Regulation of scatter factor production via a soluble inducing factor.

Scatter factor (SF) (also known as hepatocyte growth factor [HGF]) is a fibroblast-derived cytokine that stimulates motility, proliferation, and morphogenesis of epithelia. SF may play major roles in development, repair, and carcinogenesis. However, the physiologic signals that regulate its production are not well delineated. We found that various human tumor cell lines that do not produce SF secrete factors that stimulate SF production by fibroblasts, suggesting a paracrine mechanism for regulation of SF production. Conditioned medium from these cell lines contained two distinct scatter factor-inducing factor SF-IF activities: a high molecular weight (> 30 kD), heat sensitive activity and a low molecular weight (< 30 kD) heat stable activity. Further studies revealed that SF-producing fibroblasts also secrete factors that stimulate their own SF production. We characterized the < 30-kD SF-IF activity from ras-3T3 (clone D4), a mouse cell line that overproduces both SF and SF-IF. The < 30-kD filtrate from ras-3T3 conditioned medium induced four- to sixfold increases in expression of SF biologic activity, immunoreactive protein, and mRNA by multiple SF-producing fibroblast lines. Ras-3T3 SF-IF activity was stable to boiling, extremes of pH, and reductive alkylation, but was destroyed by proteases. We purified ras-3T3 SF-IF about 10,000-fold from serum-free conditioned medium by a combination of ultrafiltration, cation exchange chromatography, and reverse phase chromatography. The purified protein exhibited electrophoretic mobility of about 12 kD (reduced) and 14 kD (nonreduced) by SDS-PAGE. The identity of the protein was verified by elution of biologic activity from gel slices. Purified SF-IF stimulated SF production in a physiologic concentration range (about 20-400 pM). Its properties and activities were distinct from those of IL-1 and TNF, two known inducers of SF production. We suggest that SF-IF is a physiologic regulator of SF production.

Scatter factor modulates the metastatic phenotype of the EMT6 mouse mammary tumor.

EMT6 is a transplantable mouse mammary tumor cell line that has been utilized widely as a model system to study the effects of various treatments on local tumor growth and pulmonary metastasis. In this study, we examined the cellular mechanisms by which scatter factor (SF), a fibroblast-derived cytokine that stimulates epithelial cell motility, may contribute to tumor-cell dissemination, using the EMT6 model system. In vitro, SF stimulated EMT6 cell motility, invasiveness and cell-surface expression of urokinase (an enzyme required for cell migration through tissue). SF differentially stimulated EMT6 cell adhesion to and migration onto surfaces coated with collagen I and laminin. EMT6 cells treated in vitro with SF and injected i.v. into isogeneic BALB/c-Rw mice showed a small but significant increase (1.7-fold) in lung colony formation as compared with control cells. For EMT6 cells in vitro, SF had no effect on DNA synthesis, cell proliferation, cell size distribution, or in vitro colony-forming ability. Thus, the increase in lung colonization may be due to enhanced ability of SF-treated cells to adhere to subendothelial basement membrane or to invade through tissue. Studies of the tissue distribution of SF in BALB/c-Rw mice demonstrated high levels of active factor in the lung. Thus, the presence of endogenous pulmonary SF may have reduced the degree to which SF treatment stimulated EMT6 lung colonization. Significant SF activity was also found in extracts of EMT6 tumors. Cultured EMT6 cells did not produce SF, but did produce high titers of a soluble low-molecular-weight protein activity that is capable of stimulating SF production in human fibroblasts 3- to 5-fold. EMT6 tumor extracts contained high titers of a similar SF-inducing activity. These observations suggest that SF may contribute to the invasive and metastatic phenotype of EMT6 cells via a paracrine mechanism in which tumor cells induce the production of SF in stromal fibroblasts.

Scatter factor and hepatocyte growth factor: activities, properties, and mechanism.

