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PubMed2006

Puromycin-purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction.

In vitro blood-brain barrier (BBB) models using primary rat brain microvessel endothelial cells (BMEC) are often hampered by a lack of culture purity and poor barrier properties. To address these problems, the translation inhibitor puromycin was used to purify rat BMEC cultures. BMEC purities of 99.8% were routinely attained using puromycin treatment, and this technique proved to be far superior to other purification methods of similar difficulty. In contrast to cultures without puromycin treatment, purity of puromycin-treated cultures was unaffected by initial seeding density. Next, rat BMEC monolayer transendothelial electrical resistance (TEER) was increased by glucocorticoid treatment with either corticosterone (CORT) or hydrocortisone (HC), and a corresponding decrease in monolayer permeability to small molecules was observed. Importantly, cultures treated with both puromycin and glucocorticoid attained significantly higher TEER values (CORT 168 +/- 13 Omega x cm2; HC 218 +/- 66 Omega x cm2) than those treated by the glucocorticoid alone (CORT 57 +/- 5 Omega x cm2; HC 70 +/- 2 Omega x cm2). Glucocorticoid induction resulted in BMEC morphological changes that accompanied the increases in TEER, and BMEC tight junctions exhibited improved integrity as visualized by the localization of tight junction proteins zonula occluden-1, occludin and claudin-5. The combined use of puromycin and glucocorticoid therefore provides an in vitro system that is well suited for molecular level BBB investigations.

Animals↗
PubMed1989

Cingulin: characterization and localization.

Cingulin, a protein component associated with the tight junctions of chicken intestinal epithelium, has been purified to homogeneity by a new procedure and characterized. Purified cingulin is a heat-stable elongated dimer, composed of two polypeptides of Mr 108,000 (cingulin-108), with a Stokes' radius of approximately 15 nm, and a molecular length of 130 nm +/- 32 nm. Monoclonal antibodies were used to determine the tissue distribution and subcellular localization of cingulin in a variety of avian tissues and cultured cells. Indirect immunofluorescence analysis of semi-thin frozen sections demonstrated that cingulin is localized in the junctional complex of various polarized epithelia and in the endothelium, whereas it is essentially absent from mesenchymal and myogenic cells. In permeabilized and fixed cultured chick embryo kidney cells, the antibodies stained solely the regions of contacts between the epithelial cells. Double immunofluorescent labeling of these cells with anti-cingulin and anti-vinculin antibodies showed that cingulin is localized close to the vinculin-rich cytoskeletal belt associated with adherens junctions, but is absent from focal contacts and stress fibers. In cultured kidney cells, actin was detected mainly in stress fibers and in the peripheral junctional regions, where it showed a distribution similar to that of cingulin, suggesting that actin filaments may be part of the submembrane cytoskeleton at the level of the tight junction. Indirect immunoelectron microscopic labeling of ultrathin frozen sections of chicken intestine showed that cingulin is localized along the endofacial surfaces of the tight junction (zonula occludens), and is apparently excluded from the more basal zonula adhaerens, and from the desmosomes.

Animals↗
PubMed1991

Morphology of glial blood-brain barriers.

Glial cells, in certain situations in the CNS, may become modified to form the structural basis of the blood-brain barrier. This occurs in more primitive vertebrates, such as the elasmobranch fish, and in some higher invertebrates. In the latter, the outermost glial sheath, often called the perineurium in avascular ganglia, substitutes functionally for the vascular endothelium of higher organisms. The intercellular junctions between the lateral borders of these modified glial or perineurial cells may be of several types. In nearly all cases, adhesive and communicating (gap) junctions are found together with an occluding junctional structure. The latter is assumed to be the morphologic basis of the observed blood-brain barrier. It varies in nature and may be one in which the adjacent cell membranes fuse, partially or completely, to form a classical tight junction, or it may be one in which the cell membranes remain separated by a distinct intercellular cleft. If the latter, the cleft may be straddled by columns or septal ribbons, between which a charged matrix substance may be found. Restrictive linker junctions, recently found to be the basis of the interglial barrier in cephalopod CNS, as well as that of myriapods, are characterized by cross-striations or columns which, in combination with charged residues, inherent either in them or in the associated extracellular matrix, slow down the entry of exogenous molecules. Septate junctions, which occur between glial cells in certain other invertebrates, exhibit intercellular septal ribbons, which do not prohibit paracellular transport of all substances but may slow down the passage of some by virtue of charged moieties. There is an association of cytoskeletal components with these septate, linker, and tight junctions; the role of the cytoskeleton in tight junctions, which can be seen by freeze fracture to be based on simple ridges in insects or a more complex network of them in arachnids, may also be important in the regulation of paracellular permeability. The structural details of the junctions in different groups are summarized and their physiologic implications discussed.

