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.

The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1.

Transforming growth factor-beta1 (TGFbeta1), a major promoter of myofibroblast differentiation, induces alpha-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes alpha-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFbeta1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of alpha-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se alpha-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFbeta1-triggered enhancement of alpha-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A-containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFbeta1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.

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.

Somatomedin peptide distribution and somatomedin-binding protein content in cord plasma: comparison to normal and hypopituitary plasma.

To characterize the macromolecular forms of somatomedin (SM) in human newborn plasma, we have studied the molecular weight distribution of endogenous SM peptides as well as the content and distribution of the acid-stable and the unsaturated SM-binding proteins (SMBP) in cord blood from 13 normal term infants. The radioreceptor assayable SM peptide content was significantly reduced in newborns compared with that in normal adults. Furthermore, 50% of the SM content of newborns circulated as part of a 50,000 mol wt complex, in contrast to adult plasma where the majority of SM peptide content is found in the 150,000 mol wt range. Unsaturated SMBP was strikingly elevated in newborns (mean +/- SRM, 2.75 +/- 1.73 U/ml) compared to adult values of 0.63 +/- 0.24. Sephacryl-200 chromatography demonstrated that the unsaturated SMBP is found in the 40,000-50,000 mol wt region in newborns, adults, and GH-deficient children, although adults appear to have a secondary peak of unsaturated SMBP in the 150,000 mol wt region. Assay of the acid-stable SMBP indicated similar levels in newborns (1.15 +/- 0.26 U/ml) and adults (1.18 +/- 0.47) and strikingly lower values in GH-deficient subjects. The molecular weight of the acid-stable SMBP of newborns, adults, and hypopituitary dwarfs appeared to be similar, measuring approximately 60,000. We conclude that despite low levels of SM peptides, human cord plasma contains normal levels of the acid-stable SMBP and elevated of the unsaturated SMBP. The role of these binding proteins in cord plasma remains uncertain, but suggests that other SM peptides may be important in fetal growth.

Transforming growth factor beta regulates the levels of different fibronectin isoforms in normal human cultured fibroblasts.

Fibronectin (FN) polymorphism is caused by alternative splicing patterns in at least three regions of the primary transcript of a single gene. Using a monoclonal antibody (Mab) specific for an FN segment (ED-A), that can be included or omitted from the molecule depending on the pattern of splicing, we have examined whether transforming growth factor beta (TGF-beta) and dexamethasone, which are both known to increase the level of total FN, regulate the levels of different FN isoforms. We found that, while dexamethasone does not significantly change the ratio between the total FN and the ED-A containing FN, TGF-beta preferentially increases the expression of the FN isoform containing the ED-A sequence.

Transforming growth factor-beta regulates the splicing pattern of fibronectin messenger RNA precursor.

Fibronectin (FN) polymorphism is caused by alternative splicing patterns in at least three regions (ED-A, ED-B and IIICS) of the primary transcript of a single gene. Using monoclonal antibodies, we previously demonstrated that transforming growth factor-beta (TGF-beta) preferentially increases the accumulation of the FN isoforms containing the ED-A sequence in cultured normal human fibroblasts [Balza et al., (1988) FEBS Lett. 228, 42-44]. To determine the basis of this effect, we have examined through S1 nuclease analysis, the levels of ED-A- and ED-B-containing mRNAs in cultured normal human skin fibroblasts before and after TGF-beta treatment. These experiments have shown that TGF-beta increases the relative amount of m-RNA for ED-A- and ED-B-containing FN isoforms. These data demonstrate that a growth factor may regulate the splicing pattern of a pre-mRNA.

Localization of the cellular-fibronectin-specific epitope recognized by the monoclonal antibody IST-9 using fusion proteins expressed in E. coli.

Here we report on a monoclonal antibody (IST-9) which distinguishes between human cellular and plasma fibronectin. Using beta-galactosidase-fibronectin fusion proteins expressed in E. coli we have demonstrated that this monoclonal antibody is specific for a fibronectin segment (ED) which can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Furthermore, using the same fusion proteins we have been able to localize precisely the epitopes of two other monoclonal antibodies (IST-1 and IST-2), specific for the heparin-binding domain 5 of fibronectin.

Structural differences in the cell binding region of human fibronectin molecules isolated from cultured normal and tumor-derived human cells.

Fibronectins isolated from human plasma (pFN) and from the conditioned media of normal (N-cFN) and tumor (T-cFN) human cells were compared by cathepsin D digestion followed by immunostaining of released fragments with the monoclonal antibody 3E3, specific for the cell binding site. Two different staining patterns were obtained, one specific for pFN and N-cFN, the second common to fibronectins from the 3 different kinds of tumors studied. This indicates structural differences between N-cFN and T-cFN in the cell binding region of the fibronectin molecule.

