Hepatocyte growth factor (HGF) is a plasminogen-like protein thought to be a humoral mediator of liver regeneration. A 145-kilodalton tyrosyl phosphoprotein observed in rapid response to HGF treatment of intact target cells was identified by immunoblot analysis as the beta subunit of the c-met proto-oncogene product, a membrane-spanning tyrosine kinase. Covalent cross-linking of 125I-labeled ligand to cellular proteins of appropriate size that were recognized by antibodies to c-met directly established the c-met product as the cell-surface receptor for HGF.
Summary The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of motility, invasiveness and self-renewal traits. During both normal development and tumor pathogenesis this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment. The signals that are responsible for inducing an EMT and maintaining the resulting cellular state have been unclear. We describe three signaling pathways, involving Transforming Growth Factor (TGF)-β as well as canonical and non-canonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal state. Downregulation of endogenously synthesized inhibitors of autocrine signals in epithelial cells enables the induction of the EMT program. Conversely, disruption of autocrine signaling by added inhibitors of these pathways inhibits migration and self-renewal in primary mammary epithelial cells, and inhibits tumorigenicity and metastasis by their transformed derivatives.
Keratinocyte growth factor (KGF) is a human mitogen that is specific for epithelial cells. The complementary DNA sequence of KGF demonstrates that it is a member of the fibroblast growth factor family. The KGF transcript was present in stromal cells derived from epithelial tissues. By comparison with the expression of other epithelial cell mitogens, only KGF, among known human growth factors, has the properties of a stromal mediator of epithelial cell proliferation.
A growth factor specific for epithelial cells was identified in conditioned medium of a human embryonic lung fibroblast cell line. The factor, provisionally termed keratinocyte'growth factor (KGF) because of its predominant activity on this cell type, was purified to homogeneity by a combination of ultrafiltration, heparin-Sepharose affinity chromatography, and hydrophobic chromatography on a C4 reversed-phabe HPLC column. KGF was both acid and heat labile and consisted of a single polypeptide chain of =28 kDa. Purified KGF was a potent mitogen for epithelial cells, capable of stimulating DNA synthesis in quiescent BALB/MK epidermal keratinocytes by >500-fold with activity detectable at 0.1 nM and maximal at 1.0 nM. Lack of mitogenic activity on either fibroblasts or endothelial cells indicated that KGF possessed a target cell specificity distinct from any previously characterized growth factor. Microsequencing' revealed an amino-terminal squence containing no significant homology to any known protein. The release of this growth factor by human embryonic fibroblasts raises the possibility that KGF may play a role in mesenchymal stimlation of normal epithelial cell proliferation.Growth factors are important mediators of intercellular communication. These potent molecules are generally released by qne cell type and act to influence proliferation of other cell types (1). Interest in growth factors has been heightened by evidence of their potential involvement in neoplasia.'The v-sis transforming gene of simian sarcoma virus encodes a protein that is homologous to the B chain of platelet-derived growth factor (2,3). Moreover, a number of oncogenes are homologues of genes encoding growth factor receptors (4). Thus, increased understanding of growth factors and their receptor-mediated signal-transduction pathways is likely to provide insights into mechanisms of both normal and malignant cell growth.Recognizing that the vast majority of human malignancies are derived from epithelial tissues (5), we sought to identify growth factors specific for these cell types. In this communication, we report the purification to homogeneity of such a growth factor released by a human embryonic lung fibroblast line. Our demonstration of its unique N-terminal amino acid sequence aInd epithelial cell specificity distinguishes this mitogen from any previously described growth factor. METHODS AND MATERIALSCell Culture. M426 human embryonic fibroblasts (6), BALB/MK mouse epidermal keratinocytes (7), and NIH 3T3 mouse embryonic fibroblasts (8) were established in this laboratory. CCL208 rhesus monkey bronchial epithelial cells (9) were obtained from the American Type Culture Collection, and the B5/589 human mammary epithelial cell line, prepared as described (10), was a gift from M. Stampfer (Lawrence Berkeley Laboratory). Primary cultures of human saphenous vein endothelial cells were prepared and maintained as described elsewhere (11). Epidermal growth factor (EGF) and insulin were from Collaborative Research, and transforming gro...
Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domain homologous to the putative Wntbinding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields ϳ3 mg of purified protein/liter of conditioned medium. Using this recombinant protein, we demonstrated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cysteine-rich domain retained the ability to bind Wg. Cross-linking experiments performed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked complex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also observed a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optimal concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exerted a biphasic effect on Wg activity in an armadillo stabilization assay, increasing armadillo level at low concentrations but reducing it at higher concentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.
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