Animals↗
PubMed2006

Involvement of MAPKs in endostatin-mediated regulation of blood-retinal barrier function.

PURPOSE: This study aimed to evaluate the effects of endostatin on tight junction (TJ) integrity in retinal microvascular endothelial cells (RMECs) in vitro and in vivo. Moreover, it was hypothesized that endostatin-induced occludin upregulation regulated VEGF165-mediated increases in endothelial cell permeability and involved activation of the MAPK signaling cascade. Endostatin is a 20-kDa fragment of collagen XVIII that has been shown to be efficacious in the eye by preventing retinal neovascularization. Endostatin is a specific inhibitor of endothelial cell proliferation, migration, and angiogenesis and has been reported to reverse VEGF-mediated increases in vasopermeability and to promote integrity of the blood-retinal barrier (BRB). In order to determine the mechanism of endostatin action on BRB integrity, we have examined the effects of endostatin on a number of intracellular pathways implicated in endothelial cell physiology. METHODS: C57/Bl6 mice were injected with VEGF165 and/or endostatin, and the distribution of occludin staining was determined using retinal flatmounts. Western blot analysis of RMECs treated with VEGF165 and/or endostatin was used to determine changes in occludin expression and p38 MAPK and extracellular regulated kinase (ERK1/ERK2 MAPK) activation, while FD-4 flux across the RMEC monolayer was used to determine changes in paracellular permeability. RESULTS: Endostatin prevented the discontinuous pattern of occludin staining observed at the retinal blood vessels of mice administered an intraocular injection of VEGF165. It was shown that endostatin activated p38 MAPK 5 min after addition to RMECs and continued to do so for approximately 30 min. Endostatin was also shown to activate ERK1/ERK2 5 min after addition and continued to do so, albeit with less potency, up to and including 15 min after addition. Inhibition of p38 MAPK and ERK1/ERK2 prevented endostatin's ability to upregulate levels of occludin expression. Inhibition of these key signaling molecules was shown to prevent endostatin's ability to protect against VEGF165-mediated increases in paracellular permeability in vitro. However, it appears that p38 MAPK may play a more important role in VEGF-mediated permeability, as inhibition of ERK1/ERK2 will not prevent VEGF165-mediated permeability compared with control (untreated) cells or cells treated with both a p38 MAPK inhibitor and VEGF165. CONCLUSIONS: Occludin is important for the maintenance of tight junction integrity in vivo. In a p38 MAPK and ERK1/ERK2 dependent manner, endostatin was shown to upregulate the levels of expression of the tight junction protein occludin. Inhibition of these key MAPK components may prevent endostatin's ability to decrease VEGF165-induced paracellular permeability.

Angiogenesis Inhibitors↗
PubMed1978

Ion transport across the isolated intestinal mucosa of the winter flounder, Pseudopleuronectes americanus. I. Functional and structural properties of cellular and paracellular pathways for Na and Cl.

