Glucocorticoid down-regulation of fascin protein expression is required for the steroid-induced formation of tight junctions and cell-cell interactions in rat mammary epithelial tumor cells

Glucocorticoid hormones, which are physiological regulators of mammary epithelium development, induce the formation of tight junctions in rat Con8 mammary epithelial tumor cells. We have discovered that, as part of this process, the synthetic glucocorticoid dexamethasone strongly and reversibly down-regulated the expression of fascin, an actin-bundling protein that also interacts with the adherens junction component beta-catenin. Ectopic constitutive expression of full-length mouse fascin containing a Myc epitope tag (Myc-fascin) in Con8 cells inhibited the dexamethasone stimulation of transepithelial electrical resistance, disrupted the induced localization of the tight junction protein occludin and the adherens junction protein beta-catenin to the cell periphery, and prevented the rearrangement of the actin cytoskeleton. Ectopic expression of either the carboxyl-terminal 213 amino acids of fascin, which includes the actin and beta-catenin-binding sites, or the amino-terminal 313 amino acids of fascin failed to disrupt the glucocorticoid induction of tight junction formation. Mammary tumor cells expressing the full-length Myc-fascin remained generally glucocorticoid responsive and displayed no changes in the levels or protein-protein interactions of junctional proteins or the amount of cytoskeletal associated actin filaments. However, a cell aggregation assay demonstrated that the expression of Myc-fascin abrogated the dexamethasone induction of cell-cell adhesion. Our results implicate the down-regulation of fascin as a key intermediate step that directly links glucocorticoid receptor signaling to the coordinate control of junctional complex formation and cell-cell interactions in mammary tumor epithelial cells.     

Requirement for Ras and phosphatidylinositol 3-kinase signaling uncouples the glucocorticoid-induced junctional organization and transepithelial electrical resistance in mammary tumor cells.

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 beta-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.     

Glucocorticoid down-regulation of RhoA is required for the steroid-induced organization of the junctional complex and tight junction formation in rat mammary epithelial tumor cells

In Con8 mammary epithelial tumor cells, we have documented previously that the synthetic glucocorticoid dexamethasone induces the reorganization of the tight junction and adherens junction (apical junction) and stimulates the monolayer transepithelial electrical resistance (TER), which is a reliable in vitro measurement of tight junction sealing. Western blots demonstrated that dexamethasone treatment down-regulated the level of the RhoA small GTPase prior to the stimulation of the monolayer TER. To test the role of RhoA in the steroid regulation of apical junction dynamics functionally, RhoA levels were altered in Con8 cells by transfection of either constitutively active (RhoA.V14) or dominant negative (RhoA.DN19) forms of RhoA. Ectopic expression of constitutively active RhoA disrupted the dexamethasone-stimulated localization of zonula occludens-1 and beta-catenin to sites of cell-cell contact, inhibited tight junction sealing, and prevented the complete formation of the F-actin ring structure at the apical side of the cell monolayer. In a complementary manner, dominant negative RhoA caused a precocious organization of the tight junction, adherens junction, and the F-actin rings in the absence of steroid, whereas the monolayer TER remained glucocorticoid-responsive. Taken together, our results demonstrate that the glucocorticoid down-regulation of RhoA is a required step in the steroid signaling pathway which controls the organization of the apical junctional complex and the actin cytoskeleton in mammary epithelial cells.     

Rnd3/RhoE induces tight junction formation in mammary epithelial tumor cells

Glucocorticoid hormones stimulate adherens and tight junction formation in Con8 mammary epithelial tumor cells through a multistep process in which the membrane organization of structural apical junction proteins and tight junction sealing is controlled by specific signal transduction components. We have previously shown that dexamethasone stimulation of apical junction formation requires down-regulation of the small GTPase RhoA. Here we identified Rnd3/RhoE, a GTPase-deficient Rho family member and RhoA antagonist, as a key regulator of apical junction dynamics. Exogenously expressed Rnd3/RhoE co-localized with actin at the cell periphery and induced the localization of the adherens junction protein beta-catenin and the tight junction protein ZO-1 to sites of cell-cell contact, and led to the formation of highly sealed tight junctions. Treatment with glucocorticoids was not required to achieve complete apical junction remodeling. Consistent with Rnd3/RhoE acting as an antagonist of RhoA, expression of Rnd3/RhoE rescued the disruptive effects of constitutively active RhoA on apical junction organization. Our results demonstrate a new role for the Rho family member Rnd3/RhoE in regulating the assembly of the apical junction complex and tight junction sealing.     

