Fibronectin Protein Levels Research Articles

Induction of extracellular matrix gene expression in normal human keratinocytes by transforming growth factor β is altered by cellular differentiation

Changes in epithelial substrata have been related to the cellular capacity for proliferation and to changes in cellular behavior. The effect of TGFβ1 on the expression of the basement membrane genes, fibronectin, laminin B1, and collagen α1(IV), was examined. Northern analysis revealed that treatment of normal human epidermal keratinocytes with 100 pMTGFβ1 increased the expression of each extracellular matrix (ECM) gene within 4 h of treatment. Maximal induction was reached within 24 h after treatment. The induction of ECM mRNA expression was dose dependent and was observed at doses as low as 1–3 pMTGFβ1. Incremental doses of TGFβ1 also increased cellular levels of fibronectin protein in undifferentiated keratinocytes and resulted in increased secretion of fibronectin. Squamous-differentiated cultures of keratinocytes expressed lower levels of the extracellular matrix RNAs than did undifferentiated cells. Treatment of these differentiated cells with TGFβ1 induced the expression of fibronectin mRNA to levels seen in TGFβ-treated, undifferentiated keratinocytes but only marginally increased the expression of collagen α1(IV) and laminin B1 mRNA. The increased fibronectin mRNA expression in the differentiated keratinocytes was also reflected by increased accumulation of cellular and secreted fibronectin protein. The inclusion of cycloheximide in the protocol indicated that TGFβ induction of collagen α1(IV) mRNA was signaled by proteins already present in the cells but that TGFβ required the synthesis of a protein(s) to fully induce expression of fibronectin and laminin B1 mRNA. The differential regulation of these genes in differentiated cells may be important to TGFβ action in regulating reepithelialization.

Utility of Ascitic Fluid Analysis in Patients with Malignancy-Related Ascites

One-hundred and thirty-three consecutive ascitic patients hospitalized in our Liver Unit were prospectively investigated, to define the accuracy of ascitic fluid analysis in identifying malignancy. Patients with extrahepatic cancer and peritoneal carcinomatosis were characterized by positive cytology and higher ascitic levels of fibronectin, lactic dehydrogenase, carcinoembryonic antigen, and total protein than both patients with uncomplicated cirrhosis and patients with cirrhosis and liver cancer. Ascitic cytology, fibronectin, and lactic dehydrogenase (LDH) were the most sensitive and specific markers of extrahepatic malignancy. In contrast, none of these markers was useful in identifying patients with primary liver cancer complicating cirrhosis. For them, the only alteration of the ascitic fluid was an elevated alpha-fetoprotein concentration. The sensitivity, specificity, and accuracy of ascitic alpha-fetoprotein for detecting liver cancer were 87%, 95%, and 94%, respectively. Combining cytology with the determinations of fibronectin (or LDH) and alpha-fetoprotein in ascitic fluid satisfactorily differentiated 28 of 32 cases of malignancy-related ascites, with very low incidence of false-positives (4-6%). Therefore, in view of the frequent difficulties in detecting liver cancer as a complication of cirrhosis in patients with ascites, it is advisable to determine all these three markers in the same ascitic sample.

Regulation of plasma fibronectin biosynthesis by glucocorticoids in chick hepatocyte cultures.