The isolated intestinal mucosa of the flounder, Pseudopleuronectes americanus, when bathed in a 20 mM HCO3-Ringer's solution bubbled with 1% CO2 in O2, generated a serosa-negative PD and, when short-circuited, absorbed Cl at almost 3 times the rate of Na. Reducing HCO3 to 5 mM decreased the net Cl flux by more than 60%. The following results suggest that, despite the PD, Na and Cl transport processes are nonelectrically coupled: replacing all Na with choline abolished both the PD and net Cl flux; replacing all Cl with SO4 and mannitol abolished the PD and the net Na flux; and adding ouabain (to 0.5 mM) abolished the PD and the net Cl flux. Nearly all of the unidirectional serosa-to-mucosa Cl flux (JClsm) seemed to be paracellular since it varied with PD and Cl concentration in a manner consistent with simple diffusion. JClsm was only about one-fourth of JNasm, suggesting that the paracellular pathway is highly cation-selective. The data can be explained by the following model: (i) Na and Cl uptake across the brush border are coupled 1 : 1; Na is pumped into the lateral space and Cl follows passively, elevating the salt concentration there; (ii) the tight junction is permeable to Na but relatively impermeable to Cl; and (iii) resistance to Na diffusion is greater in the lateral space (considered in its entirety) than in the tight junction. If these assumptions are correct, the serosa-negative transmural PD is due mainly to a salt diffusion potential across the tight junction and, under short-circuit condition, most of the Na pumped into the lateral space diffuses back into the luminal solution, whereas most of the Cl enters the serosal solution. Morphological features of the epithelium support this interpretation: the cells are unusually long (60 micrometer); there is little distension of the apical 12 micrometer of the lateral space during active fluid absorption; and distension distal to this region is intermittently constricted by desmosomes.

Animals↗
PubMed1996

Epithelial barrier integrity during in vitro wound repair of the airway epithelium.

The surface epithelium of the airway mucosa forms a continuous barrier to a wide number of noxious substances present in the lumen. The restoration of the barrier integrity after injury represents a key issue in the defense capacity of the airway epithelium. Using an in vitro wound repair model of the airway epithelium, we investigated the dynamic of the restoration of the epithelial barrier integrity during the wound repair process. Airway epithelial cells in culture were chemically wounded by sodium hydroxide. The immunolocalization of zonula occludens 1 (ZO-1), a cytoplasmic protein associated with the tight junctions, was examined during the wound repair process. Junctional integrity was examined by analyzing the transepithelial resistance (TER) and the permeability to [3H]mannitol and by visualizing the permeability to lanthanum nitrate during 5 days after injury. Immediately after injury, we simultaneously observed a 36.7% decrease in the TER and a 74.9% rise in the permeability to [3H]mannitol. In addition, lanthanum nitrate penetrated in the intercellular spaces in the repairing areas, which was also characterized by the absence of ZO-1 staining, as opposed to nonrepairing cells. TER and [3H]mannitol flux values as well as lanthanum nitrate and ZO-1 localizations were found to be similar to those observed in confluent cultures only 1 to 2 days after complete wound closure. This study demonstrates that using our culture model, confluent airway epithelial cells form a continuous and efficient barrier with tight junctions. Epithelial integrity is affected immediately after injury and is completely restored within 1 to 2 days after wound closure. During such a period of time, the airway epithelium may remain exposed to the noxious effect of environment in vivo, which can prevent the epithelial barrier restoration by modifying tight junction formation.

Cell Membrane Permeability↗
PubMed1999

Ocular cell monolayers cultured on biodegradable substrates.

The aim of this study was to culture retinal pigment epithelial (RPE) and corneal endothelial cells on biodegradable substrates for future use in monolayer transplantation in the eye. The biodegradable polymers, poly-l-lactic (PLLA) and poly-dl-lactic-co-glycolic acid (85:15) (PLGA) (both of molecular weight 105 kd) were the biomaterials used. All materials were seeded with either pig/human retinal pigment epithelial cells or rabbit corneal endothelial cells and were maintained in tissue culture conditions. Upon confluency, the cell density was calculated and cell viability determined. All monolayers were stained with phalloidin-rhodamine for F-actin and antibodies to the tight junction (zonula occludens) protein, ZO1, to demonstrate the presence of tight junctions. The final cell density of human RPE monolayers on PLLA films was 2950 cells/mm(2) (+/-185). The final cell density of pig RPE on PLLA and PLGA film was 2350 cells/mm(2) (+/-152 and 178, respectively). Rabbit corneal endothelial cells had a final cell density of 2650 cells/mm(2) (+/-164). F-actin staining revealed a circumferential ring of actin filaments in all of the cells grown on substrates. ZO(1) immunohistochemistry demonstrated staining along the lateral cell borders of all cell types. The successful culture of retinal pigment epithelial and corneal endothelial monolayers on these substrates may have potential for transplanting cell monolayers in the eye to improve vision.

