chondrogenesis. Most fibronectin mRNA in other mesenchymal tissues contains exon IIIA but little exon IIIB (B-A+). Culturing of chondrocytes (cartilage-producing cells) results in loss of exon IIIB from fibronectin mRNA (B-A-). Manipulation of culture conditions to produce more adhesive chondrocytes (treatment with hyaluronidase, transformation with Rous sarcoma virus, and treatment with retinoic acid) increases the amount of fibronectin mRNA containing exon IIIA. These results suggest that exon IIIB may mediate the interactions of chondrocytes with the unique components of the cartilage matrix and exon IIIA may play a role in chondrocyte adhesion.     

A family of fibronectin mRNAs in human normal and transformed cells

Previously, two fibronectin mRNAs, generated by alternative splicing of the extra domain (ED) and type III connecting segments (IIICS) sequences, have been described in a human transformed cell line and in human liver, respectively. We now report on a family of fibronectin mRNAs identified by Northern blotting analysis in two normal human fibroblast strains (HEL 299 and Flow 7000) and five transformed cell lines (8387 and HT-1080, fibrosarcomas; G-361, melanoma; JEG-3, choriocarcinoma; and RD, rhabdomyosarcoma). Seven different fibronectin mRNA forms with electrophoretic mobilities ranging between 8.6 and 7.7 kb were identified. Each cell line contains three (HEL 299, Flow 7000 and 8387) or two (HT-1080, G-361, JEG-3 and RD) fibronectin mRNAs species with characteristic size. In all cell lines we detected one fibronectin mRNA form which lacks the ED sequence (ED- fibronectin mRNA) and one or two fibronectin mRNAs containing it (ED+ fibronectin mRNA). These data show that the presence of ED+ and ED- fibronectin mRNAs is a general feature of all cells tested. Moreover, the fibronectin mRNA pattern is characteristic of the cell type analyzed, suggesting the occurrence of specifically programmed splicing mechanisms in each cell line.     

Different expression of cell surface fibronectin in spontaneously and X-ray transformed cells

The presence of cell surface fibronectin was examined by indirect immunofluorescence in 8 groups of related rat fibroblast lines expressing an in vitro transformed phenotype. The transformed cells were selected for anchorage independent growth either after X-ray treatment (X-ray transformed cells) or from control cultures (spontaneously transformed cells). All transformed fibroblasts of the latter class showed reduced expression of fibronectin at the cell surface, whereas most of the X-ray transformed derivatives exhibited a fibronectin-positive phenotype, like the untransformed parents. Moreover, from the fibronectin-negative spontaneously transformed cells, ouabain-resistant variants were isolated, the majority of which had regained the capacity to form an extracellular matrix of fibronectin. These results emphasize the variability in the properties of transformed cells and suggest that the properties of in vitro transformed cells may depend on the cause of transformation.     

Bone and cartilage formation by skeletal muscle derived cells

In adult individuals when most tissues have progressively lost the ability to regenerate, bone maintains the potential for a continuous self remodeling. The bone marrow has been so far the main recognized source of osteoprogenitor cells that contribute to the turnover of the skeletal scaffold. The possibility though exists that a pool of osteoprogenitor cells resides within other adult tissues and in particular, as reported previously, in other connective tissues such as fat and skeletal muscle. In an attempt to identify an alternative source of osteoprogenitor cells other than bone marrow we looked into the skeletal muscle. A plastic adhering cell population, from now on referred to as skeletal muscle derived cells (SMDCs), was obtained from biopsies of human skeletal muscle. SMDCs were clonogenic and displayed a fibroblast-like morphology. The isolated cell population had a mesenchymal origin as indicated by abundant expression of type I collagen, fibronectin, and vimentin and appeared heterogeneous. SMDCs were positive for alpha smooth actin, and to a lesser extent for desmin and alpha sarcomeric myosin, two specific markers of the myogenic phenotype. Surprisingly though SMDCs expressed early markers of an osteogenic commitment as indicated by positive staining for alkaline phosphatase, osteopontin, and osteonectin. Under the appropriate stimuli, these cells deposited in vitro a mineralized bone matrix and a proteoglycan rich matrix. In addition, SMDCs cultured in the presence of low serum and insulin differentiated towards adipocytes developing abundant lipid droplets in the cytoplasm. Furthermore SMDCs formed three-dimensional bone tissue in vivo when implanted in an immunodeficient mouse, and a mature cartilage rudiment when maintained as a pellet culture. In summary, we report the isolation and characterization of a cell population from the human skeletal muscle not only able to express in vitro specific markers of distinct mesenchymal lineages (adipogenic, chondrogenic, and osteogenic), but most importantly, able to complete the differentiation pathway leading to the formation of bone and cartilage. In this respect SMDCs resemble bone marrow stromal cells (BMSCs).     

