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Requirement for Ras and Phosphatidylinositol 3-Kinase Signaling Uncouples the Glucocorticoid-induced Junctional Organization and Transepithelial Electrical Resistance in Mammary Tumor Cells*

  • Paul L. Woo
    Footnotes
    Affiliations
    From the Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California, Berkeley, California 94720-3200
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  • Dixie Ching
    Affiliations
    From the Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California, Berkeley, California 94720-3200
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  • Yi Guan
    Affiliations
    From the Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California, Berkeley, California 94720-3200
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  • Gary L. Firestone
    Footnotes
    Affiliations
    From the Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California, Berkeley, California 94720-3200
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grant DK-42799 (to G. L. F.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Recipient of a predoctoral fellowship supported by National Institutes of Health National Research Service Grant CA-09041.
    § To whom all correspondence should be addressed: Dept. of Molecular and Cell Biology, 591 LSA, University of California at Berkeley, Berkeley, CA 94720-3200. Tel.: 510-642-8319; Fax: 510-643-6791; E-mail: [email protected]
Open AccessPublished:November 12, 1999DOI:https://doi.org/10.1074/jbc.274.46.32818
      In Con8 rat mammary epithelial tumor cells, the synthetic glucocorticoid dexamethasone stimulates the remodeling of the apical junction (tight and adherens junctions) and the transepithelial electrical resistance (TER), which reflects tight junction sealing. Indirect immunofluorescence revealed that dexamethasone induced the recruitment of endogenous Ras and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase to regions of cell-cell contact, concurrently with the stimulation of TER. Expression of dominant-negative RasN17 abolished the dexamethasone stimulation in TER, whereas, dexamethasone induced the reorganization of tight junction and adherens junction proteins, ZO-1 and β-catenin, as well as F-actin, to precise regions of cell-cell contact in a Ras-independent manner. Confocal microscopy revealed that RasN17 and the p85 regulatory subunit of PI 3-kinase co-localized with ZO-1 and F-actin at the tight junction and adherens junction, respectively. Treatment with either of the PI 3-kinase inhibitors, wortmannin or LY294002, or the MEK inhibitor PD 098059, which prevents MAPK signaling, attenuated the dexamethasone stimulation of TER without affecting apical junction remodeling. Similar to dominant-negative RasN17, disruption of both Ras effector pathways using a combination of inhibitors abolished the glucocorticoid stimulation of TER. Thus, the glucocorticoiddependent remodeling of the apical junction and tight junction sealing can be uncoupled by their dependence on Ras and/or PI 3-kinase-dependent pathways, implicating a new role for Ras and PI 3-kinase cell signaling events in the steroid control of cell-cell interactions.
      MAGUK
      membrane-associated guanylate kinase
      TER
      transepithelial electrical resistance
      Dex
      dexamethasone
      ZO-1
      zonula occludens-1
      MAPK
      mitogen-activated protein kinase
      ERK
      extracellular regulated kinase
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PIPES
      piperazine-N,N′-bis(2-ethanesulfonic acid
      PKC
      protein kinase C
      PBS
      phosphate-buffered saline
      FITC
      fluorescein isothiocyanate
      The apical junctional complex, which consists of the tight junction and the adherens junction, controls intercellular adhesion and the permeability properties involved in epithelial cell-cell interactions. The tight junction, a specialized structure located at the apex of the junctional complex, restricts the lateral diffusion of lipids and membrane proteins, and thereby physically defines the border between the apical and basolateral compartments (
      • Cereijido M.
      • Valdes J.
      • Shoshani L.
      • Contreras R.G.
      ). Moreover, tight junctions form a regulated barrier for the diffusion of solutes through the paracellular pathway to control the microenvironment on each side of the epithelium (
      • Madara J.L.
      ). Immediately basal to the tight junction is the adherens junction that is responsible for intercellular adhesion between neighboring cells, a process critical for the proper organization and physiological function of the tissue (). Both these intercellular junctions have been proposed to associate with the perijunctional actin cytoskeleton and signaling molecules through multiprotein complexes to form an integrated functional unit.
