Requirement of regulated activation of Ras for response of MDCK cells to hepatocyte growth factor/scatter factor

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering, migration, and branching tubule formation of MDCK cells. To examine the role of the Ras protein in the HGF/SF-induced responses, we constructed MDCK cell clones expressing either inducible dominant-negative Ras or constitutively activated Ras and analyzed their effects on responses of cells to HGF/SF. Induced expression of dominant-negative Ras prevented cell dissociation required for cell scattering, migration, and cystic formation as well as branching morphology required for branching tubule formation. Constitutively activated Ras induced cell dissociation, but not a scattered fibroblastic morphology even in the presence of HGF/SF. MDCK cells expressing constitutively activated Ras migrated at a level similar to that of wild-type MDCK cells stimulated by HGF/SF. MDCK cells expressing constitutively activated Ras showed disorganized growth in three-dimensional culture and did not form the branching tubule structures. These results indicate that activation of the Ras protein is essential for the cell scattering, migration, and branching tubule formation of MDCK cells induced by HGF/SF, and a properly regulated activation is required for some stages of the HGF/SF-induced responses of MDCK cells.     

Rac1 protects epithelial cells against anoikis

Rho family members play a critical role in malignant transformation. Anchorage-independent growth and the ability to avoid apoptosis caused by loss of anchorage (anoikis) are important features of transformed cells. Here we show that constitutive activation of Rac1 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. Constitutively active Rac1-V12 decreases DNA fragmentation and caspase activity by 50% in MDCK cells kept in suspension. In addition, expression of Rac1-V12 in MDCK cells in suspension conditions causes an increase in the number of surviving cells. We also investigated the signaling pathways that are activated by Rac1 to stimulate cell survival. We show that expression of Rac1-V12 in MDCK cells in suspension stimulates a number of signaling cascades that have been implicated in the control of cell survival, including the p42/44 ERK, p38, protein kinase B, and nuclear factor kappaB pathways. Using specific chemical or protein inhibitors of these respective pathways, we show that Rac1-mediated cell survival strongly depends on phosphatidylinositol 3-kinase activity and that activation of ERK, p38, and NF-kappaB are largely dispensable for Rac1 survival signaling. In conclusion, these studies demonstrate that Rac1 can suppress apoptosis in epithelial cells in anchorage-independent conditions and suggest a potential role for Rac1-mediated survival signaling in cell transformation.     

Matrix metalloproteinase 13 (MMP13) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1), regulated by the MAPK pathway, are both necessary for Madin-Darby canine kidney tubulogenesis

A classic model of tubulogenesis utilizes Madin-Darby canine kidney (MDCK) cells. MDCK cells form monoclonal cysts in three-dimensional collagen and tubulate in response to hepatocyte growth factor, which activates multiple signaling pathways, including the mitogen-activated protein kinase (MAPK) pathway. It was shown previously that MAPK activation is necessary and sufficient to induce the first stage of tubulogenesis, the partial epithelial to mesenchymal transition (p-EMT), whereas matrix metalloproteinases (MMPs) are necessary for the second redifferentiation stage. To identify specific MMP genes, their regulators, tissue inhibitors of matrix metalloproteinases (TIMPs), and the molecular pathways by which they are activated, we used two distinct MAPK inhibitors and a technique we have termed subtraction pathway microarray analysis. Of the 19 MMPs and 3 TIMPs present on the Canine Genome 2.0 Array, MMP13 and TIMP1 were up-regulated 198- and 169-fold, respectively, via the MAPK pathway. This was confirmed by two-dimensional and three-dimensional real time PCR, as well as in MDCK cells inducible for the MAPK gene Raf. Knockdown of MMP13 using short hairpin RNA prevented progression past the initial phase of p-EMT. Knockdown of TIMP1 prevented normal cystogenesis, although the initial phase of p-EMT did occasionally occur. The MMP13 knockdown phenotype is likely because of decreased collagenase activity, whereas the TIMP1 knockdown phenotype appears due to increased apoptosis. These data suggest a model, which may also be important for development of other branched organs, whereby the MAPK pathway controls both MDCK p-EMT and redifferentiation, in part by activating MMP13 and TIMP1.     