Biosynthesis of fibronectin by human embryonal fibroblasts transformed by SV-40 virus

Fibronectin biosynthesis by human embryonic fibroblasts transformed with virus SV-40 was studied in intact cells and in a cell-free protein synthesizing system on free and membrane-bound polyribosomes isolated from these cells. It was found that fibronectin release from transformed fibroblasts into the culturing medium was decreased 4.5-fold, while its per cent content--2-fold. The amount of fibronectin precipitated by antibodies in the course of an immunoprecipitation reaction in transformed cells appeared to be somewhat higher than in normal cells, although when expressed on a per cent basis this content was decreased only 1.5-fold. However, the content of fibronectin monomer with Mr = 220 kD exceeded that in normal fibroblast cell material 1.6 times. Study on fibronectin biosynthesis in a cell-free system revealed that in transformed cells 45% of fibronectin is synthesized on free polyribosomes as compared to 13% in normal fibroblasts. It is assumed that the decreased fibronectin biosynthesis in human fibroblasts transformed with virus SV-40 results in spatial uncoupling of polyribosomes and membrane structures responsible for protein transport from the cell, as a result of which a significant part of fibronectin synthesized by transformed fibroblasts undergoes intracellular degradation.     

The cartilage-specific fibronectin isoform has a high affinity binding site for the small proteoglycan decorin

Binding of fibronectin to the small proteoglycan decorin plays an important role in cell differentiation and cell migration. The cartilage-specific (V+C)(-) fibronectin isoform, in which nucleotides that normally encode the protein segments V, III(15), and I(10) are spliced out, is one of the major splice variants present in cartilage matrices. Full-length and truncated cDNA constructs were used to express recombinant versions of fibronectin. Results demonstrated that the (V+C)(-) isoform has a higher affinity for decorin. Dissociation constants for decorin and fibronectin interaction were calculated to be 93 nm for the V(+)C(+) isoform and 24 nm and 223 nm for (V+C)(-) fibronectin. Because heparin competed with decorin competitively, binding of decorin to fibronectin likely occurs at a heparin-binding region. We propose that alternative splicing of the V and C regions changes the global conformation of fibronectin in such a way that it opens an additional decorin-binding site. This conformational change is responsible for the higher affinity of the (V+C)(-) fibronectin isoform for decorin.     

Human articular chondrocytes produce IL-7 and respond to IL-7 with increased production of matrix metalloproteinase-13

Introduction

Fibronectin fragments have been found in the articular cartilage and synovial fluid of patients with osteoarthritis and rheumatoid arthritis. These matrix fragments can stimulate production of multiple mediators of matrix destruction, including various cytokines and metalloproteinases. The purpose of this study was to discover novel mediators of cartilage destruction using fibronectin fragments as a stimulus.

Methods

Human articular cartilage was obtained from tissue donors and from osteoarthritic cartilage removed at the time of knee replacement surgery. Enzymatically isolated chondrocytes in serum-free cultures were stimulated overnight with the 110 kDa α5β1 integrin-binding fibronectin fragment or with IL-1, IL-6, or IL-7. Cytokines and matrix metalloproteinases released into the media were detected using antibody arrays and quantified by ELISA. IL-7 receptor expression was evaluated by flow cytometry, immunocytochemical staining, and PCR.