Fibronectin concentration in the plasma of patients with malignant and benign breast disease.

Fibronectin concentration was determined in plasma from 97 patients with benign or malignant breast disease and from 62 controls. Median plasma fibronectin concentration (microgram FN/ml plasma) appeared to be significantly higher in patients with non-metastatic or metastatic breast cancer as compared to age-matched controls (P less than 0.01 and P less than 0.03, respectively); however, statistical significance disappeared when results were expressed as a function of total plasma protein content (microgram FN/mg total plasma protein). In patients with benign breast disease plasma fibronectin values were not significantly different from control levels. Our data indicate that the clinical usefulness of measuring FN in breast cancer patients appears to be very limited.

Fibrillary co-deposition of laminin-5 and large unspliced tenascin-C in the invasive front of oral squamous cell carcinoma in vivo and in vitro.

PURPOSE: Invasion of oral squamous cell carcinoma (OSCC) is associated with laminin-5 (Ln-5) synthesis, focal Ln-5 loss from the basement membrane (BM), and Ln-5 depositions in the stroma beneath invading carcinoma cell complexes. METHODS: The study is focused on the laminin-5 matrix reorganisation within the stroma of the OSCC invasive front outside the basement membrane region as well as in OSCC-fibroblast co-culture in relation to unspliced tenascin-C (Tn-CL) and ED-B+ fibronectin (ED-B+ fn) using confocal laser scanning microscopy. RESULTS: In vivo, Ln-5 was demonstrated as fibrillary deposition in the invasive front. It was co-localised to Tn-CL. In pure OSCC cultures, Ln-5 was synthesised and deposited as a spot-like matrix. Fibrillary structures were not found. In contrast, in the OSCC-fibroblast co-culture, a fibrillary Ln-5 matrix organisation was revealed within the interface of OSCC cell-fibroblast complexes exclusively in co-distribution with Tn-CL and ED-B+ fn. CONCLUSION: At least in vitro, a carcinoma cell-stroma fibroblast interaction is indispensable for fibrillary Ln-5/Tn-CL matrix organisation. Behind the parallels to the initial basement membrane formation in organotypic cultures, the fibrillary multiprotein complexes at the OSCC cell-fibroblast interface are suggested as provisional basement membrane fragments with a possible supportive role for invasive tumour behaviour.

Altered fibronectin distribution in cultured fibroblasts from patients with Ehlers-Danlos syndrome.

Using the immunofluorescence technique we have studied the distribution of fibronectin in cultured fibroblasts from patients affected by Ehlers-Danlos Syndrome (EDS) types I, II and VI. In these cells the amount of fibronectin production is reduced with respect to normal fibroblasts; moreover fibronectin fibers are shorter, thicker, mainly pericellular and show intracytoplasmic accumulation. The altered fibronectin distribution may be the result of altered interactions of fibronectin with extracellular matrix components, either due to abnormal fibronectin or to changes in other extracellular matrix molecules (e.g. collagens, hyaluronic acid).

Steady-state levels of different tenascin mRNAs in various normal human tissues.

Northern blot analysis of TN mRNA from different human tissues shows two major bands of about 6 and 8 kb which correspond to two different mRNAs generated by alternative splicing of the primary transcript. In liver, pancreas and kidney only the 6 kb TN mRNA was detectable. The highest levels of 8 kb TN mRNA were observed in placenta and skin representing 30% and 52% of total TN mRNA, respectively. In all other tissues tested the 8 kb TN mRNA represented less than 20% of total TN mRNA.

Extracellular pH controls pre-mRNA alternative splicing of tenascin-C in normal, but not in malignantly transformed, cells.

In cultured normal human fibroblasts, 2 main tenascin-C (TN-C) isoforms are generated by alternative splicing of the single TN-C primary transcript, 8 type III repeats being included or omitted in the mRNA. In these cultured cells, small pH variations of the culture medium (from 7.2 to 6.8) strikingly modify the alternative splicing pattern of the TN-C primary transcript. We report that malignantly transformed cells do not respond to extracellular pH variations as normal cells do. Indeed, malignantly transformed cells kept in culture media at pH values from 6.6 to 7.6 show no variations in the splicing pattern of the TN-C primary transcript and accumulate almost exclusively the large TN-C mRNA. These observations may explain the preferential accumulation in vivo of the large TN-C isoform in the extracellular matrix of different types of neoplasia.

Differential expression of the fibronectin isoform containing the ED-B oncofetal domain in normal human fibroblast cell lines originating from different tissues.

