Coronavirus M Protein Trafficking in Epithelial Cells Utilizes a Myosin Vb Splice Variant and Rab10.

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). Co-expressed M proteins and MYO5B+D co-localized with endogenous Rab10 and Rab11a. We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays. One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly and its interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV-CoV, MERS-CoV and SARS-CoV-2 M proteins also blocked colocalization with MYO5B+D. The knockdown of Rab10 blocked the co-localization of M proteins with MYO5B+D and was rescued by re-expression of CFP-Rab10. Our results suggest that CoV M proteins traffic through Rab10-containing systems, in association with MYO5B+D.

YAP drives cell competition by activating choline metabolism.

Cell competition is a phenomenon that eliminates unfit cells from cell society, a function vital for maintaining cellular and organismal homeostasis. We previously showed that Madin-Darby canine kidney (MDCK) epithelial cells expressing the active form of the transcriptional coactivator Yes-associated protein (YAP) are apically extruded when surrounded by normal MDCK cells. Although we demonstrated that the arachidonic acid (AA) cascade is involved in YAP-dependent apical extrusion, the metabolic events leading to this outcome remained unclear. Here, we present the results of metabolomic analysis that identified phosphatidylcholine (PC) biosynthesis as the most significant player in this process. Removal of the PC biosynthetic components choline and methionine from culture medium inhibited YAP-dependent apical extrusion. Inhibition of either choline uptake or metabolic cycles involving choline or methionine also decreased YAP-dependent apical extrusion. At the molecular level, active YAP induced expression of the genes encoding glycerophosphocholine phosphodiesterase 1 (GPCPD1) and lecithin-cholesterol acyltransferase (LCAT), which are involved in choline metabolism. Our results indicate that YAP-dependent cell competition depends on YAP-mediated activation of the choline metabolic cycle.

Specific substrates composed of collagen and fibronectin support the formation of epithelial cell sheets by MDCK cells lacking α-catenin or classical cadherins.

The effect of the extracellular matrix substrates on the formation of epithelial cell sheets was studied using MDCK cells in which the α-catenin gene was disrupted. Although the mutant cells did not form an epithelial cell sheet in conventional cell culture, the cells formed an epithelial cell sheet when they were cultured on or in a collagen gel; the same results were not observed when cells were cultured on collagen-coated cover glasses or culture dishes. Moreover, the cells cultured on the cell culture inserts coated with fibronectin, Matrigel, or vitronectin formed epithelial cell sheets, whereas the cells cultured on cover glasses coated with these proteins did not form the structure, implying that the physical and chemical features of the substrates exert a profound effect on the formation of epithelial cell sheets. MDCK cells lacking the expression of E- and K-cadherins displayed similar properties. When the mutant MDCK cells were cultured in the presence of blebbistatin, they formed epithelial cell sheets, suggesting that myosin II was involved in the formation of these sheets. These cell sheets showed intimate cell-cell adhesion, and electron microscopy confirmed the formation of cell junctions. We propose that specific ECM substrates organize the formation of basic epithelial cell sheets, whereas classical cadherins stabilize cell-cell contacts and promote the formation of structures.

Comprehensive N-glycosylation analysis of the influenza A virus proteins HA and NA from adherent and suspension MDCK cells.

Glycosylation is considered as a critical quality attribute for the production of recombinant biopharmaceuticals such as hormones, blood clotting factors, or monoclonal antibodies. In contrast, glycan patterns of immunogenic viral proteins, which differ significantly between the various expression systems, are hardly analyzed yet. The influenza A virus (IAV) proteins hemagglutinin (HA) and neuraminidase (NA) have multiple N-glycosylation sites, and alteration of N-glycan micro- and macroheterogeneity can have strong effects on virulence and immunogenicity. Here, we present a versatile and powerful glycoanalytical workflow that enables a comprehensive N-glycosylation analysis of IAV glycoproteins. We challenged our workflow with IAV (A/PR/8/34 H1N1) propagated in two closely related Madin-Darby canine kidney (MDCK) cell lines, namely an adherent MDCK cell line and its corresponding suspension cell line. As expected, N-glycan patterns of HA and NA from virus particles produced in both MDCK cell lines were similar. Detailed analysis of the HA N-glycan microheterogeneity showed an increasing variability and a higher complexity for N-glycosylation sites located closer to the head region of the molecule. In contrast, NA was found to be exclusively N-glycosylated at site N73. Almost all N-glycan structures were fucosylated. Furthermore, HA and NA N-glycan structures were exclusively hybrid- and complex-type structures, to some extent terminated with alpha-linked galactose(s) but also with blood group H type 2 and blood group A epitopes. In contrast to the similarity of the overall glycan pattern, differences in the relative abundance of individual structures were identified. This concerned, in particular, oligomannose-type, alpha-linked galactose, and multiantennary complex-type N-glycans.