Discoidin domain receptor 1 activation suppresses alpha2beta1 integrin-dependent cell spreading through inhibition of Cdc42 activity

Upregulation and overexpression of discoidin domain receptor 1 (DDR1) have been implied in the regulation of kidney development and progression of cancers. Our previous studies with Mardin-Darby canine kidney (MDCK) cells showed that overexpression of DDR1 inhibited cell spreading, whereas dominant negative DDR1 promoted cell spreading on collagen-coated dish. Cell spreading is an important characteristic for cell differentiation and survival. However, little is known about the molecular mechanisms underlying the role of DDR1 in cell spreading. We have found here a novel signaling pathway of DDR1 consisting of Cdc42 that regulates the assembly and disassembly of cytoskeleton and cell spreading in MDCK cells. Cell spreading involves the organization of cytoskeleton that is mainly regulated by Rho-family GTPases. We assessed the activity of Rho-family GTPases and transfected MDCK cells with constitutively active or dominant negative GTPases, and quantified the extent of cell spreading. These results showed that DDR1 decreased the filamentous actin ratio and Rac1/Cdc42 activities, but had no effects on RhoA activity. Neither constitutively active nor dominant negative Rac1 altered DDR1-inhibited cell spreading. Constitutively active Cdc42 could rescue the DDR1-inhibited cell spreading, whereas dominant negative Cdc42 inhibited cell spreading, indicating that DDR1-inhibited cell spreading is Cdc42 dependent. With the use of alpha(2)beta(1) integrin blocking antibody, we showed that collagen-induced Cdc42 activation was mediated by alpha(2)beta(1) integrin. Moreover, ectopic FAK expression enhanced the Cdc42 activity. Reducing FAK activity by dominant negative FAK (FRNK) markedly abolished the Cdc42 activity. These findings show that DDR1a/b activation inhibits cell spreading through suppressing alpha(2)beta(1) integrin-mediated Cdc42 activation.     

Translocation of MARCKS and reorganization of the cytoskeleton by PMA correlates with the ion selectivity,the confluence,and transformation state of kidney epithelial cell lines

The role of protein kinase C (PKC) in the regulation of the cytoskeleton of epithelial cells with tightly sealed contacts, poor contacts, and without contacts were investigated by incubating them with a protein kinase C activator phorbol myristoyl acetate (PMA). The morphology and organization of the membrane skeleton and stress fibers as well as the localization of an actin-bundling PKC substrate MARCKS in confluent MDCK cells originating from the distal tubulus of dog kidney, LLC-PK1 cells originating from the proximal tubulus of pig kidney, src-transformed MDCK cells, epidermoid carcinoma A431 cells, and MDCK cells grown in low calcium medium (LC medium) in low density were visualized with phase contrast and immunofluorescence microscopy. Four different responses to the PMA-treatment in actin-based structures of cultured epithelial cells were observed: 1) disintegration of the membrane skeleton in confluent MDCK cells; 2) depolymerization of the stress fibers in confluent MDCK and LLC-PK1 cells; 3) formation of the membrane skeleton in A431 cells, and 4) formation of the stress fibers and membrane skeleton in LC-MDCK cells. Thus, it seems that in fully confluent tightly sealed epithelium, activation of PKC has a deleterious effect on actin-based structures, whereas in cells without contacts or loose contacts, activation of PKC by PMA results in improvement of actin-based cytoskeletal structures. The main difference between the two kidney cell lines used is their selectivity to ion transport: the monolayer of LLC-PK1 cells is anion selective and MDCK cells cation selective. We propose a model where alterations in the ionic milieu within the MDCK cells by means of cation channels affect the disintegration of the membrane skeleton. The distribution of MARCKS followed the distribution of fodrin in both cell lines upon PMA-treatment, suggesting that phosphorylation of MARCKS by PKC may contribute in the regulation of the integrity of the membrane skeleton. J. Cell. Physiol. 181:83–95, 1999. © 1999 Wiley-Liss, Inc.     

{beta}1 Integrin and IL-3R coordinately regulate STAT5 activation and anchorage-dependent proliferation

We previously demonstrated that integrin-dependent adhesion activates STAT5A, a well known target of IL-3-mediated signaling. Here, we show that in endothelial cells the active beta1 integrin constitutively associates with the unphosphorylated IL-3 receptor (IL-3R) beta common subunit. This association is not sufficient for activating downstream signals. Indeed, only upon fibronectin adhesion is Janus Kinase 2 (JAK2) recruited to the beta1 integrin-IL-3R complex and triggers IL-3R beta common phosphorylation, leading to the formation of docking sites for activated STAT5A. These events are IL-3 independent but require the integrity of the IL-3R beta common. IL-3 treatment increases JAK2 activation and STAT5A and STAT5B tyrosine and serine phosphorylation and leads to cell cycle progression in adherent cells. Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry. Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression. Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.     

Control of adhesion-dependent cell survival by focal adhesion kinase

The interactions of integrins with extracellular matrix proteins can activate focal adhesion kinase (FAK) and suppress apoptosis in normal epithelial and endothelial cells; this subset of apoptosis has been termed "anoikis." Here, we demonstrate that FAK plays a role in the suppression of anoikis. Constitutively activated forms of FAK rescued two established epithelial cell lines from anoikis. Both the major autophosphorylation site (Y397) and a site critical to the kinase activity (K454) of FAK were required for this effect. Activated FAK also transformed MDCK cells, by the criteria of anchorage-independent growth and tumor formation in nude mice. We provide evidence that this transformation resulted primarily from the cells' resistance to anoikis rather than from the activation of growth factor response pathways. These results indicate that FAK can regulate anoikis and that the conferral of anoikis resistance may suffice to transform certain epithelial cells.     