Actins↗
PubMed1985

Characterization of a 36,000-dalton protein from the surface of Madin-Darby canine kidney cells involved in cell attachment and spreading.

We have identified and immunochemically characterized a 36,000-dalton membrane glycoprotein from Madin-Darby canine kidney cells. This protein is surface-labeled by lactoperoxidase-mediated iodination and metabolically labeled by [35S]methionine. It binds to Concanavalin A and incorporates 2-D-3H-mannose residues, thus indicating it is a glycoprotein. Rabbit polyclonal antibodies against this protein evenly decorate the external surface of trypsinized, unpolarized cells. The external apical surface of confluent monolayers, grown under culture conditions in which the tight junctions are closed and the cells have acquired polarity, is also evenly stained. The basolateral aspects of the external surface are stained only when the tight junctions are opened by removal of Ca++ or when the antibody has access to the monolayer from the basal side, which indicates an even distribution of this antigen on the surface of polarized cells. The antibody has no inhibitory effect on the opening and resealing of tight junctions in dense cultures, but does inhibit the attachment and spreading of cells on a substrate, which then blocks the establishment of a confluent functional monolayer.

Animals↗
PubMed2006

Functional interaction between the ZO-1-interacting transcription factor ZONAB/DbpA and the RNA processing factor symplekin.

Epithelial tight junctions participate in the regulation of gene expression by controlling the activity of transcription factors that can interact with junctional components. One such protein is the Y-box transcription factor ZONAB/DbpA that binds to ZO-1, a component of the junctional plaque. Symplekin, another nuclear protein that can associate with tight junctions, functions in the regulation of polyadenylation and thereby promotes gene expression. Here, we addressed the question of whether these two proteins interact and whether this is of functional relevance. We demonstrate that ZONAB/DbpA and symplekin form a complex in kidney and intestinal epithelial cells that can be immunoprecipitated and that exists in the nucleus. The interaction between ZONAB/DbpA and symplekin can be reconstituted with recombinant proteins. In reporter gene assays in which ZONAB/DbpA functions as a repressor, symplekin functionally interacts with ZONAB/DbpA, indicating that symplekin can also promote transcriptional repression. RNAi experiments indicate that symplekin depletion reduces the nuclear accumulation and the transcriptional activity of ZONAB/DbpA in colon adenocarcinoma cells, resulting in inhibition of proliferation and reduced expression of the ZONAB/DbpA-target gene cyclin D1. Our data thus indicate that symplekin and ZONAB/DbpA cooperate in the regulation of transcription, and that they promote epithelial proliferation and cyclin D1 expression.

Animals↗
PubMed1984

A freeze-fracture study of ruffle-ended post-secretory ameloblasts.

The post-secretory portion of the rat incisor enamel organ was prepared for routine transmission electron microscopy and freeze-fracture replication in order to define further the structural surface features of the ruffle-ended ameloblasts. Surface views of the distal plasma membrane of the ruffle-ended ameloblasts revealed a well-developed zonula occludens junction with from six to ten rows of tight junctional strands. Gap junctions were also observed just proximal to the tight junctional strands. The membranes of the ruffled border contained a rich supply of intramembrane particles (IMP). The IMPs were approximately 7 to 8 nm in diameter and preferentially located on the P-face profiles of the membrane. The density of IMPs on the membranes of the ruffled border was higher than that on the lateral borders of the cell. It is suggested that the IMPs of the ruffled border may represent enzymatic proteins in the basal cell membrane of absorptive ameloblasts. In addition, the large, highly-developed zonula occludens appeared structurally capable of sealing the intercellular spaces between the ruffle-ended ameloblasts.