Cell-extracellular matrix interactions in the fluidic phase direct the topology and polarity of self-organized epithelial structures.

INTRODUCTION: In vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as scaffolds to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell-ECM interactions for the spatiotemporal coordination to different ECM at the tissue scale is not fully understood. METHODS: Here, using in vitro assay with engineered MDCK cells expressing H2B-mCherry (nucleus) and gp135/Podocalyxin-GFP (apical marker), we show in multi-dimensions that such coordination for epithelial morphogenesis can be determined by cell-soluble ECM interaction in the fluidic phase. RESULTS: The coordination depends on the native topology of ECM components such as sheet-like basement membrane (BM) and type I collagen (COL) fibres: scaffold formed by BM (COL) facilitates a close-ended (open-ended) coordination that leads to the formation of lobular (tubular) epithelium. Further, cells form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side, and time-lapse two-photon scanning imaging reveals the polarization occurring early and maintained through the lobular expansion. During polarization, gp135-GFP was converged to the apical surface collectively in the lobular/tubular structures, suggesting possible intercellular communications. Under suspension culture, the polarization was impaired with multi-lumen formation in the tubules, implying the importance of ECM biomechanical microenvironment. CONCLUSION: Our results suggest a biophysical mechanism for cells to form polarity and coordinate positioning at tissue scale, and in engineering epithelium through cell-soluble ECM interaction and self-assembly.

Influenza A Viruses Enter Host Cells via Extracellular Ca2+ Influx-Involved Clathrin-Mediated Endocytosis.

Influenza A virus (IAV) is internalized into its host cells by endocytosis, which involves many cellular proteins and molecules. In this study, we focus on the function of calcium ion (Ca2+) in IAV endocytosis. We have found that IAV infection is accompanied by the increase in concentration of cytosolic Ca2+, which is mainly attributed to the influx of extracellular Ca2+. When Ca2+ influx is abolished, IAV internalization will be markedly suppressed, but the virus attachment to its host cells will be unaffected. Extracellular Ca2+ influx is essential to the clathrin-mediated endocytosis (CME) of IAVs but dispensable to the clathrin-independent endocytosis of the virus and is dispensable to the CME of transferrin or low-density lipoprotein as a control. Ca2+ influx might participate in the dynamin-promoted membrane fission in the CME of IAVs. Our study highlights that IAVs enter host cells via extracellular Ca2+ influx-involved clathrin- and dynamin-dependent endocytosis, which will facilitate better understanding of IAV infection and development of anti-influenza drugs.

A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells.

Selective protein distribution on distinct plasma membranes is important for epithelial cell function. To date, how proteins are directed to specific epithelial cell surface is not fully understood. Here we report a conserved DSSDE motif in LDL-receptor (LDLR) modules of corin (a transmembrane serine protease) and CD320 (a receptor for vitamin B12 uptake), which regulates apical membrane targeting in renal epithelial cells. Altering this motif prevents specific apical corin and CD320 expression in polarized Madin-Darby canine kidney (MDCK) cells. Mechanistic studies indicate that this DSSDE motif participates in a Rab11a-dependent mechanism that specifies apical sorting. In MDCK cells, inhibition of Rab11a, but not Rab11b, expression leads to corin and CD320 expression on both apical and basolateral membranes. Together, our results reveal a novel molecular recognition mechanism that regulates LDLR module-containing proteins in their specific apical expression in polarized renal epithelial cells.

Epithelial layer unjamming shifts energy metabolism toward glycolysis.