Alpha 1-adrenergic receptor-mediated phosphoinositide hydrolysis and prostaglandin E2 formation in Madin-Darby canine kidney cells. Possible parallel activation of phospholipase C and phospholipase A2

alpha 1-Adrenergic receptors mediate two effects on phospholipid metabolism in Madin-Darby canine kidney (MDCK-D1) cells: hydrolysis of phosphoinositides and arachidonic acid release with generation of prostaglandin E2 (PGE2). The similarity in concentration dependence for the agonist (-)-epinephrine in eliciting these two responses implies that they are mediated by a single population of alpha 1-adrenergic receptors. However, we find that the kinetics of the two responses are quite different, PGE2 production occurring more rapidly and transiently than the hydrolysis of phosphoinositides. The antibiotic neomycin selectively decreases alpha 1-receptor-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis without decreasing alpha 1-receptor-mediated arachidonic acid release and PGE2 generation. In addition, receptor-mediated inositol trisphosphate formation is independent of extracellular calcium, whereas release of labeled arachidonic acid is largely calcium-dependent. Moreover, based on studies obtained with labeled arachidonic acid, receptor-mediated generation of arachidonic acid cannot be accounted for by breakdown of phosphatidylinositol monophosphate, phosphatidylinositol bisphosphate, or phosphatidic acid. Further studies indicate that epinephrine produces changes in formation or turnover of several classes of membrane phospholipids in MDCK cells. We conclude that alpha 1-adrenergic receptors in MDCK cells appear to regulate phospholipid metabolism by the parallel activation of phospholipase C and phospholipase A2. This parallel activation of phospholipases contrasts with models described in other systems which imply sequential activation of phospholipase C and diacylglycerol lipase or phospholipase A2.     

Regulation of epithelial tubule formation by Rho family GTPases

Previous work has established that the integrin signal transduction pathway plays an important role in the regulation of epithelial tubule formation. Furthermore, it has been demonstrated that Rho-kinase, an effector of the Rho signaling pathway, is an important downstream modulator of collagen-mediated renal and mammary epithelial tubule morphogenesis. In the present study, MDCK cells that expressed mutant dominant-negative, constitutively active Rho family GTPases were used to provide further insight into Rho-GTPase signaling and the regulation of epithelial tubule formation. Using collagen gel overlays on MDCK cells as a model system, we observed phosphorylated myosin light chain (pMLC) at the leading edge of migrating lamellipodia. This epithelial remodeling led to the formation of multicellular branching epithelial tubular structures with extensive tight junctions. However, in cells expressing dominant-negative RhoN19, MLC phosphorylation, epithelial remodeling, and tubule formation were inhibited. Instead, only small apical lumens with a solitary tight junctional ring were observed, providing further evidence that Rho signaling through Rho-kinase is important in the regulation of epithelial tubule formation. Because the present model for the Rho signaling pathway proposes that Rac plays a prominent but reciprocal role in cell regulation, experiments were conducted using cells that expressed constitutively active RacV12. When incubated with collagen gels, RacV12-expressing cells formed small apical lumens with simple tight junctions, suggesting that Rac1 signaling also has a prominent role in the regulation of epithelial morphogenesis. Complementary collagen gel overlay experiments with wild-type MDCK cells demonstrated that endogenous Rac1 activation levels decreased over a time course consistent with lamellipodia and tubule formation. Under these conditions, Rac1 was initially localized to the basolateral membrane. However, after epithelial remodeling, activated Rac1 was observed primarily in lamellipodia. These studies support a model in which Rac1 and RhoA are important modulators of epithelial tubule formation. Rac signaling; Rho signaling; tight junction; adherens junction     

Mechanisms of activation and action of mDial in the formation of parallel stress fibers in MDCK cells

mDial is a downstream target molecule of Rho small G protein and regulates the formation of parallel stress fibers in MDCK cells. mDial consists of at least one Rho-binding domain (RBD), one FH3 domain (FH3D), one coiled-coil domain (CCD), one FH1 domain (FH1D), one FH2 domain (FH2D), and another CCD in this order from the N-terminus to the C-terminus. We constructed various deletion mutants of mDial and investigated the mechanisms of its activation and action by measuring the formation of parallel stress fibers in MDCK cells. We show here that at least FH1D and second CCD are essential for the formation of parallel stress fibers. Furthermore, we present the evidence suggesting that mDial has another domain which interacts with RBD, that this interaction masks FH1D and second CCD and keeps mDial inactive, and that the binding of Rho to RBD opens this folded structure, resulting in the activation of mDial.