Scatter factor (SF) was first identified as a fibroblast-derived protein which disperses (i.e., "scatters") cohesive colonies of epithelium. SF-like proteins were found in human smooth muscle cell conditioned medium, amniotic fluid, and placental tissue. SFs markedly stimulate migration of epithelial, carcinoma, and vascular endothelial cell types at picomolar concentrations. Hepatocyte growth factors (HGFs) were originally described as platelet- and serum-derived proteins which stimulate hepatocyte DNA synthesis. Partial amino acid sequence data for mouse and human SFs indicate significant homology with HGFs. We used biological, biochemical, and immunological assays to evaluate and compare the activities, properties, and mechanisms of action of mouse SF, human SF (fibroblast or placenta derived), and recombinant human HGF (hrHGF). We report the following findings: (a) mouse SF exhibits species-related differences in biological activities relative to the human factors; (b) human SF and hrHGF show significant overlap in biological activities (i.e., hrHGF stimulates motility of multiple normal and carcinoma cell types, whereas human SF stimulates DNA synthesis in several normal cell types); (c) the three factors contain common antigenic determinants; and (d) all three proteins stimulate rapid phosphorylation of tyrosine residues on the c-met protooncogene protein product (the putative receptor for HGF) and on another protein with Mr 110,000. A few biological and immunological differences between human SFs and hrHGF were observed. These may reflect minor variations in amino acid sequence or posttranslational modification related to the sources of the factors. Taken as a whole, our findings suggest that by structural, functional, immunological, and mechanistic criteria, human SF and human HGF are essentially identical.

Protein kinase C down-regulation enhances cAMP-mediated induction of urokinase-type plasminogen activator mRNA in LLC-PK1 cells.

Expression of the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells can be induced by signals mediated by both cAMP-dependent protein kinase (PKA) and Ca(2+)- and phospholipid-dependent protein kinase (PKC). We have utilized the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) to down-regulate PKC, in order to test for an effect on the PKA-mediated induction of the uPA gene expression. Incubation of cells for 24 h with 100 ng/ml TPA caused a marked decrease of PKC protein, both in cytosolic and particulate fractions, and an 85% reduction of total PKC activity. After down-regulation of PKC, uPA mRNA accumulation induced by 8-Br-cAMP was 5-10-fold higher than in control cells. Both uPA mRNA stability and uPA gene transcription rates induced by 8-Br-cAMP were increased by PKC down-regulation (6- and 1.8-fold, respectively). Although total PKA activity was reduced by 20% in extracts from PKC-depleted cells, activation of PKA by 8-Br-cAMP was 2.5-fold higher than in control cells. This enhanced activation of PKA in PKC-depleted cells also occurred in response to other cAMP derivatives and to cAMP induced endogenously by the activation of adenylate cyclase with forskolin, but was not due to down-regulation-associated changes in the rate of cAMP synthesis. Our results demonstrate that in LLC-PK1 cells, down-regulation of PKC results in an enhanced induction of uPA gene expression by cAMP-mediated signals without alterations in adenylate cyclase activity, suggesting a mechanism distal to adenylate cyclase.

Staurosporine stimulates expression of the urokinase-type (u-PA) plasminogen activator in LLC-PK1 cells.

In LLC-PK1 porcine epithelial cells, the urokinase-type plasminogen activator (u-PA) mRNA and protein can be induced either by stimulation of the protein kinase C (PKC) pathway using a tumor promoter (PMA) or by stimulation of the protein kinase A (PKA) pathway with calcitonin (SCT). By contrast, addition of 10(-7) M staurosporine, an inhibitor of PKC, to LLC-PK1 cells also stimulated urokinase production. In contrast to the in vitro situation (where staurosporine inhibited PKC activity), in the cell-culture system the microbial agent caused an early translocation of PKC and inhibited PKA. Addition of staurosporine together with PMA or with SCT further increased urokinase mRNA and protein synthesis. Maximal stimulation was obtained when all 3 agents were added together. We thus assume that in LLC-PK1 cells the PKA and PKC signal-transferring pathways can function independently.

Interaction of gastrin with transferrin: effects of ferric ions.