Selective glucocorticoid control of Rho kinase isoforms regulate cell-cell interactions

The two Rho kinase isoforms ROCK1 and ROCK2 are downstream effectors of the small GTPase RhoA, although relatively little is known about potential isoform specific functions or the selective control of their cellular activities. Using Con8 rat mammary epithelial cells, we show that the synthetic glucocorticoid dexamethasone strongly stimulates the level of ROCK2 protein, which accounts for the increase in total cellular ROCK2 activity, whereas, steroid treatment down-regulated ROCK1 specific kinase activity without altering ROCK1 protein levels. In Con8 cells, the glucocorticoid induced formation of tight junctions requires the steroid-mediated down-regulation RhoA and function of the RhoA antagonist Rnd3. Treatment with the ROCK inhibitor Y-27632 ablated both the glucocorticoid-induced and Rnd3-mediated stimulation in tight junction sealing. Taken together, our results demonstrate that the expression and activity of ROCK1 and ROCK2 can be uncoupled in a signal-dependent manner, and further implicate a new function for ROCK2 in the steroid control of tight junction dynamics.     

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     

Occludin is required for apoptosis when claudin�Cclaudin interactions are disrupted

Disruption of tight junctions is often seen during pathogen infection, inflammation, and tumor progression. Mislocalization of the tight junction proteins occludin and claudin in mammary epithelial monolayers leads to apoptosis through the extrinsic pathway. To further investigate the mechanism of this response, a normal mammary epithelial cell line (EpH4) as well as primary mammary epithelial cells were treated with a claudin-disrupting mimic peptide, DFYNP (aspartic acid–phenylalanine–tyrosine–asparagine–proline). Using fluorescent indicators, we found that caspase-3 activation, resulting from treatment with DFYNP, was restricted to EpH4 and primary mammary epithelial cells with mislocalized claudin-4. Mislocalized claudin-4 and occludin were colocalized in non-junctional puncta, and both molecules were found in the death-inducing signaling complex (DISC) where they colocalized with Fas, fas-associated protein with death domain (FADD), active caspase-8 and caspase-3 at distinct apical domains. Importantly, caspase-3 activation was totally repressed in primary mammary epithelial cells from occludin null mice. Thus, the apoptotic response appears to be initiated by the movement of occludin to the DISC suggesting that this molecule has signaling properties that initiate cell death when its tight junction location is disrupted.     

Extracellular signal-regulated kinases 1/2 control claudin-2 expression in Madin-Darby canine kidney strain I and II cells

The tight junction of the epithelial cell determines the characteristics of paracellular permeability across epithelium. Recent work points toward the claudin family of tight junction proteins as leading candidates for the molecular components that regulate paracellular permeability properties in epithelial tissues. Madin-Darby canine kidney (MDCK) strain I and II cells are models for the study of tight junctions and based on transepithelial electrical resistance (TER) contain "tight" and "leaky" tight junctions, respectively. Overexpression studies suggest that tight junction leakiness in these two strains of MDCK cells is conferred by expression of the tight junction protein claudin-2. Extracellular signal-regulated kinase (ERK) 1/2 activation by hepatocyte growth factor treatment of MDCK strain II cells inhibited claudin-2 expression and transiently increased TER. This process was blocked by the ERK 1/2 inhibitor U0126. Transfection of constitutively active mitogen-activated protein kinase/extracellular signal-regulated kinase kinase into MDCK strain II cells also inhibited claudin-2 expression and increased TER. MDCK strain I cells have higher levels of active ERK 1/2 than do MDCK strain II cells. U0126 treatment of MDCK strain I cells decreased active ERK 1/2 levels, induced expression of claudin-2 protein, and decreased TER by approximately 20-fold. U0126 treatment also induced claudin-2 expression and decreased TER in a high resistance mouse cortical collecting duct cell line (94D). These data show for the first time that the ERK 1/2 signaling pathway negatively controls claudin-2 expression in mammalian renal epithelial cells and provide evidence for regulation of tight junction paracellular transport by alterations in claudin composition within tight junction complexes.     

Deciliation Is Associated with Dramatic Remodeling of Epithelial Cell Junctions and Surface Domains

Stress-induced shedding of motile cilia (autotomy) has been documented in diverse organisms and likely represents a conserved cellular reaction. However, little is known about whether primary cilia are shed from mammalian epithelial cells and what impact deciliation has on polarized cellular organization. We show that several chemically distinct agents trigger autotomy in epithelial cells. Surprisingly, deciliation is associated with a significant, but reversible increase in transepithelial resistance. This reflects substantial reductions in tight junction proteins associated with “leaky” nephron segments (e.g., claudin-2). At the same time, apical trafficking of gp80/clusterin and gp114/CEACAM becomes randomized, basal-lateral delivery of Na,K-ATPase is reduced, and expression of the nonciliary apical protein gp135/podocalyxin is greatly decreased. However, ciliogenesis-impaired MDCK cells do not undergo continual junction remodeling, and mature cilia are not required for autotomy-associated remodeling events. Deciliation and epithelial remodeling may be mechanistically linked processes, because RNAi-mediated reduction of Exocyst subunit Sec6 inhibits ciliary shedding and specifically blocks deciliation-associated down-regulation of claudin-2 and gp135. We propose that ciliary autotomy represents a signaling pathway that impacts the organization and function of polarized epithelial cells.     