Plasma fibronectin is an acute-phase reactant synthesized by hepatocytes. Glucocorticoids are one of the major factors implicated in controlling the hepatic acute-phase response. To study the regulatory effects of glucocorticoids on plasma fibronectin biosynthesis, a model chick hepatocyte culture system under serum- and hormone-free conditions was used. In the presence of either dexamethasone or corticosterone, secreted plasma fibronectin increased maximally to 2.8-fold above basal levels. The stimulatory effect of the hormones was maintained only in their continuous presence, since plasma fibronectin production dropped to near basal levels within 16 h of glucocorticoid withdrawal. Pulse-chase studies indicated that pretreatment of cells with dexamethasone stimulated the level of secreted plasma fibronectin but had no effect on its rate of secretion. The increase in plasma fibronectin production by dexamethasone was abolished in a dose-dependent manner by the addition of progestin, an antagonist of dexamethasone known to compete specifically for the liver glucocorticoid receptor. Actinomycin D and alpha-amanitin, which are inhibitors of transcription, also blocked the early dexamethasone effect on plasma fibronectin synthesis. Slot blot hybridization of total RNA samples from dexamethasone-treated cultures revealed a 6-fold stimulatory rise in fibronectin mRNA during the first 6 h of treatment, which later declined and was no longer evident at 48 and 72 h. However, fibronectin mRNA levels were elevated to about the same extent in control and dexamethasone-treated cells at the later time points. During the same time period (0 to 72 h), plasma fibronectin protein levels rose and remained elevated. Evaluation of pulse-chase experiments following pretreatment with hormone for 48 h demonstrated that equal amounts of plasma fibronectin were translated by dexamethasone-treated and control cells, but only 42% of labeled plasma fibronectin was secreted by control cells compared with 93% for dexamethasone-treated cells. These findings suggest that the early phase of glucocorticoid regulation of plasma fibronectin biosynthesis occurs at the transcriptional level and is mediated through the specific action of the glucocorticoid receptor. A later phase of glucocorticoid-stimulated plasma fibronectin biosynthesis results from modulation of post-translational processing events leading to secretion of an increased amount of newly translated plasma fibronectin polypeptides.

Altered beta-actin gene expression in phorbol myristate acetate-treated chondrocytes and fibroblasts.

Phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, was shown to have opposite effects on the cellular morphology and steady-state levels of beta-actin mRNA in embryonic chicken muscle fibroblasts and sternal chondrocytes. When fibroblasts were treated with PMA, they formed foci of densely packed cells, ceased to adhere to culture plates, and had significantly reduced levels of beta-actin mRNA and protein. Conversely, when treated with PMA, floating chondrocytes attached to culture dishes, spread out, and began to accumulate high levels of beta-actin mRNA and proteins. In the sternal chondrocytes the stimulation of the beta-actin mRNA production was accompanied by increased steady-state levels of fibronectin mRNAs and protein. These alterations were concomitant with a fivefold reduction in type II collagen mRNA and a cessation in its protein production. After fibronectin and actin mRNAs and proteins reached their maximal levels, type I collagen mRNA and protein synthesis were turned on. Removal of PMA resulted in reduced beta-actin mRNA levels in chondrocytes and in a further alteration in the cell morphology. These observed correlations between changes in cell adhesion and morphology and beta-actin expression suggest that the effect of PMA on cell shape and adhesion may result in changes in the microfilament organization of the cytoskeleton which ultimately lead to changes in the extracellular matrix produced by the cells.

Altered β-Actin Gene Expression in Phorbol Myristate Acetate-Treated Chondrocytes and Fibroblasts

Phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, was shown to have opposite effects on the cellular morphology and steady-state levels of beta-actin mRNA in embryonic chicken muscle fibroblasts and sternal chondrocytes. When fibroblasts were treated with PMA, they formed foci of densely packed cells, ceased to adhere to culture plates, and had significantly reduced levels of beta-actin mRNA and protein. Conversely, when treated with PMA, floating chondrocytes attached to culture dishes, spread out, and began to accumulate high levels of beta-actin mRNA and proteins. In the sternal chondrocytes the stimulation of the beta-actin mRNA production was accompanied by increased steady-state levels of fibronectin mRNAs and protein. These alterations were concomitant with a fivefold reduction in type II collagen mRNA and a cessation in its protein production. After fibronectin and actin mRNAs and proteins reached their maximal levels, type I collagen mRNA and protein synthesis were turned on. Removal of PMA resulted in reduced beta-actin mRNA levels in chondrocytes and in a further alteration in the cell morphology. These observed correlations between changes in cell adhesion and morphology and beta-actin expression suggest that the effect of PMA on cell shape and adhesion may result in changes in the microfilament organization of the cytoskeleton which ultimately lead to changes in the extracellular matrix produced by the cells.