In development of an embryo, healing of a wound, or progression of a carcinoma, a requisite event is collective epithelial cellular migration. For example, cells at the advancing front of a wound edge tend to migrate collectively, elongate substantially, and exert tractions more forcefully compared with cells many ranks behind. With regards to energy metabolism, striking spatial gradients have recently been reported in the wounded epithelium, as well as in the tumor, but within the wounded cell layer little is known about the link between mechanical events and underlying energy metabolism. Using the advancing confluent monolayer of MDCKII cells as a model system, here we report at single cell resolution the evolving spatiotemporal fields of cell migration speeds, cell shapes, and traction forces measured simultaneously with fields of multiple indices of cellular energy metabolism. Compared with the epithelial layer that is unwounded, which is non-migratory, solid-like and jammed, the leading edge of the advancing cell layer is shown to become progressively more migratory, fluid-like, and unjammed. In doing so the cytoplasmic redox ratio becomes progressively smaller, the NADH lifetime becomes progressively shorter, and the mitochondrial membrane potential and glucose uptake become progressively larger. These observations indicate that a metabolic shift toward glycolysis accompanies collective cellular migration but show, further, that this shift occurs throughout the cell layer, even in regions where associated changes in cell shapes, traction forces, and migration velocities have yet to penetrate. In characterizing the wound healing process these morphological, mechanical, and metabolic observations, taken on a cell-by-cell basis, comprise the most comprehensive set of biophysical data yet reported. Together, these data suggest the novel hypothesis that the unjammed phase evolved to accommodate fluid-like migratory dynamics during episodes of tissue wound healing, development, and plasticity, but is more energetically expensive compared with the jammed phase, which evolved to maintain a solid-like non-migratory state that is more energetically economical.

Effects of high-dose uric acid on cellular proteome, intracellular ATP, tissue repairing capability and calcium oxalate crystal-binding capability of renal tubular cells: Implications to hyperuricosuria-induced kidney stone disease.

Hyperuricosuria is associated with kidney stone disease, especially uric acid (UA) and calcium oxalate (CaOx) types. Nevertheless, detailed mechanisms of hyperuricosuria-induced kidney stone formation remained unclear. This study examined changes in cellular proteome and function of renal tubular cells after treatment with high-dose UA for 48-h. Quantitative proteomics using 2-DE followed by nanoLC-ESI-ETD MS/MS tandem mass spectrometry revealed significant changes in levels of 22 proteins in the UA-treated cells. These proteomic data could be confirmed by Western blotting. Functional assays revealed an increase in intracellular ATP level and enhancement of tissue repairing capability in the UA-treated cells. Interestingly, levels of HSP70 and HSP90 (the known receptors for CaOx crystals) were increased in apical membranes of the UA-treated cells. CaOx crystal-cell adhesion assay revealed significant increase in CaOx-binding capability of the UA-treated cells, whereas neutralization of the surface HSP70 and/or HSP90 using their specific monoclonal antibodies caused significant reduction in such binding capability. These findings highlighted changes in renal tubular cells in response to high-dose UA that may, at least in part, explain the pathogenic mechanisms of hyperuricosuria-induced mixed kidney stone disease.

Leader-cell-driven epithelial sheet fingering.

Collective cell migration is crucial in many biological processes such as wound healing, tissue morphogenesis, and tumor progression. The leading front of a collective migrating epithelial cell layer often destabilizes into multicellular finger-like protrusions, each of which is guided by a leader cell at the fingertip. Here, we develop a subcellular-element-based model of this fingering instability, which incorporates leader cells and other related properties of a monolayer of epithelial cells. Our model recovers multiple aspects of the dynamics, especially the traction force patterns and velocity fields, observed in experiments on Madin-Darby canine kidney cells. Our model predicts the necessity of the leader cell and its minimal functions for the formation and maintenance of a stable finger pattern. Meanwhile, our model allows for an analysis of the role of supracellular actin cable on the leading front, predicting that while this observed structure helps maintain the shape of the finger, it is not required in order to form a finger. In addition, we also study the driving instability in the context of continuum active fluid model, which justifies some of our assumptions in the computational approach. In particular, we show that in our model no finger protrusions would emerge in a phenotypically homogenous active fluid and hence the role of the leader cell and its followers are often critical.

