Loss of MDCK cell alpha 2 beta 1 integrin expression results in reduced cyst formation, failure of hepatocyte growth factor/scatter factor-induced branching morphogenesis, and increased apoptosis

1995 ◽  
Vol 108 (11) ◽  
pp. 3531-3540 ◽  
Author(s):  
E.U. Saelman ◽  
P.J. Keely ◽  
S.A. Santoro
Keyword(s):  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Antisense Rna ◽  
Mdck Cells ◽  
Cyst Formation ◽  
Branching Morphogenesis ◽  
Integrin Subunit ◽  
Scatter Factor ◽  
Beta 1 Integrin ◽  
Hepatocyte Growth

Cellular interactions with collagen in a model of kidney tubulogenesis were investigated using Madin-Darby canine kidney (MDCK) cells in an in vitro morphogenetic system. MDCK cells adhered to collagen types I and IV in a Mg(2+)-dependent manner, typical of the alpha 2 beta 1 integrin. Collagen-Sepharose affinity chromatography and immunoblotting demonstrated the presence and collagen binding activity of the alpha 2 beta 1 integrin on MDCK cells. To assess the function of alpha 2 beta 1 integrin, MDCK cells were transfected with a plasmid pRSV alpha 2′ which allowed the expression of alpha 2-integrin subunit antisense RNA. Three G418-resistant clones showing reduced adhesion to collagen, stable genomic integration of the antisense construct, decreased alpha 2-integrin subunit mRNA and decreased alpha 2-integrin subunit protein expression were selected for analysis in morphogenetic experiments. MDCK cells and plasmid-only control transfectants, cultured in three-dimensional collagen type I gels, showed normal cyst formation, whereas the antisense RNA transfectants showed increased apoptosis and formed small rudimentary cysts. Stimulation with hepatocyte growth factor/scatter factor-containing 3T3 fibroblast-conditioned medium or recombinant hepatocyte growth factor/scatter factor resulted in extensive branching of the preformed control cysts whereas the surviving small cysts formed by antisense expressing cells increased in size but failed to elongate and branch upon stimulation. We conclude that alpha 2 beta 1 integrin collagen interactions play a crucial role in the hepatocyte growth factor/scatter factor-induced tubulogenesis and branching morphogenesis of MDCK cells in collagen gels as well as an important role in cell survival.

Effect of scatter factor and hepatocyte growth factor on motility and morphology of mdck cells

1992 ◽  
Vol 28 (5) ◽  
pp. 364-368 ◽  
Author(s):  
Yuan LI ◽  
Ansamma Joseph ◽  
Madhu M. Bhargava ◽  
Eliot M. Rosen ◽  
Toshikazu Nakamura ◽  
...  
Keyword(s):  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Mdck Cells ◽  
Scatter Factor ◽  
Hepatocyte Growth

scholarly journals Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros.

1995 ◽  
Vol 128 (1) ◽  
pp. 171-184 ◽  
Author(s):  
A S Woolf ◽  
M Kolatsi-Joannou ◽  
P Hardman ◽  
E Andermarcher ◽  
C Moorby ◽  
...  
Keyword(s):  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Early Development ◽  
Branching Morphogenesis ◽  
Mesenchymal Cells ◽  
T Antigen ◽  
Ureteric Bud ◽  
Scatter Factor ◽  
Hepatocyte Growth

Several lines of evidence suggest that hepatocyte growth factor/scatter factor (HGF/SF), a soluble protein secreted by embryo fibroblasts and several fibroblast lines, may elicit morphogenesis in adjacent epithelial cells. We investigated the role of HGF/SF and its membrane receptor, the product of the c-met protooncogene, in the early development of the metanephric kidney. At the inception of the mouse metanephros at embryonic day 11, HGF/SF was expressed in the mesenchyme, while met was expressed in both the ureteric bud and the mesenchyme, as assessed by reverse transcription PCR, in situ hybridization, and immunohistochemistry. To further investigate the expression of met in renal mesenchyme, we isolated 13 conditionally immortal clonal cell lines from transgenic mice expressing a temperature-sensitive mutant of the SV-40 large T antigen. Five had the HGF/SF+/met+ phenotype and eight had the HGF/SF-/met+ phenotype. None had the HGF/SF+/met- nor the HGF/SF-/met- phenotypes. Thus the renal mesenchyme contains cells that express HGF/SF and met or met alone. When metanephric rudiments were grown in serum-free organ culture, anti-HGF/SF antibodies (a) inhibited the differentiation of metanephric mesenchymal cells into the epithelial precursors of the nephron; (b) increased cell death within the renal mesenchyme; and (c) perturbed branching morphogenesis of the ureteric bud. These data provide the first demonstration for coexpression of the HGF/SF and met genes in mesenchymal cells during embryonic development and also imply an autocrine and/or paracrine role for HGF/SF and met in the survival of the renal mesenchyme and in the mesenchymal-epithelial transition that occurs during nephrogenesis. They also confirm the postulated paracrine role of HGF/SF in the branching of the ureteric bud.

