Differential roles for actin polymerization and a myosin II motor in assembly of the epithelial apical junctional complex

Differentiation and polarization of epithelial cells depends on the formation of the apical junctional complex (AJC), which is composed of the tight junction (TJ) and the adherens junction (AJ). In this study, we investigated mechanisms of actin reorganization that drive the establishment of AJC. Using a calcium switch model, we observed that formation of the AJC in T84 intestinal epithelial cells began with the assembly of adherens-like junctions followed by the formation of TJs. Early adherens-like junctions and TJs readily incorporated exogenous G-actin and were disassembled by latrunculin B, thus indicating dependence on continuous actin polymerization. Both adherens-like junctions and TJs were enriched in actin-related protein 3 and neuronal Wiskott-Aldrich syndrome protein (N-WASP), and their assembly was prevented by the N-WASP inhibitor wiskostatin. In contrast, the formation of TJs, but not adherens-like junctions, was accompanied by recruitment of myosin II and was blocked by inhibition of myosin II with blebbistatin. In addition, blebbistatin inhibited the ability of epithelial cells to establish a columnar phenotype with proper apico-basal polarity. These findings suggest that actin polymerization directly mediates recruitment and maintenance of AJ/TJ proteins at intercellular contacts, whereas myosin II regulates cell polarization and correct positioning of the AJC within the plasma membrane.     

Differential regulation of three glucose transporter genes in a renal epithelial cell line

Three hexose transporter genes, the Na(+)/glucose cotransporters SGLT1 and SGLT3 (formerly SAAT1/pSGLT2) and the facilitative transporter GLUT1, are expressed in a renal epithelial cell line with proximal tubule characteristics. A number of studies have demonstrated that SGLT1 expression is coupled to the cellular differentiation state and is also negatively regulated by its substrate glucose. In the present study, we demonstrate that SGLT3 mRNA expression is relatively unaffected by conditions promoting dedifferentiation (reseeding to a subconfluent density, activation of protein kinase C) or differentiation (confluent cell density, activation of protein kinase A) nor was expression sensitive to hyperglycemic glucose levels in the medium. We further demonstrate that protein kinase A and protein kinase C exert opposing effects on GLUT1 and SGLT1 mRNA levels in polarized cell monolayers, indicating that GLUT1 mRNA is also highly regulated in polarized epithelial cells by agents affecting cell differentiation. The relatively constitutive expression of SGLT3 mRNA suggests a novel role for this low-affinity Na(+)/glucose cotransporter, to provide concentrative glucose uptake under hyperglycemic conditions where expression of high-affinity glucose cotransporter SGLT1 mRNA is significantly downregulated.     

Absence of gap junctional complexes in two established renal epithelial cell lines (LLC-PK1 and MDCK)

The type of junctions present in the membranes of the two renal epithelial cell lines, LLC-PK1 and MDCK, and of subcultured porcine aortic endothelial (PAE) cells have been studied by freeze-fracture. No gap junctions were observed in the two renal cell lines, while they were numerous in the endothelial cells. Tight junctions were abundant in LLC-PK1 and MDCK cells and varied in numbers of ridges from one to ten. ONly a few simple tight junctions unconnected with gap junctions were observed in PAE cells. The occurrence of gap junctions in these cells correlates with their ability to form intercellular communicating channels.     

Endocytosis of the apical junctional complex: mechanisms and possible roles in regulation of epithelial barriers

Tight junctions (TJ) and adherens junctions (AJ) regulate cell-cell adhesion and barrier function of simple polarized epithelia. These junctions are positioned in the apical end of the lateral plasma membrane and form the so-called apical junctional complex (AJC). Although initially seen as purely structural features, the AJC is now known to play important roles in cell differentiation and proliferation. The AJC is a highly dynamic entity, undergoing rapid remodeling during normal epithelial morphogenesis and under pathologic conditions. There is growing evidence that remodeling of the AJC is mediated by internalization of junctional proteins. This review summarizes what is known about endocytic pathways, intracellular destinations and signaling cascades involved in internalization of AJC proteins. Potential biological roles for AJC endocytosis in maintaining functional apical junctions, reversible opening of epithelial barrier and disruption of intercellular adhesion are also discussed.     