      Assembly of the tight junction requires the initial engagement of cell-cell contacts at the adherens junction, a process that is mediated by the calcium-dependent intercellular adhesion between E-cadherin molecules and the formation of an intracellular protein complex that includes E-cadherin, α-catenin, β-catenin, γ-catenin (plakoglobin), and the actin cytoskeleton (
      • Gumbiner B.
      • Stevenson B.
      • Grimaldi A.
      ,
      • Ozawa M.
      • Baribault H.
      • Kemler R.
      ). Activation of cadherin-mediated cell-cell adhesion triggers a series of molecular events that can lead to the recruitment of tight junction components to the points of cell-cell contact and assembly into a complex to form a functional unit capable of providing a tight paracellular seal to the epithelium (
      • Siliciano J.D.
      • Goodenough D.A.
      ). The tight junction complex consists of several classes of protein components that includes transmembrane proteins, intracellular peripheral membrane proteins, and potential cell signaling molecules. At present, 3 transmembrane protein families, occludin (
      • Furuse M.
      • Hirase T.
      • Itoh M.
      • Nagafuchi A.
      • Yonemura S.
      • Tsukita S.
      • Tsukita S.
      ), 8 members of the claudin family (
      • Furuse M.
      • Fujita K.
      • Hiiragi T.
      • Fujimoto K.
      • Tsukita S.
      ,
      • Morita K.
      • Furuse M.
      • Fujimoto K.
      • Tsukita S.
      ), and junctional adhesion molecule (
      • Martin-Padura I.
      • Lostaglio S.
      • Schneemann M.
      • Williams L.
      • Romano M.
      • Fruscella P.
      • Panzeri C.
      • Stoppacciaro A.
      • Ruco L.
      • Villa A.
      • Simmons D.
      • Dejana E.
      ), have been identified to reside at the tight junction. Although little is known about the claudin protein family or junctional adhesion molecule, occludin is thought to provide paracellular barrier function through its extracellular domain (
      • Wong V.
      • Gumbiner B.M.
      ). The cytoplasmic tail of occludin interacts with a complex of related peripheral membrane proteins, ZO-1, ZO-2, and ZO-3, three members of the membrane-associated guanylate kinase (MAGUK)1 protein family (
      • Mitic L.L.
      • Anderson J.M.
      ). Each of these proteins contain three PDZ (PSD95/SAP90, discs-large, ZO-1) domains (
      • Ranganathan R.
      • Ross E.M.
      ), a Src homology (SH3) domain, and a region similar to guanylate kinase. It has been proposed that these protein-binding modules allow MAGUK members to coordinate the localization and clustering of transmembrane and peripheral membrane proteins. In doing so, MAGUK proteins at the tight junction may provide a bridge connecting the cytoskeleton or intracellular signaling pathways to transmembrane proteins, such as occludin, thereby regulating the extracellular tight junction seal (
      • Anderson J.M.
      ). In this regard, several potential cell signaling molecules, such as PKC-ζ (
      • Dodane V.
      • Kachar B.
      ), atypical PKC isotype-specific interacting protein (
      • Izumi Y.
      • Hirose T.
      • Tamai Y.
      • Hirai S.
      • Nagashima Y.
      • Fujimoto T.
      • Tabuse Y.
      • Kemphues K.J.
      • Ohno S.
      ), the heterotrimeric G protein subunit (
      • Denker B.M.
      • Saha C.
      • Khawaja S.
      • Nigam S.K.
      ), and the Ras target AF-6 (
      • Yamamoto T.
      • Harada N.
      • Kano K.
      • Taya S.
      • Canaani E.
      • Matsuura Y.
      • Mizoguchi A.
      • Ide C.
      • Kaibuchi K.
      ), have been localized to the tight junction. Although conceptually intriguing, their functional role in regulating tight junction assembly and/or permeability properties has not been characterized.
      The physiological plasticity and tissue-specific regulation of assembly and function of epithelial cell tight junctions implicate a complex set of signal transduction pathways that likely target and control the apical junctional complex. For example, in various cell types the permeability properties of the tight junction can be influenced by growth factors, intracellular calcium, calmodulin, protein kinase C, receptor and nonreceptor tyrosine kinases, heterotrimeric G proteins, lipid second messengers, and phospholipase C (
      • Balda M.S.