scholarly journals The von Hippel-Lindau Tumor Suppressor Gene Inhibits Hepatocyte Growth Factor/Scatter Factor-Induced Invasion and Branching Morphogenesis in Renal Carcinoma Cells

1999 ◽  
Vol 19 (9) ◽  
pp. 5902-5912 ◽  
Author(s):  
Shahriar Koochekpour ◽  
Michael Jeffers ◽  
Paul H. Wang ◽  
Changning Gong ◽  
Gregory A. Taylor ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tumor Suppressor ◽  
Hepatocyte Growth Factor ◽  
Tumor Suppressor Gene ◽  
Branching Morphogenesis ◽  
Suppressor Gene ◽  
Scatter Factor ◽  
Invasive Phenotype ◽  
Von Hippel Lindau ◽  
Hepatocyte Growth

ABSTRACT Loss of function in the von Hippel-Lindau (VHL) tumor suppressor gene occurs in familial and most sporadic renal cell carcinomas (RCCs). VHL has been linked to the regulation of cell cycle cessation (G0) and to control of expression of various mRNAs such as for vascular endothelial growth factor. RCC cells express the Met receptor tyrosine kinase, and Met mediates invasion and branching morphogenesis in many cell types in response to hepatocyte growth factor/scatter factor (HGF/SF). We examined the HGF/SF responsiveness of RCC cells containing endogenous mutated (mut) forms of the VHL protein (VHL-negative RCC) with that of isogenic cells expressing exogenous wild-type (wt) VHL (VHL-positive RCC). We found that VHL-negative 786-0 and UOK-101 RCC cells were highly invasive through growth factor-reduced (GFR) Matrigel-coated filters and exhibited an extensive branching morphogenesis phenotype in response to HGF/SF in the three-dimensional (3D) GFR Matrigel cultures. In contrast, the phenotypes of A498 VHL-negative RCC cells were weaker, and isogenic RCC cells ectopically expressing wt VHL did not respond at all. We found that all VHL-negative RCC cells expressed reduced levels of tissue inhibitor of metalloproteinase 2 (TIMP-2) relative to the wt VHL-positive cells, implicating VHL in the regulation of this molecule. However, consistent with the more invasive phenotype of the 786-0 and UOK-101 VHL-negative RCC cells, the levels of TIMP-1 and TIMP-2 were reduced and levels of the matrix metalloproteinases 2 and 9 were elevated compared to the noninvasive VHL-positive RCC cells. Moreover, recombinant TIMPs completely blocked HGF/SF-mediated branching morphogenesis, while neutralizing antibodies to the TIMPs stimulated HGF/SF-mediated invasion in vitro. Thus, the loss of the VHL tumor suppressor gene is central to changes that control tissue invasiveness, and a more invasive phenotype requires additional genetic changes seen in some but not all RCC lines. These studies also demonstrate a synergy between the loss of VHL function and Met signaling.

scholarly journals Expression of a human hepatocyte growth factor/scatter factor cDNA in MDCK epithelial cells influences cell morphology, motility, and anchorage-independent growth

1992 ◽  
Vol 117 (4) ◽  
pp. 889-894 ◽  
Author(s):  
Y Uehara ◽  
N Kitamura
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Hepatocyte Growth Factor ◽  
Cell Motility ◽  
Mdck Cells ◽  
Soft Agar ◽  
Scatter Factor ◽  
Anchorage Independent Growth ◽  
Surface Receptors ◽  
Hepatocyte Growth

The addition of exogenous hepatocyte growth factor (HGF)/scatter factor (SF) to MDCK epithelial cells results in fibroblastic morphology and cell motility. We generated HGF/SF producing MDCK cells by transfection with an expression plasmid containing human HGF/SF cDNA. Production of HGF/SF by these cells induced a change from an epithelial to a fibroblastic morphology and increased cell motility. In addition, the HGF/SF producing cells acquired efficient anchorage-independent growth in soft agar but did not form tumors in nude mice. The morphological change and the stimulation of the anchorage-independent growth were prevented by anti-HGF/SF antibody, suggesting that the factor is secreted and then exerts its effects through cell surface receptors.

Branching morphogenesis of human mammary epithelial cells in collagen gels

1994 ◽  
Vol 107 (12) ◽  
pp. 3557-3568 ◽  
Author(s):  
F. Berdichevsky ◽  
D. Alford ◽  
B. D'Souza ◽  
J. Taylor-Papadimitriou
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Hepatocyte Growth Factor ◽  
Mammary Epithelial Cells ◽  
Branching Morphogenesis ◽  
Mammary Epithelial ◽  
Collagen Gels ◽  
Scatter Factor ◽  
Human Mammary Epithelial ◽  
Hepatocyte Growth