High glucose induces phosphorylation and oxidation of mitochondrial proteins in renal tubular cells: A proteomics approach.

Mitochondrial dysfunction has been thought to play roles in the pathogenesis of diabetic nephropathy (DN). However, precise mechanisms underlying mitochondrial dysfunction in DN remained unclear. Herein, mitochondria were isolated from renal tubular cells after exposure to normal glucose (5.5 mM glucose), high glucose (25 mM glucose), or osmotic control (5.5 mM glucose + 19.5 mM mannitol) for 96 h. Comparative proteomic analysis revealed six differentially expressed proteins among groups that were subsequently identified by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and confirmed by Western blotting. Several various types of post-translational modifications (PTMs) were identified in all of these identified proteins. Interestingly, phosphorylation and oxidation were most abundant in mitochondrial proteins whose levels were exclusively increased in high glucose condition. The high glucose-induced increases in phosphorylation and oxidation of mitochondrial proteins were successfully confirmed by various assays including MS/MS analyses. Moreover, high glucose also increased levels of phosphorylated ezrin, intracellular ATP and ROS, all of which could be abolished by a p38 MAPK inhibitor (SB239063), implicating a role of p38 MAPK-mediated phosphorylation in high glucose-induced mitochondrial dysfunction. These data indicate that phosphorylation and oxidation of mitochondrial proteins are, at least in part, involved in mitochondrial dysfunction in renal tubular cells during DN.

Prostaglandin E2 and its receptor EP2 trigger signaling that contributes to YAP-mediated cell competition.

Cell competition is a biological process by which unfit cells are eliminated from "cell society." We previously showed that cultured mammalian epithelial Madin-Darby canine kidney (MDCK) cells expressing constitutively active YAP were eliminated by apical extrusion when surrounded by "normal" MDCK cells. However, the molecular mechanism underlying the elimination of active YAP-expressing cells was unknown. Here, we used high-throughput chemical compound screening to identify cyclooxygenase-2 (COX-2) as a key molecule triggering cell competition. Our work shows that COX-2-mediated PGE2 secretion engages its receptor EP2 on abnormal and nearby normal cells. This engagement of EP2 triggers downstream signaling via an adenylyl cyclase-cyclic AMP-PKA pathway that, in the presence of active YAP, induces E-cadherin internalization leading to apical extrusion. Thus, COX-2-induced PGE2 appears a warning signal to both abnormal and surrounding normal cells to drive cell competition.

Leflunomide increased the renal exposure of acyclovir by inhibiting OAT1/3 and MRP2.

Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (LEF) and the antiviral drug acyclovir to reduce the high risk of infection. Acyclovir is a substrate of organic anion transporter (OAT) 1/3 and multidrug resistance-associated protein (MRP) 2. Considering the extraordinarily long half-life of LEF's active metabolite teriflunomide (TER) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir. Here we used a specific MRP inhibitor MK571 and probenecid (OAT1/3 and MRP2 inhibitor) to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. We showed that LEF and probenecid, but not MK571 significantly increased the plasma concentration of acyclovir. However, kidney and liver exposures of acyclovir were increased when coadministered with LEF, probenecid or MK571. The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid, whereas it was increased to as much as 14.5-fold by MK571. Consistently, these drugs markedly decreased the urinary excretion of acyclovir. TER (0.5-100 µmol/L) dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC50 value of 4.91 µmol/L. TER (5 µmol/L) significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells. These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2, which increased its kidney/plasma ratio and renal injury risk. However, the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells' uptake of acyclovir and increased the plasma concentration.

Primary cilia and the exocyst are linked to urinary extracellular vesicle production and content.