      • Gonzalez-Mariscal L.
      • Contreras R.G.
      • Macias-Silva M.
      • Torres-Marquez M.E.
      • Garcia-Sainz J.A.
      • Cereijido M.
      ,
      • Madara J.L.
      • Parkos C.
      • Colgan S.
      • Nusrat A.
      • Atisook K.
      • Kaoutzani P.
      ,
      • Balda M.S.
      • Gonzalez-Mariscal L.
      • Matter K.
      • Cereijido M.
      • Anderson J.M.
      ,
      • Buse P.
      • Woo P.L.
      • Alexander D.B.
      • Cha H.H.
      • Reza A.
      • Sirota N.D.
      • Firestone G.L.
      ,
      • Takeda H.
      • Tsukita S.
      ,
      • Woo P.L.
      • Cha H.H.
      • Singer K.L.
      • Firestone G.L.
      ). Regulatory proteins belonging to the Ras superfamily of small GTPases consisting of the Ras, Rho, and Rab subfamilies, which transduce intracellular signals from a variety of extracellular stimuli, have been proposed to play a role in cell-cell interactions. Recently, Rac and Rho have been shown to be critical for the establishment and maintenance of intercellular adhesion (
      • Braga V.M.
      • Machesky L.M.
      • Hall A.
      • Hotchin N.A.
      ,
      • Takaishi K.
      • Sasaki T.
      • Kotani H.
      • Nishioka H.
      • Takai Y.
      ), and have been implicated as regulators of tight junction assembly and permeability properties (
      • Nusrat A.
      • Giry M.
      • Turner J.R.
      • Colgan S.P.
      • Parkos C.A.
      • Carnes D.
      • Lemichez E.
      • Boquet P.
      • Madara J.L.
      ,
      • Jou T.S.
      • Schneeberger E.E.
      • Nelson W.J.
      ,
      • Gopalakrishnan S.
      • Raman N.
      • Atkinson S.J.
      • Marrs J.A.
      ). Rho is also involved in the sphingosine 1-phosphate induction of cadherins and the formation of well developed adherens junctions in HEK293 fibroblast cells (
      • Lee M.
      • Van Brocklyn J.R.
      • Thangada S.
      • Liu C.H.
      • Hand A.R.
      • Menzeleev R.
      • Spiegel S.
      • Hla T.
      ). Studies in Drosophila have attributed a role for Rac1 in organizing perijunctional actin at the adherens junction of the wing disc epithelium (
      • Eaton S.
      • Auvinen P.
      • Luo L.
      • Jan Y.N.
      • Simons K.
      ). Moreover, Rac-dependent signaling at cell junctions appears to be cell-type specific, because activated Rac, or its exchange factor, Tiam1, induce invasion of T lymphoma cells but suppress invasion in epithelial cells by increasing its adhesive properties (
      • Hordijk P.L.
      • ten Klooster J.P.
      • Van der Kammen R.A.
      • Michiels F.
      • Oomen L.C.
      • Collard J.G.
      ). Rab proteins, such as rab13 (
      • Zahraoui A.
      • Joberty G.
      • Arpin M.
      • Fontaine J.J.
      • Hellio R.
      • Tavitian A.
      • Louvard D.
      ) and rab3B (
      • Weber E.
      • Berta G.
      • Tousson A.
      • St. John P.
      • Green M.W.
      • Gopalokrishnan U.
      • Jilling T.
      • Sorscher E.J.
      • Elton T.S.
      • Abrahamson D.R.
      • Kirk K.L.
      ), have been found to be concentrated at the tight junctions, although they are generally thought to function in the control of vesicle targeting to the plasma membrane. Aberrant activation of oncogenic Ras proteins in epithelial cells is characterized by mesenchymal/fibroblastic morphology with a perturbation of the adherens junction (
      • Schoenenberger C.A.
      • Zuk A.
      • Kendall D.
      • Matlin K.S.
      ,
      • Kinch M.S.
      • Clark G.J.
      • Der C.J.
      • Burridge K.
      ). However, little is known about the role of cellular Ras in the regulation of epithelial junctional complex or whether the physiological stimuli that control cell-cell interactions can exert their effects through Ras signaling pathways.