Ameloblasts↗
PubMed1995

Water pathways across a reconstituted epithelial barrier formed by Caco-2 cells: effects of medium hypertonicity.

Caco-2 cells, originated in a human colonic cancer, are currently used as model systems to study transepithelial transports. To further characterize their water permeability properties, clone P1 Caco-2 cells were cultured on permeable supports. At confluence, the transepithelial net water movement (Jw), mannitol permeability (Ps), and electrical resistance (R) were simultaneously measured. The observed results were correlated with transmission and freeze-fracture electron microscopy studies and compared with those obtained, in similar experimental conditions, in a typical mammalian epithelial barrier: the rabbit rectum. When the serosal solution was made hypertonic (50 mM polyethylene glycol-PEG), the spontaneously observed secretory Jw rapidly reversed, became absorptive and then stabilized. Simultaneously, the R values dropped and Ps went up. In the case of the rabbit rectal epithelium, a similar treatment did not elicit significant changes in the water permeability during the first 20 min following the osmotic challenge while there was a significant increase in the transepithelial resistance. After exposure to serosal hypertonicity, several morphological modifications developed in the Caco-2 cells: Localized dilations in the intercellular spaces and vacuoles in the cytoplasm appeared. Nevertheless, most cells remained in contact and no evidence of cell shrinking was observed. Simultaneously, the tight-junction structure was more or less disorganized. The filament network lost its sharpness and "omega" figures appeared, bordering the intercellular spaces. In some cases the tight-junction network was completely disrupted. In the case of the rabbit rectum the structural modifications were completely different: Serosal hypertonicity rapidly induced cell shrinking and the opening of the intercellular spaces, with no noticeable change in the tight-junction structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Electrical and freeze-fracture analysis of the effects of ionic cadmium on cell membranes of human proximal tubule cells.

We previously reported that cell cultures of human proximal tubule (HPT) cells respond to ionic cadmium in a manner consistent with well-defined Cd(2+)-elicited responses reported for in vivo systems. However, one unique finding was that the transepithelial electrical resistance and tight junction sealing strands were altered as a result of Cd2+ exposure at micromolar concentrations. These alterations are reexamined in detail in the present report to determine whether the Cd(2+)-induced alterations are specific alterations in the tight junction structure or reflect a general alteration in the cell membrane. Exhaustive analysis of tight junction sealing strands demonstrated no significant alterations due to Cd2+ exposure, even at the concentration that elicited a significant reduction in transepithelial resistance. Further analysis of intramembrane particle distribution demonstrated a significant increase in apical intramembrane particles, indicating that Cd2+ exposure altered the characteristics of the apical cell membrane. Overall, the results were consistent with evidence of Cd(2+)-induced alteration in the apical cell membrane of the HPT cell.

Cadmium↗
PubMed2005

Effects of lysophosphatidylcholine on monolayer cell permeability of human coronary artery endothelial cells.