Fibronectin (FN) polymorphism is due both to alternative splicing of three sequences (ED-A, ED-B, and IIICS) of the primary transcript and to post-translational modifications. The FN isoform containing the ED-B sequence (B-FN), while having an extremely restricted distribution in normal adult tissues, has a high expression in fetal and tumor tissues. On a panel of non-fetal skin, fetal skin, and fetal lung fibroblast cell lines we have studied, through S1-nuclease protection analysis, the expression of the ED-B containing FN mRNA as well as the expression of the ED-B containing FN isoform through immunoblotting and immunofluorescence techniques, using domain specific monoclonal antibodies. The results show that the expression of B-FN in the different fibroblast cell lines has an extremely great variability depending on the developmental stage of the donor and on the tissue of origin. Moreover, we found that SV-40-transformed fibroblasts present a higher expression of B-FN mRNA with respect to their normal counterparts. An increase in the relative amount of the B-FN isoform in normal human fibroblasts was also obtained by treatment with transforming growth factor-beta.

Expression of tenascin and of the ED-B containing oncofetal fibronectin isoform in human cancer.

Tenascin (TN) and the oncofetal ED-B containing fibronectin isoform (B-FN) have been reported to be stromal markers of a number of malignancies. Here we report on studies of the distribution of TN and B-FN in normal adult tissues and in benign and malignant tumors, as well as on the levels of the B-FN mRNA in cultured fetal and non-fetal human fibroblasts originating from different tissues. B-FN has an extremely restricted distribution in normal adult tissues, is not expressed in benign tumors, but is greatly expressed in a high percentage of malignant tumors. On the contrary, human TN in normal adult tissues is less restricted than what has previously been reported and it is largely expressed in a number of both benign and malignant tumors. Moreover, we observed a great variability in the relative amount of B-FN mRNA among the 17 normal human fibroblast cell lines tested. We found very low levels in non-fetal skin fibroblasts and higher levels in fetal lung fibroblasts. We also found differences in the relative amounts of B-FN mRNA between fibroblast cell lines originating from the skin and the lung of the same subject.

Elution of fibronectin proteolytic fragments from a hydroxyapatite chromatography column. A simple procedure for the purification of fibronectin domains.

Human plasma fibronectin is composed of at least five distinct domains which we refer to as Hep-1/Fib-1, Gel, Cell, Hep-2 and Fib-2 depending on their affinity for heparin (Hep), gelatin (Gel), the cell surface (Cell) or fibrin (Fib). These domains are aligned from the NH2 to the COOH terminus in the above order and can be separated from each other by mild proteolytic digestion. We have studied the elution of fibronectin thermolysin digest from a hydroxyapatite column using a linear gradient (0.5-190 mM) of sodium phosphate buffer. The five major fibronectin domains were eluted from the hydroxyapatite chromatography column in the following order: Gel, Fib-2, Cell, Hep-1/Fib-1 and Hep-2. They were identified on the basis of their molecular mass, affinity to different macromolecules and reaction with domain-specific monoclonal antibodies. All domains except the Cell and Hep-2 domains eluted as single homogeneous peaks. The Cell domain eluted as two different peaks and the Hep-2 domain eluted as four different peaks. This is the first time that heterogeneity of these two domains has been observed. Since chromatography of a fibronectin thermolysin digest on a hydroxyapatite column provides a good separation of the five major fibronectin domains, we have elaborated a procedure in which each fibronectin domain is purified by no more than two steps; hydroxyapatite and molecular exclusion chromatography. Fractionation of fibronectin proteolytic digest on a hydroxyapatite chromatography column should be of great value in the comparative analysis of fibronectin from different sources and in the study of fibronectin heterogeneity. Its use in combination with molecular exclusion chromatography offers a simple and high-yield method for the purification of large amounts of fibronectin domains.

Effects of a Met-enkephalin analogue and naloxone infusion on anterior pituitary hormone secretion in acromegaly.

The effect of a long-acting analogue of Met-enkephalin (Damme) and naloxone on anterior pituitary hormone secretion has been investigated in 14 acromegalic patients. Damme produced a progressive fall in circulating LH and cortisol and stimulated prolactin release in normoprolactinaemic patients. Naloxone infusion significantly stimulated gonadotrophin and cortisol secretion without modifying basal prolactin release both in normo-and hyperprolactinamic patients. GH levels remained unchanged during naloxone and Damme infusion. The data suggest that endogenous opiate receptors do not play a major role in modulating GH hypersecretion in acromegaly.

Acetylcholinesterase in normal and malignant human cells.

Acetylcholinesterase (AChE) activity was determined in normal and malignant human cell lines by histochemical methods. In normal human fibroblasts, no AChE activity could be demonstrated by any histochemical technique or substrate. Enzymic activity was observed in HT-1080 human fibrosarcoma cells, RD 2 human rhabdomyosarcoma cells, and SW 311 human colon carcinoma cells. Activity was localized around the nuclear envelope, in the cytoplasm and associated with the cortical region of most cells. The specificity of the reaction was shown through the use of specific cholinesterase inhibitors.