      To further elucidate the mechanisms of signal transmission required for the regulation of the tight junction, we have been utilizing the Con8 mammary tumor epithelial cell line to investigate the hormonal control of cell-cell interactions (
      • Buse P.
      • Woo P.L.
      • Alexander D.B.
      • Cha H.H.
      • Reza A.
      • Sirota N.D.
      • Firestone G.L.
      ). Con8 cells grow as poorly differentiated monolayers, exhibiting deficient cell adhesion and poor tight junction organization (
      • Wong V.
      • Ching D.
      • McCrea P.D.
      • Firestone G.L.
      ). Our previous studies have shown that treatment with the synthetic glucocorticoid dexamethasone induces the assembly and function of the tight junction, concurrent with an induction of a G1 cell cycle arrest (
      • Buse P.
      • Woo P.L.
      • Alexander D.B.
      • Cha H.H.
      • Reza A.
      • Sirota N.D.
      • Firestone G.L.
      ,
      • Goya L.
      • Maiyar A.C.
      • Ge Y.
      • Firestone G.L.
      ). Dexamethasone stimulates an increase in the transepithelial electrical resistance (TER) of the epithelial monolayer, which directly correlated with a decrease in paracellular permeability to radioactive tracers across the epithelium (
      • Buse P.
      • Woo P.L.
      • Alexander D.B.
      • Cha H.H.
      • Reza A.
      • Sirota N.D.
      • Firestone G.L.
      ), thus verifying the steroid induction of tight junction barrier properties. In addition, glucocorticoids induce the reorganization of the apical junction leading to the recruitment of tight junction and adherens junction proteins to their respective location (
      • Buse P.
      • Woo P.L.
      • Alexander D.B.
      • Cha H.H.
      • Reza A.
      • Sirota N.D.
      • Firestone G.L.
      ,
      • Wong V.
      • Ching D.
      • McCrea P.D.
      • Firestone G.L.
      ). Given the intimate association of certain growth factor signaling components with structural proteins at the cell junction, we investigated the potential roles of cellular Ras and PI 3-kinase in the cell signaling pathways by which glucocorticoids regulate mammary tumor cell-cell interactions. Our results demonstrate that Ras and PI 3-kinase are recruited to regions of cell-cell contact as a consequence of the glucocorticoid-induced membrane reorganization event. Moreover, inhibition of Ras and PI 3-kinase pathways abolishes or attenuates the glucocorticoid-mediated enhancement of the tight junction seal without altering the remodeling of the cell junction, which suggests that these two events can be uncoupled by their requirements for growth factor signaling pathways.

      DISCUSSION

      A complex array of intracellular cell signaling pathways cooperate to regulate the adhesive and permeability properties of the adherens and tight junctions by coordinately targeting components of the apical junctional complex. As a result, dynamic changes in cell-cell interactions can occur in response to specific extracellular stimuli that are involved in the development, organization, and function of differentiated epithelia. In Con8 mammary epithelial tumor cells, glucocorticoid receptor signaling induces the remodeling of tight junction and adherens junction proteins from a disorganized distribution to an organized state typified by a continuous belt of staining surrounding each cell. Subsequently, glucocorticoids then stimulate the barrier property of the tight junction resulting in the increase in TER. Our results have uncovered a previously uncharacterized cross-talk between glucocorticoid receptor and growth factor receptor signaling pathways in which Ras-dependent signals are required for glucocorticoids to enhance the integrity of the tight junction (barrier function) at a step after the steroid-regulated remodeling of the apical junctional complex (see model in Fig. 10). Following the inhibition of cellular Ras function, glucocorticoids effectively reorganize the distribution of ZO-1, β-catenin, and F-actin to sites of cell-cell contact, whereas, the glucocorticoid-mediated stimulation of TER was abolished. Treatment with inhibitors of MEK or PI 3-kinase, two known downstream components of Ras effector pathways selectively attenuated the glucocorticoid enhancement of an electrically tight cell monolayer. Thus, the glucocorticoid-mediated remodeling of the apical junction and the barrier function of the tight junction can be uncoupled by their dependence on Ras signaling and its downstream effector pathways.