Results

IL-7 was found to be produced by chondrocytes treated with fibronectin fragments. Compared with cells isolated from normal young adult human articular cartilage, increased IL-7 production was noted in cells isolated from older adult tissue donors and from osteoarthritic cartilage. Chondrocyte IL-7 production was also stimulated by combined treatment with the catabolic cytokines IL-1 and IL-6. Chondrocytes were found to express IL-7 receptors and to respond to IL-7 stimulation with increased production of matrix metalloproteinase-13 and with proteoglycan release from cartilage explants.

Conclusion

These novel findings indicate that IL-7 may contribute to cartilage destruction in joint diseases, including osteoarthritis.     

Behavior of cells seeded in isolated fibronectin matrices

Cell-free fibronectin matrix (FN-matrix) isolated from chick embryo fibroblasts was used to study cell-matrix interaction. After 24 h, most fibroblastic cells, including those without cell surface fibronectin, adopted bipolar fusiform morphology. Cells grew in parallel arrays and aligned with each other apparently along FN-matrix. Since the orientation of fibronectin fibers was determined by chick embryo fibroblasts, our results suggested that intercellular organization of "matrix-using" cell type may be influenced by "matrix-producing" cell type. Whereas the elongation and alignment effects induced by FN-matrix have been detected in fibroblasts (both normal and transformed), myoblast, aortic endothelial cells, neural cell lines (B103 and RT4D1), and cardiac muscle cells, similar effects are not detected in bone marrow hemopoietic cells, circulating lymphocytic T and B cells, and sympathetic neurons. For epithelial cells, FN-matrix has varying effects. Elongation and alignment effects are detected only in transformed epithelial cells with a great reduction in keratin expression. The morphology of normal or transformed epithelial cells with abundant keratin appears unaffected by FN-matrix. FN-matrix reduced the growth of several transformed fibroblastic lines up to 25%, but did not restore the appearance of actin stress fibers and the normal migratory activities of Rous sarcoma virus-transformed rat cells.     

The response of testicular leukocytes to lipopolysaccharide-induced inflammation: further evidence for heterogeneity of the testicular macrophage population

The majority of macrophages in the rat testis can be identified by the tissue-resident macrophage marker ED2. A smaller population of intratesticular macrophages do not express the ED2 antigen but are positive for the monocyte/macrophage marker ED1. Treatment of adult rats with the inflammatory stimulus lipopolysaccharide (LPS) had no effect on the number of testicular resident (ED2(+)) macrophages but caused a transient increase in ED1(+)ED2(-) monocyte-like macrophages (an average three-fold increase 12 h later). In both control and LPS-treated rat testes, a majority of macrophages that expressed ED1 and all Leydig cells were immuno-positive for the inducible isoform of nitric oxide synthase (iNOS). However, less than 6% of ED2(+) macrophages showed any iNOS expression, even after LPS treatment. This deficiency was confirmed by the finding that isolated ED2(+) testicular macrophages (>98% pure) stimulated with LPS did not produce NO in vitro. In contrast, resident macrophages from the peritoneum showed the expected NO response, and purified Leydig cells produced significant NO regardless of the presence or absence of LPS. Collectively, these data indicate the presence of at least two macrophage subsets in the adult rat testis: (1) the ED2(+) resident macrophages, which do not alter following LPS-treatment and mostly do not express iNOS or produce NO in response to an inflammatory stimulus, and (2) the ED1(+)ED2(-) monocyte-like macrophages, which increase in number after LPS-treatment and express iNOS even in the absence of exogenous inflammatory stimulation. It is highly probable that these different subsets have different functional roles within the testis.     

Fibronectin expression is determined by the genotype of the transformed parental cells in heterokaryons between normal and transformed fibroblasts

The expression of fibronectin, a cell surface-associated transformation-sensitive glycoprotein, was studied in hetero- and homokaryons of normal and SV40-transformed human fibroblasts. In immunofluorescence, fibroblast homokaryons had an intense surface-associated and intracelluar fibronectin fluorescence similar to that of normal fibroblasts. Transformed cells and their homokaryons had a minimal surface-associated and a weak intracellular fibronectin fluorescence. In heterokaryons formed between transformed and normal fibroblasts, the expression of fibronectin fell within 24 h to the level of the transformed cell homokaryons. The change was detectable already at 3 h after fusion and was gene-dose dependent. These results show that the transformed genotype determines fibronectin expression in the heterokaryons.