Trichomonas vaginalis perturbs the junctional complex in epithelial cells

Trichomonas vaginalis, a protist parasite of the urogenital tract in humans, is the causative agent of trichomonosis, which in recent years have been associated with the cervical cancer development. In the present study we analyzed the modifications at the junctional complex level of Caco-2 cells after interaction with two isolates of T. vaginalis and the influence of the iron concentration present in the parasite＇ s culture medium on the interaction effects. Our results show that T. vaginalis adheres to the epithelial cell causing alterations in the junctional complex, such as： （a） a decrease in transepithelial electrical resistance; （b） alteration in the pattern of junctional complex proteins distribution as observed for E-cadherin, occludin and ZO-1; and （c） enlargement of the spaces between epithelial cells. These effects were dependent on （a） the degree of the parasite virulence isolate, （b） the iron concentration in the culture medium, and （c） the expression of adhesin proteins on the parasite surface.     

Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines

Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.     

alpha-Actinin localization in the junctional complex of intestinal epithelial cells

We have used antibody to chicken gizzard alpha-actinin to identify and localize this molecule in chicken intestinal epithelium. The antibody binds only to alpha-actinin when tested against a crude extract of chicken gizzard. Extracts of purified epithelial cells contain a molecule which has a subunit molecular weight of 100,000 on sodium dodecyl sulphate gels and which is able to inhibit the interaction of alpha-actinin antibody and 125I-labeled chicken gizzard alpha-actinin. By indirect immunofluorescence, alpha-actinin is localized in the apical portion of chicken intestinal epithelial cells. Ethanol-fixed cryostat sections of intestine taken through the apical portion of the epithelial cells and in a plane perpendicular to the long axis of the cells show that alpha-actinin is organized in a polygonal pattern which corresponds to the outlines of the polygonally packed epithelial cells. We interpret the data as indicating that alpha-actinin is a component of the tight junction (zonula occludens) and/or the belt desmosome (zonula adherens), both of which are membrane structures known to encircle the cell and to be confined to its apical portion.     

Differential regulation of gamma-glutamyltransferase mRNAs in four human tumour cell lines.

Human gamma-glutamyltransferase (GGT) belongs to a multigenic family and at least three mRNAs are transcribed from the gene that codes for an active enzyme. Four human tumour cell lines (HepG2, LNCap, HeLa and U937) with different GGT levels were used to investigate how GGT activity, total GGT mRNA and each individual GGT mRNA subtype responded to tumour necrosis factor-alpha (TNF-alpha), 12-O-tetradecanoylphorbol 13-acetate (TPA) or sodium butyrate treatment. Butyrate reduced the GGT activity in HepG2 cells, and the level of total GGT mRNA accordingly, whereas TNF-alpha and TPA did not alter these parameters. In LNCap cells, TNF-alpha, TPA, and butyrate reduced the activity as well as the level of GGT total mRNA. In HeLa cells no significant changes were observed either in activity or in mRNA level whereas TPA induced both GGT activity and mRNA levels in U937 cells. The distribution of each GGT mRNA subtype (A, B and C) was found to be cell specific: type B mRNA was the major form in HepG2 cells, while type A was the major form in LNCap and HeLa, type A and type C were expressed almost at the same level in U937 cells. The GGT mRNA subtypes were also differently modulated in these cells after TNF-alpha, TPA or butyrate treatment, suggesting that they are regulated by distinct and cell type specific mechanisms.     

Biochemical properties of endothelin converting enzyme in renal epithelial cell lines.

This is the first report clearly demonstrating the presence of endothelin (ET) converting enzyme (ECE) in non-vascular cells (renal epithelial cell lines, MDCK and LLC-PK1). ECEs derived from these epithelial cells were very similar to the endothelial ECE in the following biochemical properties: 1) The optimum pH was 7.0; 2) the Km value for big ET-1 was approximately 30 microM; 3) the enzyme was potently inhibited by EDTA, o-phenanthroline and phosphoramidon; and 4) the enzyme did not convert big ET-2 or big ET-3. These data suggest that phosphoramidon-sensitive ECE is involved in the processing of big ET-1 to ET-1 in the renal tubule.     