The sedimentation behavior of 125I-labeled gastrin has been studied as a function of Fe3+ ion concentration and pH. Both sedimentation velocity and sedimentation equilibrium experiments indicated that high-molecular-weight Fe3+-gastrin complexes were formed at pH 5.0 and pH 7.4. Self-association of gastrin alone was observed at pH values below 5.0. 125I-labeled gastrin bound to human serum apotransferrin at pH 7.4. Scatchard analysis of the gastrin-apotransferrin complex gave a Kd of approximately 6.4 microM at 37 degrees C, with two binding sites per molecule of apotransferrin. No significant binding of gastrin to diferric transferrin was observed under the same conditions. The binding of gastrin to apotransferrin was inhibited by NaCl. The results are consistent with the hypothesis that gastrin and transferrin act synergistically in the uptake of dietary iron by the gastrointestinal tract.

Phosphorylation of human growth hormone by the epidermal growth factor-stimulated tyrosine kinase.

In the present study, we have demonstrated that human growth hormone (hGH) can be phosphorylated by the epidermal growth factor (EGF)-stimulated tyrosine kinase of A431 cell membranes. Phosphotyrosine was the predominant phosphoamino acid released from phosphorylated hGH on partial acid hydrolysis. All five tyrosine-containing tryptic peptides of hGH are also phosphorylated by the EGF-stimulated tyrosine kinase. The highest phosphate incorporation was found for peptide T4 (residues 20-38), which is distinguished by a high frequency of acidic amino acids. The phosphorylated peptides have been characterized by HPLC and two-dimensional mapping on paper. Comparison with the labeled peptides obtained on tryptic digestion of phosphorylated hGH suggests that tyrosine phosphorylation is restricted to two tryptic peptides, T4 (tyrosine-28 or -35) and T6 (tyrosine-42). It is suggested that the absence of early insulin-like activity in the naturally occurring Mr 20,000 variant of hGH, which has an internal deletion spanning residues 32-46, may be a consequence of the loss of the tyrosine phosphorylation sites at residues 35 and 42.

Phosphorylation of gastrin-17 by epidermal growth factor-stimulated tyrosine kinase.

Tyrosine phosphorylation seems to be a key event in the control of cellular growth. Several viral transforming proteins, including the src protein of Rous sarcoma virus, the p120 protein of Abelson leukaemia virus and the middle T antigen of polyoma virus, are phosphorylated by associated tyrosine kinases. The levels of kinase activity correlate with the transforming efficiency of the virus. The receptors for epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin are also phosphorylated by associated tyrosine kinase activities, which are stimulated by EGF, PDGF and insulin, respectively. The EGF-stimulated kinase and the src protein share similar substrate specificity for tyrosines immediately C-terminal to a sequence of acidic amino acids. Such a sequence is also found adjacent to the phosphotyrosine of middle T antigen, and in the homologous region of the hormone gastrin, adjacent to a tyrosine which is sulphated in approximately half the gastrin isolated from gastric mucosa. Reports that gastrin acts as a growth factor for cells of the gastrointestinal tract suggested that phosphorylation of this tyrosine might be physiologically more relevant than sulphation. We report here that synthetic human gastrin 17 is phosphorylated by the EGF-stimulated tyrosine kinase of A431 cell membranes. The Km values of 53-87 and 223-547 microM obtained in the presence and absence of EGF, respectively, are the lowest reported so far for this enzyme.

Two forms of murine epidermal growth factor: rapid separation by using reverse-phase HPLC.

Epidermal growth factor (EGF) has been isolated from acid extracts of C57BL6/J mouse submaxillary glands by using hydrophobic chromatography. High yields of EGF in large amounts (10 mg) can be isolated reliably from the acid extract of the glands in less than 4 hr. The reverse-phase HPLC techniques used to purify the EGF initially yielded what appeared to be a single homogeneous EGF molecule. However, ion pairing reagents (e.g., heptafluorobutyric acid) altered the chromatographic properties, revealing two distinct species: EGF-alpha and EGF-beta. The apparent molecular weights, isoelectric points, and antigenic properties of EGF-alpha and EGF-beta were identical, and both forms stimulated a mitogenic response in 3T3 cells. Analysis of different preparations of purified EGF (commercial and experimental) indicated the presence of EGF-alpha and EGF-beta in constant proportion. Previous EGF binding studies must have used mixtures of 125I-labeled EGF-alpha and 125I-labeled EGF-beta. The two molecules appear to compete for an identical receptor on the cell surface.