BACKGROUND: Lysophosphatidylcholine (LysoPC) is a product of phosphatidylcholine hydrolysis by phospholipase A2, which is associated with atherosclerosis. However, the underlying molecular mechanisms are still unclear. The purpose of this study was to determine the effects of LysoPC on monolayer permeability of human coronary artery endothelial cells (HCAECs). METHODS: HCAECs were cultured with LysoPC in a dose- and time-dependent manner. Monolayer permeability was studied by using a transwell system with a Texas-Red-labeled dextran tracer. The messenger RNA and protein levels of endothelial tight junction proteins were determined with the use of real-time reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. Superoxide anion levels were determined with the use of fluorescent dye dihydroethidium-based flow cytometry analysis. Activation of mitogen-activated protein kinases was determined by performing Bio-Plex immunoassay. RESULTS: LysoPC (30 micromol/L) increased monolayer permeability by 53% and decreased the messenger RNA levels of zonula occludens-1, occludin, claudin-1, and junctional adhesion molecule by 44%, 53%, 50%, and 52%, respectively, compared with controls (P < .05). Western blot analysis showed reduced protein levels of these tight junction molecules. LysoPC (15 and 30 micromol/L) also increased superoxide anion production by 54% and 58%, respectively, compared with controls (P < .05). Antioxidant seleno-L-methionine (20 and 30 micromol/L) inhibited LysoPC (30 micromol/L)-induced permeability by 42% and 68%, respectively (P < .05). Furthermore, LysoPC (30 micromol/L) activated c-Jun N-terminal kinase and p38 phosphorylation, but not extracellular signal-related kinase 1/2, within 5 to 10 minutes. CONCLUSIONS: LysoPC increases monolayer permeability and reduces the expression of tight junction molecules in HCAECs through oxidative stress and activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. The antioxidant can effectively block LysoPC-induced endothelial permeability.

Cadherins↗
PubMed2001

Protein kinase C regulates the phosphorylation and cellular localization of occludin.

Occludin is an integral membrane phosphoprotein specifically associated with tight junctions, contributing to the structure and function of this intercellular seal. Occludin function is thought to be regulated by phosphorylation, but no information is available on the molecular pathways involved. In the present study, the involvement of the protein kinase C pathway in the regulation of the phosphorylation and cellular distribution of occludin has been investigated. Phorbol 12-myristate 13-acetate and 1,2-dioctanoylglycerol induced the rapid phosphorylation of occludin in Madin-Darby canine kidney cells cultured in low extracellular calcium medium with a concomitant translocation of occludin to the regions of cell-cell contact. The extent of occludin phosphorylation as well as its incorporation into tight junctions induced by protein kinase C activators or calcium switch were markedly decreased by the protein kinase C inhibitor GF-109203X. In addition, in vitro experiments showed that the recombinant COOH-terminal domain of murine occludin could be phosphorylated by purified protein kinase C. Ser(338) of occludin was identified as an in vitro protein kinase C phosphorylation site using peptide mass fingerprint analysis and electrospray ionization tandem mass spectroscopy. These findings indicate that protein kinase C is involved in the regulation of occludin function at tight junctions.

Alkaline Phosphatase↗
PubMed2006

[Effects of sense vascular endothelial growth factor cDNA transfection on mononuclear chemotaxis protein-1 expression in rat pulmonary microvessel endothelial cells].

OBJECTIVE: To study the transformation characteristics of tight junction of microvessel endothelial cells in bleomycin (BLM) induced pulmonary fibrosis (PF), and the effects of vascular endothelial growth factor (VEGF) on mononuclear chemotaxis protein-1 (MCP-1) mRNA expression in pulmonary microvessel endothelial cells (EC). METHODS: Forty healthy rats were equally divided into a control and an experimental group in random. In the experimental group, PF were induced by BLM application. In each group, 10 rats were instilled by lanthanum sal at 3, 7, 14 and 28 d after BLM application, and lung samples were made and observed by transmission electron microscopy. The pulmonary microvessel EC of the other 10 rats in each group were preserved by tissue culture methods at the same time, and the cells were transfected with sense VEGF cDNA, and then MCP-1 mRNA expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) technique. RESULTS: Blood vessel endothelial cells of the control group were intact. The basement membrane was shown as a continuous line. Granules of lanthanum sal did not cross the tight junction of endothelial cells. The width of the junction gap in rats of the experimental group treated at different times was increased and lanthanum particles of high density were seen in the gap junction, particularly on day 3, and were distributed in the area of subendothelium. The MCP-1 mRNA expression in VEGF transfected microvessel EC was significantly higher than that of the control group (1.21 +/- 0.22 vs 0.36 +/- 0.06, P < 0.05). CONCLUSION: In BLM induced PF, the opening of the tight junction of EC, and the high expression of MCP-1 induce inflammatory cell infiltration and cytokine over-secretion, which in turn enhances proliferation of fibroblasts, one of the important factors underlying lung fibrosis.