Induction of fascin spikes in breast cancer cells by activation of the insulin-like growth factor-I receptor

Insulin-like growth factor-I receptor (IGF-IR) signaling contributes to the formation of mammary carcinomas and has chiefly been studied with regard to the proliferative and anti-apoptotic effects of IGF-IR signaling. However, IGF-IR activation also affects the actin cytoskeleton and alterations in cell migratory behavior are of known importance for the malignant conversion and metastasis of epithelial cells. The actin-binding protein fascin is found in cell projections and spikes that are involved in the locomotion of mesenchymal cells. Fascin expression is typically low in normal epithelial cells, but is markedly upregulated in several types of carcinomas. Here, we also demonstrate increased fascin expression in breast carcinoma cell lines and adopt MCF-7 human mammary carcinoma cells that over-express wild-type or kinase-inactivated forms of the IGF-IR as a model system to test the hypothesis that IGF-IR activation induces fascin projections. We show that the time-dependent dissociation of cell colonies that occurs upon receptor activation by IGF-I involves the formation of dynamic, fascin-containing lateral cell projections that co-localize with ruffling membranes in association with protrusive activity and cell migratory phenotype. The molecular mechanism of these effects is completely dependent on IGF-IR tyrosine kinase activity and is mediated by a phosphatidylinositol (PI) 3-kinase-dependent process. In demonstrating transduction of fascin spike assembly by activation of a peptide growth factor receptor, these novel data reveal a wide role for fascin spikes in cell motility and provide new insight into the complex effects of IGF-IR signaling on actin cytoskeletal organization.     

Suppression of rat mammary tumor cell growth in vitro by glucocorticoids requires serum proteins. Characterization of wild type and glucocorticoid-resistant epithelial tumor cells

CON8 is a single-cell derived subclone of the 13762NF transplantable, hormone-responsive rat mammary tumor that proliferates rapidly in serum-free medium. Addition of either glucocorticoids or calf serum alone caused a slight stimulation of CON8 proliferation. However, glucocorticoids required the presence of specific serum proteins to strongly suppress CON8 cell growth. Furthermore, the anchorage-independent growth of CON8 cells was significantly reduced in the presence of glucocorticoids and serum. We have designated this serum activity GMGSF, for glucocorticoid modulating growth suppression factor. Inhibition of cell growth was limited to steroids with strong glucocorticoid biological activity, while exposure to the glucocorticoid antagonist RU38486 prevented this response. Half-maximal growth inhibition and half-maximal expression of a glucocorticoid-inducible gene product (2 nM) occurred slightly below the half-maximal receptor binding of [3H]dexamethasone (10nM). We have also selected a variant mammary epithelial tumor cell line, derived from CON8, denoted 8RUV7, whose proliferation and soft agar colony formation failed to be suppressed by glucocorticoids in the presence of serum. These glucocorticoid-resistant variant cells possess functional glucocorticoid receptors, competently produce the glucocorticoid-responsive gene product plasminogen activator inhibitor, and along with CON8 cells express milk fat globule protein antigens on their cell surface, indicative of their mammary epithelial cell character. We are using this variant line to genetically dissect the molecular mechanism of the glucocorticoid/GMGSF growth suppression pathway in mammary epithelial tumor cells.     

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation.     

Na,K-ATPase inhibition alters tight junction structure and permeability in human retinal pigment epithelial cells

Na,K-ATPase regulates avariety of transport functions in epithelial cells. In cultures ofhuman retinal pigment epithelial (RPE) cells, inhibition of Na,K-ATPaseby ouabain and K⁺ depletion decreased transepithelialelectrical resistance (TER) and increased permeability of tightjunctions to mannitol and inulin. Electrophysiological studiesdemonstrated that the decrease in TER was due to an increase inparacellular shunt conductance. At the light microscopy level, thisincreased permeability was not accompanied by changes in thelocalization of the tight junction proteins ZO-1, occludin, andclaudin-3. At the ultrastructural level, increased tight junctionpermeability correlated with a decrease in tight junction membranecontact points. Decreased tight junction membrane contact points andincreased tight junction permeability were reversible inK⁺-repletion experiments. Confocal microscopy revealed thatin control cells, Na,K-ATPase was localized at both apical andbasolateral plasma membranes. K⁺ depletion resulted in alarge reduction of apical Na,K-ATPase, and after K⁺repletion the apical Na,K-ATPase recovered to control levels. Theseresults suggest a functional link exists between Na,K-ATPase and tightjunction function in human RPE cells.