      Figure thumbnail gr10
      Figure 10Model of the glucocorticoid receptor-regulated process that controls apical junction remodeling and tight junction sealing. We propose that glucocorticoid receptor signaling events that regulate tight junction dynamics induce the remodeling of the apical junction, which serves to recruit structural and cell signaling proteins to the cell junction. In a subsequent step, the tight junction sealing process requires the Ras, MEK/MAPK, and PI 3-kinase pathways.
      The signal transduction system that regulates tight junction permeability is relatively uncharacterized, although the presence of three members of the MAGUK protein family, ZO-1, ZO-2, and ZO-3, suggests that a complex network of small molecule-protein and protein-protein interactions occur in a sequential and regulated manner. A pressing challenge in the study of tight junction dynamics has been to understand the regulatory pathways by which the tight junction structure can respond to extracellular stimuli. Increasing evidence suggests that the specificity of distinct cellular responses is contingent upon the correct spatial organization of a defined repertoire of cell signaling components localized at the junctional complex. Our results demonstrate that Ras and PI 3-kinase are recruited to and highly concentrated at regions of cell-cell contact during the glucocorticoid-dependent reorganization of the intercellular junction in mammary tumor cells. Earlier studies have shown that Ras is localized to the cell periphery in v-Ha-Ras-transformed and v-Ki-Ras transformed Madin-Darby canine kidney cells (
      • Schoenenberger C.A.
      • Zuk A.
      • Kendall D.
      • Matlin K.S.
      ,
      • Furth M.E.
      • Davis L.J.
      • Fleurdelys B.
      • Scolnick E.M.
      ). Our results further show that the dominant-negative RasN17 is also recruited to the junctional complex and specifically co-localizes with ZO-1 and F-actin at the tight junction and adherens junction, respectively, demonstrating that the inability of RasN17 to bind GTP and, thus its effectors, does not alter its proper distribution to the plasma membrane. That the p85 subunit of PI 3-kinase also co-localizes with ZO-1 and F-actin in dexamethasone-treated Con8 cells raises the question of whether the recruitment of PI 3-kinase to the junctional complex is through the direct binding to activated Ras. Our results demonstrate that the PI 3-kinase translocation to cellular junctions occurs in a Ras-independent mechanism, since p85 was still capable of co-localizing with dominant-negative RasN17 along the lateral junction. However, it remains possible that the mechanism by which Ras and PI 3-kinase are localized to the junction is not mutually exclusive, and that normally Ras can activate PI 3-kinase once they are near each other.
      The biological activity of both Ras and PI 3-kinase is dependent on their correct recruitment to the plasma membrane. A likely explanation for the targeting of Ras and PI 3-kinase to the lateral junction is for the proper presentation of these cell-signaling molecules to their upstream activators. Consistent with this mechanism, growth factor receptors, such as the scatter factor/hepatocyte growth factor receptor and epidermal growth factor-receptor, function and are concentrated along the lateral junction (
      • Crepaldi T.
      • Pollack A.L.
      • Prat M.
      • Zborek A.
      • Mostov K.
      • Comoglio P.M.
      ,
      • Dempsey P.J.
      • Meise K.S.
      • Yoshitake Y.
      • Nishikawa K.
      • Coffey R.J.
      ). The adaptor protein, Shc, which mediates the association of tyrosine kinase receptors with the Grb-2/Sos complex involved in Ras activation can also interact with phosphotyrosyl residues on cadherins through its SH2 domain (
      • Xu Y.
      • Guo D.F.
      • Davidson M.
      • Inagami T.
      • Carpenter G.
      ). In addition, activation of the Ras guanine nucleotide exchange factor, Ras-GRF2, by calcium influx caused its recruitment to intercellular junctions in kidney epithelial cells (
      • Fam N.P.
      • Fan W.T.
      • Wang Z.
      • Zhang L.J.
      • Chen H.
      • Moran M.F.