Animals↗
PubMed1982

Cellular interconnections in the young mouse ovary. Freeze-fracture study.

Intercellular junctions in the young mouse ovary were examined by electron microscopy utilizing freeze-fracture and thin-sectioning techniques. Projections from the granulosa cells adjacent to the oocyte (GI) traverse the zona pellucida and form small gap junctions on the oocyte surface. On the P-face of these cells, the junctional aggregations are occasionally associated with linear strands of particles. In contrast, large gap junctional areas are frequently observed between the more peripherally located granulosa cells (GE) and are also present in the theca interna (TI) cell layer surrounding the follicles. Three types of tight junctional strands are discernible on the P-face of theca externa cells (TE): angularly zigzag strands consisting of intermittently distributed intramembranous particles on wide ridges, intermediate zigzag strands consisting of more continuously distributed particles, and wavy strands consisting of rather fused particles. Tight junctional strands are also present in the middle of grooves on the E-face of endothelial cells of blood vessels. In the germinal epithelial cell layer, tight junctional strands appear to be discrete and form a less anastomosing network.

Animals↗
PubMed2006

Histamine-induced phosphorylation of the regulatory light chain of myosin II disrupts the barrier integrity of corneal endothelial cells.

PURPOSE: To investigate histamine-induced changes in the phosphorylation of myosin light chain (MLC) and its influence on the barrier integrity of corneal endothelial cells through altered contractility of the actin cytoskeleton. METHODS: Experiments were performed in cultured bovine corneal endothelial cells (BCECs). Phosphorylation of MLC, which increases contractility of the actin cytoskeleton through actomyosin interaction, was assessed by urea-glycerol gel electrophoresis and Western blot analysis. Immunocytochemistry was used to locate phosphorylated MLC in relation to tight junctions. Phosphorylation of the 17-kDa PKC-potentiated inhibitory protein of type 1 protein phosphatase (CPI-17), which inhibits MLC phosphatase, was studied using Western blot analysis. The cortical actin cytoskeleton was visualized by staining with Texas-red phalloidin. Barrier integrity was determined by quantifying horseradish peroxidase (HRP; 44 kDa) flux across cells grown on porous filters. RESULTS: RT-PCR and Western blot analysis confirmed the expression of Galphaq/11-coupled H1 receptors in BCECs. Exposure to histamine (100 microM; 10 minutes) led to phosphorylation of MLC (134% relative to untreated cells) and of CPI-17. Histamine also increased the flux of HRP by sevenfold and disrupted the assembly of the dense cortical actin found in resting cells. PKC activation by phorbol 12-myristate 13-acetate (PMA; 100 nM; 30 minutes) caused phosphorylation of both MLC and CPI-17. The histamine-induced MLC phosphorylation was reduced by pre-exposure to either ML-7 (50 microM), an MLCK (MLC kinase) inhibitor, or chelerythrine (10 microM), an inhibitor of PKC. Cotreatment with agents that elevate cAMP in BCECs prevented the histamine-induced MLC phosphorylation and the disruption of the actin cytoskeleton, and increased HRP flux. Phosphorylated MLC in response to histamine or PMA was found in a punctate form in close proximity to ZO-1, a marker of the tight junctional complex. CONCLUSIONS: Histamine induces MLC phosphorylation by activating MLCK and partly inhibiting MLC phosphatase. The latter is facilitated by the phosphorylation of CPI-17. Localization of phosphorylated MLC in proximity to ZO-1 suggests increased contractility of the cortical actin at the tight junctional complex. This contractility oppose the tethering forces and lead to a breakdown of the barrier integrity. Last, elevated cAMP prevents histamine-induced loss of the barrier integrity, not only by blocking inactivation of MLC phosphatase but also by inactivating MLCK.

Actins↗
PubMed2005

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.

Animals↗