Secretion of endothelin and related peptides from renal epithelial cell lines

Using specific radioimmunoassays (RIAs) for endothelin (ET) and big ET, we have studied whether ET and related peptides are secreted from renal epithelial cell lines (LLCPK1 and MDCK) of non-endothelial origin. Dilution curves of extracts of conditioned media from both LLCPK1 and MDCK cell lines were parallel to those of standard porcine (p) ET and big pET in each RIA. Both cell lines incubated in serum-free medium secreted ET- and C-terminal fragment (CTF)-like immunoreactivity (LI) of big ET as a function of time. Reverse-phase HPLC coupled with both RIAs of the extracted media from both cell lines revealed a single component with ET-LI coeluting with pET(1-21) and several components with CTF-LI, one corresponding to the elution position of big pET(1-39), one to its CTF(22-39), and the others eluting earlier than CTF. These data indicate that endothelin and related peptides are synthesized by and secreted from cells other than endothelial cells.     

Differential glycosylation of MUC1 in tumors and transfected epithelial and lymphoblastoid cell lines

The membrane-bound mucin-like protein MUC1 with a specified number of tandem repeats has been expressed by transfection of the cDNAs in both the epithelial cell lines MDCK and LLC-PK1, and human lymphoblastoid cell lines T2 and C1R. The structure and glycosylation states of the MUC1 in these four lines were compared with that of the endogenous MUC1 found in the human pancreatic (HPAF) and breast (BT-20) tumor cell lines using flow cytometry and Western blot analysis with anti-MUC1 antibodies, which are either sensitive or insensitive to the glycosylation state of the tandem repeat, and pretreatment of cells with phenyl--galactosaminide, an inhibitor of mucin sialylation. A similar analysis of MUC1 expression in transfected normal and O-glycosylation defective CHO cells reveals that the addition of galactose to the core oligosaccharide structure is apparently responsible for the anomalous difference in Mr between the mature and propeptide forms of the MUC1. Both the tumor cells and the transfected lymphoblastoid cells consistently express significant steady state levels of both the heavily glycosylated mature forms and the poorly glycosylated propeptide forms of the MUC1, whereas MUC1 is found predominantly as the mature extensively glycosylated species in the transfected epithelial cells. Immunofluoresence microscopy of cross sections of the polarized epithelial cells grown on culture filter inserts reveals that the MUC1 is clearly present at the apical surface of the cells, consistent with its expression in normal tissues. Thus, the successful expression of the MUC1 by transfection of either lymphoblastoid cells or epithelial cells yields model systems both for studying the natural structure/function relationships of the protein domains within the MUC1 molecule and for further elucidating the previously reported MHC-independent T-cell recognition of the MUC1.     

Numb modulates the paracellular permeability of intestinal epithelial cells through regulating apical junctional complex assembly and myosin light chain phosphorylation

Numb is highly expressed throughout the crypt-villus axis of intestinal mucosa and functions as cell fate determinant and integrator of cell-to-cell adhesion. Increased paracellular permeability of intestinal epithelial cells is associated with the epithelial barrier dysfunction of inflammatory bowel diseases (IBDs). The apical junctional complex (AJC) assembly and myosin light chain (MLC) phosphorylation regulate adherens junctions (AJ) and tight junctions (TJ). We determined whether and how Numb modulate the paracellular permeability of intestinal epithelial cells. Caco-2 intestinal epithelial cells and their Numb-interfered counterparts were used in the study for physiological, morphological and biological analyses. Numb, expressed in intestinal epithelial cells and located at the plasma membrane of Caco-2 cells in a basolateral to apical distribution, increased in the intestinal epithelial cells with the formation of the intestinal epithelial barrier. Numb expression decreased and accumulated in the cytoplasm of intestinal epithelial cells in a DSS-induced colitis mouse model. Numb co-localized with E-cadherin, ZO-1 and Par3 at the plasma membrane and interacted with E-cadherin and Par3. Knockdown of Numb in Caco-2 cells altered the F-actin structure during the Ca²⁺ switch assay, enhanced TNFα-/INF-γ-induced intestinal epithelial barrier dysfunction and TJ destruction, and increased the Claudin-2 protein level. Immunofluorescence experiments revealed that NMIIA and F-actin co-localized at the cell surface of Caco-2 cells. Numb knockdown in Caco-2 cells increased F-actin contraction and the abundance of phosphorylated MLC. Numb modulated the intestinal epithelial barrier in a Notch signaling-independent manner. These findings suggest that Numb modulates the paracellular permeability by affecting AJC assembly and MLC phosphorylation.     