     

Nonreceptor tyrosine kinase c-Yes interacts with occludin during tight junction formation in canine kidney epithelial cells

Occludin is an integral membrane protein that is tyrosine phosphorylated when localized at tight junctions. When Ca(2+) was depleted from the culture medium, occludin tyrosine phosphorylation was diminished from Madin-Darby canine kidney epithelial cells in 2 min. This dephosphorylation was correlated with a significant reduction in transepithelial electrical resistance (TER), indicating a global loss of the tight junction barrier function. Reconstitution of Ca(2+) resulted in a robust tyrosine rephosphorylation of occludin that was temporally associated with an increase in TER. Moreover, we demonstrate in this study that occludin was colocalized with the nonreceptor tyrosine kinase c-Yes at cell junction areas and formed an immunoprecipitable complex with c-Yes in vivo. This complex dissociated when the cells were incubated in medium without Ca(2+) or treated with a c-Yes inhibitor, CGP77675. In the presence of CGP77675 after Ca(2+) repletion, occludin tyrosine phosphorylation was completely abolished and both tight junction formation and the increase of the TER were inhibited. Our study thus provides strong evidence that occludin tyrosine phosphorylation is tightly linked to tight junction formation in epithelial cells, and that the nonreceptor tyrosine kinase c-Yes is involved in the regulation of this process.     

Maintenance of the epithelial barrier in a bronchial epithelial cell line is dependent on functional E-cadherin local to the tight junctions

Tight junctions (TJ) are essential components of polarized epithelia, and E-cadherin is important for their formation and maintenance. The bronchial epithelial cell line, 16HBE14o- expresses E- and P-cadherin, but not N-cadherin. E- and P-cadherin levels changed during culture, the former increasing after confluence, and the latter were markedly reduced. All detectable E-cadherin was bound to β- and γ-catenins. We investigated involvement of E-cadherin with epithelial integrity using an E-cadherin specific, function-blocking antibody, SHE78-7. Surprisingly, apical SHE78-7 exposure caused a prompt fall in transepithelial resistance (TER), while TER remained unchanged for 8 hrs after basal exposure then dropped. SHE78-7 exposure increased epithelial permeability to mannitol, inulin, and 9.5 kDa and 77 kDa dextrans and caused fragmentation and loss of the tight junction protein, ZO-1, from the cell borders in some areas. Ultrastructural studies showed that all junctional intercellular contact was lost in the center of SHE78-7 induced lesions. Near the lesion periphery, epithelial structure was maintained, but TJs were dysfunctional as shown by ruthenium red penetration. Analysis of epithelial penetration by SHE78-7 revealed discrete, local defects in the apical barrier at the top of some cell hills that permitted rapid access of the antibody to E-cadherin near the apical surface. In contrast, after basal exposure, antibody initially engaged with E-cadherin nearer the basal surface and only accessed apical E-cadherin later. Taken together with the TER measurements, these data suggest compartmentalization of E-cadherin function within 16HBE14o- cells, with only the apical E-cadherin adjacent to the tight junctions contributing to the function of the latter.     

Autocrine Production of Insulin-like Growth Factor-I (IGF-I) Affects Paracellular Transport Across Epithelial Cells In Vitro

Autocrine production of growth factors can have significant effects on cell activity. We report for the first time that autocrine production of insulin-like growth factor-I (IGF-I) alters paracellular transport across bovine mammary epithelial cells in vitro. Paracellular transport was assessed by measuring phenol red transport across mammary alveolar cells-large T antigen (MAC-T cells) derived from parental mammary epithelial cells, cultured on porous membranes and compared with two different transfected MAC-T cell lines that constitutively secrete IGF-I. Phenol red transport was essentially blocked in parental cell culture after six days, while IGF-I secreting cells provided essentially no barrier. Surprisingly, neither co-culture studies between parental and IGF-I-secreting cells nor addition of exogenous IGF-I or IGF-binding protein-3 reversed the phenol red transport properties. IGF-I-secreting cells did however express lower levels of the junction components occludin and E-cadherin than parental cells, suggesting that localized autocrine IGF-I activity might lead to increased permeability via changes in both the tight and adherens junction protein levels.