To study the morphogenesis of human epithelial cells in vitro we have used a three-dimensional collagen matrix and a newly developed mammary epithelial cell line, 1–7 HB2. In standard medium 1–7 HB2 cells formed compact balls/spheres inside collagen type I gels, while cocultivation with various fibroblast cell lines or growth in fibroblast-conditioned media resulted in the appearance of branching structures. At least two different soluble factors secreted by fibroblasts were found to be implicated in the branching morphogenesis. Firstly, hepatocyte growth factor/scatter factor could induce branching in a concentration-dependent manner. Moreover, a polyclonal serum against hepatocyte growth factor/scatter factor completely inhibited the branching morphogenesis induced by medium conditioned by MRC-5 fibroblast cells. In contrast, a morphogenetic activity secreted by human foreskin fibroblasts was identified that appears to be different from hepatocyte growth factor/scatter factor and from a number of other well-characterized growth factors or cytokines. This model system has been used to examine the role of integrins in mammary morphogenesis. The expression of the alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 4 integrins was decreased when cells were plated on collagen gels. The addition of specific blocking monoclonal antibodies directed to the alpha 2- and beta 1-integrin subunits to growth media impaired cell-cell interactions and interfered with the formation of compact structures inside collagen gels, suggesting that the alpha 2 beta 1 integrin can control intercellular adhesion in mammary morphogenesis. In contrast one of the blocking monoclonal antibodies against the alpha 3-integrin subunit (P1B5) mimicked the effect of soluble ‘morphogens’. Our results suggest that the modulation of alpha 3 beta 1 activity may represent an important event in the induction of branching morphogenesis of human mammary epithelial cells.

scholarly journals Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells.

1995 ◽  
Vol 15 (2) ◽  
pp. 1110-1122 ◽  
Author(s):  
A J Ridley ◽  
P M Comoglio ◽  
A Hall
Keyword(s):  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Mdck Cells ◽  
Cell Junctions ◽  
Stress Fibers ◽  
Scatter Factor ◽  
Actin Reorganization ◽  
Membrane Ruffling ◽  
Additional Signal ◽  
Hepatocyte Growth

Scatter factor/hepatocyte growth factor (SF/HGF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of cell colonies followed by disruption of cell-cell junctions and subsequent cell scattering. These responses are accompanied by changes in the actin cytoskeleton, including increased membrane ruffling and lamellipodium extension, disappearance of peripheral actin bundles at the edges of colonies, and an overall decrease in stress fibers. The roles of the small GTP-binding proteins Ras, Rac, and Rho in regulating responses to SF/HGF were investigated by microinjection. Inhibition of endogenous Ras proteins prevented SF/HGF-induced actin reorganization, spreading, and scattering, whereas microinjection of activated H-Ras protein stimulated spreading and actin reorganization but not scattering. When a dominant inhibitor of Rac was injected, SF/HGF- and Ras-induced spreading and actin reorganization were prevented, although activated Rac alone did not stimulate either response. Microinjection of activated Rho inhibited spreading and scattering, while inhibition of Rho function led to the disappearance of stress fibers and peripheral bundles but did not prevent SF/HGF-induced motility. We conclude that Ras and Rac act downstream of the SF/HGF receptor p190Met to mediate cell spreading but that an additional signal is required to induce scattering.

scholarly journals Characterization of the Scatter Factor/Hepatocyte Growth Factor Gene Promoter

1995 ◽  
Vol 270 (2) ◽  
pp. 830-836 ◽  
Author(s):  
Antje Plaschke-Schlütter ◽  
Jürgen Behrens ◽  
Ermanno Gherardi ◽  
Walter Birchmeier
Keyword(s):  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Gene Promoter ◽  
Scatter Factor ◽  
Factor Gene ◽  
Growth Factor Gene ◽  
Hepatocyte Growth

scholarly journals Paracrine Regulation of Germinal Center B Cell Adhesion through the c-Met–Hepatocyte Growth Factor/Scatter Factor Pathway

1997 ◽  
Vol 185 (12) ◽  
pp. 2121-2131 ◽  
Author(s):  
Robbert van der Voort ◽  
Taher E.I. Taher ◽  
Robert M.J. Keehnen ◽  
Lia Smit ◽  
Martijn Groenink ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
T Cell ◽  
B Cells ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Germinal Center ◽  
Scatter Factor ◽  
Hepatocyte Growth

T cell–dependent humoral immune responses are initiated by the activation of naive B cells in the T cell areas of the secondary lymphoid tissues. This primary B cell activation leads to migration of germinal center (GC) cell precursors into B cell follicles where they engage follicular dendritic cells (FDC) and T cells, and differentiate into memory B cells or plasma cells. Both B cell migration and interaction with FDC critically depend on integrin-mediated adhesion. To date, the physiological regulators of this adhesion were unkown. In the present report, we have identified the c-met–encoded receptor tyrosine kinase and its ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF), as a novel paracrine signaling pathway regulating B cell adhesion. We observed that c-Met is predominantly expressed on CD38+CD77+ tonsillar B cells localized in the dark zone of the GC (centroblasts). On tonsil B cells, ligation of CD40 by CD40-ligand, induces a transient strong upregulation of expression of the c-Met tyrosine kinase. Stimulation of c-Met with HGF/SF leads to receptor phosphorylation and, in addition, to enhanced integrin-mediated adhesion of B cells to both VCAM-1 and fibronectin. Importantly, the c-Met ligand HGF/SF is produced at high levels by tonsillar stromal cells thus providing signals for the regulation of adhesion and migration within the lymphoid microenvironment.

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