      ). PDZ-containing adaptor proteins, such as those of the ZO-1 family, may serve as molecular scaffolds to selectively assemble cell signaling molecules at specialized junctions. Evidence to support this model has been described in Caenorhabditis elegans, in which a complex of PDZ-containing proteins, one of which is a member of the MAGUK family, mediates the proper localization of the epidermal growth factor receptor LET-23 to the basolateral junction through direct protein-protein interaction (
      • Kaech S.M.
      • Whitfield C.W.
      • Kim S.K.
      ). In addition, a recently cloned PDZ-containing protein in Drosophila, CNK, is thought to assemble signaling molecules in the Ras pathway to cell-cell contact regions (
      • Therrien M.
      • Wong A.M.
      • Rubin G.M.
      ). Thus, it is becoming increasingly apparent that diverse cellular responses affecting cell-cell interactions are mediated by distinct sets of regulatory proteins that are directly associated with the apical junction.
      The requirement for Ras and PI 3-kinase activity for the glucocorticoid induction of tight junction sealing indicates that a select subset of downstream targets reside at the tight junction to regulate paracellular permeability. In this regard, PKC-ζ, which can directly bind to GTP-bound Ras (
      • Diaz-Meco M.T.
      • Lozano J.
      • Municio M.M.
      • Berra E.
      • Frutos S.
      • Sanz L.
      • Moscat J.
      ) and be activated by the PI 3-kinase target, 3-phosphoinositide-dependent protein kinase-1 (
      • Le Good J.A.
      • Ziegler W.H.
      • Parekh D.B.
      • Alessi D.R.
      • Cohen P.
      • Parker P.J.
      ,
      • Chou M.M.
      • Hou W.
      • Johnson J.
      • Graham L.K.
      • Lee M.H.
      • Chen C.
      • Newton A.C.
      • Schaffhausen B.S.
      • Toker A.
      ), have been shown to be localized specifically at the tight junction in Madin-Darby canine kidney and Caco-2 epithelial cells (
      • Dodane V.
      • Kachar B.
      ), as well as in the Con8 mammary tumor cells, which have established well formed tight junctions by glucocorticoid treatment.
      V. Wong and G. L. Firestone, unpublished data.
      However, the role of PKC-ζ in the regulation of paracellular permeability has yet to be determined. In addition, the Ras effector AF-6 is specifically localized to the tight junction and may provide a link to the cytoskeleton by directly interacting with F-actin and ZO-1 (
      • Yamamoto T.
      • Harada N.
      • Kano K.
      • Taya S.
      • Canaani E.
      • Matsuura Y.
      • Mizoguchi A.
      • Ide C.
      • Kaibuchi K.
      ). PI 3-kinase has also been shown to mediate Ras-dependent actin rearrangement (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.
      • Downward J.
      ). The potential role of lipid products of PI 3-kinase as direct activators of tight junction proteins may represent a unique function for these secondary messengers. However, the fact that inhibitors of PI 3-kinase cannot completely mimic the dominant-negative Ras repression of tight junction activation suggests that PI 3-kinase may function in concert with other signaling pathways to control permeability properties.
      Inhibition of MAPK activation by treatment of the Con8 mammary tumor cells with the MEK inhibitor, PD 098059, prevented the full induction of TER by glucocorticoids. This result implicates a requirement for MAPK signaling in the glucocorticoid-stimulated tight junction sealing. It is interesting to note that other studies have shown that MAPK may be involved in the disassembly of adherens junctions by hepatocyte growth factor in Madin-Darby canine kidney cells (
      • Potempa S.
      • Ridley A.J.
      ) and decreased expression of adherens junction components in PC12 cells (
      • Lu Q.
      • Paredes M.
      • Zhang J.
      • Kosik K.S.
      ). One possible explanation for our observation that MAPK is required for the activation of tight junctions in mammary tumor cells by glucocorticoids is the alteration in the cellular location of MAPK. In the absence of glucocorticoids, MAPK is phosphorylated and translocated to the nucleus in serum-treated Con8 cells, whereas, dexamethasone treatment of the mammary tumor cells maintains MAPK primarily in a cytoplasmic compartment.
      P. Buse, S. Tran, and G. L. Firestone, submitted for publication.
      Thus, in mammary tumor cells, it is tempting to speculate that MAPK could play a role in regulating the phosphorylation and function of tight and/or adherens junction components. Intriguingly, MAPK has been shown to phosphorylate the gap junction protein connexin 43 (
      • Warn-Cramer B.J.