The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex

The role of the epithelial adhesion molecule uvomorulin in the formation of the epithelial junctional complex in the Madin-Darby canine kidney (MDCK) cell line was investigated. Experiments were carried out to determine whether specific inhibition of uvomorulin function would interfere selectively with the formation, stability, or function of the apical zonula adherens (ZA) and zonula occludens (ZO), or whether it would interfere with all forms of intercellular contact including the desmosomes. The effects of blocking antibodies and Fab fragments to uvomorulin on the formation of the junctional complex was examined with a Ca2+ switch assay for de novo junction assembly. The formation of the ZO, the ZA, and the desmosomes was assayed by fluorescence staining with an antibody to the tight junction-specific protein ZO-1, with rhodamine-phalloidin for ZA-associated actin filaments, and with an anti-desmoplakin antibody, respectively. Under different conditions and times of antibody treatment the extent of inhibition of the formation of each of the junctional elements was very similar. The ability of the cells to eventually overcome the inhibitory effect of the antibodies and form junctions correlated with the reappearance of uvomorulin at the regions of cell-cell contact. Therefore uvomorulin seems to mediate an early adhesion event between epithelial cells that is a prerequisite for the assembly of all elements of the junctional complex. In contrast, the transepithelial electrical resistance of confluent, well-established monolayers of MDCK cells grown on filters was not greatly affected by treatment with the various antibodies or Fab fragments. A small transient decrease in resistance observed with the polyclonal alpha-uvomorulin IgG may be due to a more subtle modulation of the junctional complex.     

Establishment of epithelial and fibroblast cell cultures and cell lines from primary renal cancer nephrectomies

Renal cell carcinoma (RCC) is one of the most lethal urogenital cancers and effective treatment of metastatic RCC remains an elusive target. Cell lines enable the in vitro investigation of molecular and genetic changes leading to renal carcinogenesis and are important for evaluating cellular drug response or toxicity. This study details a fast and easy protocol of establishing epithelial and fibroblast cell cultures or cell lines concurrently from renal cancer nephrectomy tissue. The protocol involves mechanical disaggregation, collagenase digestion and cell sieving for establishing epithelial cells while fibroblast cells were grown from explants. This protocol has been modified from previous published reports with additional antibiotics and washing steps added to eliminate microbial contamination from the surgical source. Cell characterisation was carried out using immunofluorescence and quantitative polymerase chain reaction. Eleven stable epithelial renal tumour cell lines of various subtypes, including rare subtypes, were established with a spontaneous immortalisation rate of 21.6% using this protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalisation. Cells of epithelial origin expressed higher expressions of epithelial markers such as pan‐cytokeratin, cytokeratin 8 and E‐cadherin whereas fibroblast cells expressed high α‐smooth muscle actin. Further mutational analysis is needed to evaluate the genetic or molecular characteristics of the cell lines.     

Differential regulation of anaphase promoting complex/cyclosome substrates by the spindle assembly checkpoint in Saccharomyces cerevisiae

The anaphase promoting complex (APC) targets proteins for degradation to promote progression through the cell cycle. Here we show that Clb5, an APCCdc20 substrate, is degraded when the spindle checkpoint is active, while other APCCdc20 substrates are stabilized, suggesting that APCCdc20 inhibition by the spindle checkpoint is substrate specific.     

Ca(2+) regulation of gap junctional coupling in lens epithelial cells

The quantitative effects of Ca²⁺signaling on gap junctional coupling in lens epithelial cells have beendetermined using either the spread of Mn²⁺ that is imagedby its ability to quench the fluorescence of fura 2 or the spread ofthe fluorescent dye Alexa Fluor 594. Gap junctional coupling wasunaffected by a mechanically stimulated cell-to-cell Ca²⁺wave. Furthermore, when cytosolic Ca²⁺ concentration(Ca) increased after the addition of the agonistATP, coupling was unaffected during the period thatCa was maximal. However, coupling decreasedtransiently ~5-10 min after agonist addition whenCa returned to resting levels, indicating that thistransient decrease in coupling was unlikely due to a direct action ofCa on gap junctions. An increase inCa mediated by the ionophore ionomycin that wassustained for several minutes resulted in a more rapid and sustaineddecrease in coupling (IC₅₀ ~300 nM Ca²⁺, Hillcoefficient of 4), indicating that an increase in Ca alone could regulate gap junctions. Thus Ca increases that occurred during agonist stimulation and cell-to-cell Ca²⁺ waves were too transient to mediate a sustaineduncoupling of lens epithelial cells.