      • Lampe P.D.
      • Kurata W.E.
      • Kanemitsu M.Y.
      • Loo L.W.M.
      • Eckhart W.
      • Lau A.F.
      ) that interacts with the ZO-1 tight junction protein (
      • Giepmans B.N.
      • Moolenaar W.H.
      ). In addition, it has been reported that MAPK can phosphorylate and activate the myosin light chain kinase (
      • Klemke R.L.
      • Cai S.
      • Giannini A.L.
      • Gallagher P.J.
      • de Lanerolle P.
      • Cheresh D.A.
      ), providing a possible mechanism in which MAPK can regulate the myosin ATPase-mediated contraction of the perijunctional actomyosin belt to influence tight junction integrity. We have also shown that inhibition of both MAPK and PI 3-kinase pathways cooperate to prevent the glucocorticoid stimulated electrical tightness of the mammary tumor cell tight junctions. The combination of kinase inhibitors did not indirectly inhibit tight junction permeability due to apoptosis, because glucocorticoids were able to provide a protective effect from the inhibition of cell survival pathways caused by these agents. These results suggest that the requirement for Ras in the barrier function of tight junctions induced by glucocorticoids may involve at least two downstream pathways, PI 3-kinase and MAPK.
      At present, it is unclear how each tight junction molecule is regulated to provide barrier function to the epithelia. One hypothesis is that the phosphorylation of the transmembrane protein, occludin, dictates the permeability properties of epithelial cells (
      • Sakakibara A.
      • Furuse M.
      • Saitou M.
      • Ando A.Y.
      • Tsukita S.
      ,
      • Wong V.
      ). Further support for the role of a kinase in the regulation of tight junctions is found in studies utilizing ATP-depletion experiments, which abolishes the barrier function of the tight junction without altering ZO-1 distribution (
      • Mandel L.J.
      • Bacallao R.
      • Zampighi G.
      ). Consistent with our observations in dominant-negative Ras expressing cells, other studies have also found that the formation of a continuous junctional belt of tight junction proteins along the cell periphery does not always correlate with the establishment of electrically tight epithelia. For example, expression of dominant-negative forms of RhoA and Rac1 can induce a leaky tight junction without an apparent effect on the distribution of ZO-1 or occludin (
      • Jou T.S.
      • Schneeberger E.E.
      • Nelson W.J.
      ). We have previously shown that enhancement of tight junction sealing in a non-transformed mammary epithelial cell line, 31EG4, by glucocorticoids occurs without a change in ZO-1 localization (
      • Woo P.L.
      • Cha H.H.
      • Singer K.L.
      • Firestone G.L.
      ,
      • Zettl K.S.
      • Sjaastad M.D.
      • Riskin P.M.
      • Parry G.
      • Machen T.E.
      • Firestone G.L.
      ,
      • Singer K.L.
      • Stevenson B.R.
      • Woo P.L.
      • Firestone G.L.
      ). Evidence for a functionally defective tight junction without alterations in tight junction morphology has also been described in rat models of colitis in which tight junction permeability increased in the intestinal and biliary epithelia without structural changes in the tight junction (
      • Lora L.
      • Mazzon E.
      • Martines D.
      • Fries W.
      • Muraca M.
      • Martin A.
      • d'Odorico A.
      • Naccarato R.
      • Citi S.
      ). Our results have dissociated two key events involved in the glucocorticoid regulation of tight junction dynamics in mammary tumor cells, the first being the induced organization of the junctional complex that occurs in a Ras-independent manner, and the second being the Ras-dependent process that leads to an increase in barrier function of the tight junction. We are currently attempting to characterize the downstream targets of Ras signaling that mediate this novel convergence point between glucocorticoid receptor and growth factor receptor cell signaling that control cell-cell interactions of mammary epithelial cells.

      Acknowledgments

      We express our appreciation to Anita C. Maiyar for critical evaluation of this manuscript and helpful experimental suggestions. We thank Tran Van, Minnie Wu, and Kenneth Oh for technical assistance.

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