The recently proposed idea of "urocrine signaling" hypothesizes that small secreted extracellular vesicles (EVs) contain proteins that transmit signals to distant cells. However, the role of renal primary cilia in EV production and content is unclear. We previously showed that the exocyst, a highly conserved trafficking complex, is necessary for ciliogenesis; that it is present in human urinary EVs; that knockdown (KD) of exocyst complex component 5 (EXOC5), a central exocyst component, results in very short or absent cilia; and that human EXOC5 overexpression results in longer cilia. Here, we show that compared with control Madin-Darby canine kidney (MDCK) cells, EXOC5 overexpression increases and KD decreases EV numbers. Proteomic analyses of isolated EVs from EXOC5 control, KD, and EXOC5-overexpressing MDCK cells revealed significant alterations in protein composition. Using immunoblotting to specifically examine the expression levels of ADP-ribosylation factor 6 (ARF6) and EPS8-like 2 (EPS8L2) in EVs, we found that EXOC5 KD increases ARF6 levels and decreases EPS8L2 levels, and that EXOC5 overexpression increases EPS8L2. Knockout of intraflagellar transport 88 (IFT88) confirmed that the changes in EV number/content were due to cilia loss: similar to EXOC5, the IFT88 loss resulted in very short or absent cilia, decreased EV numbers, increased EV ARF6 levels, and decreased Eps8L2 levels compared with IFT88-rescued EVs. Compared with control animals, urine from proximal tubule-specific EXOC5-KO mice contained fewer EVs and had increased ARF6 levels. These results indicate that perturbations in exocyst and primary cilia affect EV number and protein content.

Cell line-dependent activation and antiviral activity of T-1105, the non-fluorinated analogue of T-705 (favipiravir).

The antiviral drug T-705 (favipiravir) and its non-fluorinated analogue T-1105 inhibit the polymerases of RNA viruses after being converted to their ribonucleoside triphosphate (RTP) metabolite. We here compared the activation efficiency of T-705 and T-1105 in four cell lines that are commonly used for their antiviral evaluation. In MDCK cells, the levels of T-705-RTP were markedly lower than those of T-1105-RTP, while the opposite was seen in A549, Vero and HEK293T cells. In the latter three cell lines, T-1105 activation was hindered by inefficient conversion of the ribonucleoside monophosphate to the ribonucleoside diphosphate en route to forming the active triphosphate. Accordingly, T-1105 had better anti-RNA virus activity in MDCK cells, while T-705 was more potent in the other three cell lines. Additionally, we identified a fourth metabolite, the NAD analogue of T-705/T-1105, and showed that it can be formed by nicotinamide mononucleotide adenylyltransferase.

Annexin A2 expression and partners during epithelial cell differentiation.

The members of the annexin family of calcium- and phospholipid-binding proteins participate in different cellular processes. Annexin A2 binds to S100A10, forming a functional heterotetrameric protein that has been involved in many cellular functions, such as exocytosis, endocytosis, cell junction formation, and actin cytoskeleton dynamics. Herein, we studied annexin A2 cellular movements and looked for its partners during epithelial cell differentiation. By using immunofluorescence, mass spectrometry (MS), and western blot analyses after S100A10 affinity column separation, we identified several annexin A2-S100A10 partner candidates. The association of putative annexin A2-S100A10 partner candidates obtained by MS after column affinity was validated by immunofluorescence and sucrose density gradient separation. The results show that three proteins are clearly associated with annexin A2: E-cadherin, actin, and caveolin 1. Overall, the data show that annexin A2 can associate with molecular complexes containing actin, caveolin 1, and flotillin 2 before epithelial differentiation and with complexes containing E-cadherin, actin, and caveolin 1, but not flotillin 2 after cell differentiation. The results indicate that actin, caveolin 1, and E-cadherin are the principal protein partners of annexin A2 in epithelial cells and that the serine phosphorylation of the N-terminal domain does not play an essential role during epithelial cell differentiation.

Induced Proton Perturbation for Sensitive and Selective Detection of Tight Junction Breakdown.

Tight junctions (TJs) in the epithelial cell gap play primary roles in body defense and nutrient absorption in multicellular organisms. Standard in vitro assays lack sensitivity, selectivity, temporal resolution, and throughput for bioengineering applications. Prompted by the rigorous barrier functions of TJ, we developed a TJ assay that senses proton leaks in the cell gap using ion-sensitive field-effect transistors. Upon exposure of Madin-Darby canine kidney (MDCK) cells cultured on a gate dielectric to a calcium-chelator EGTA, ammonia-assisted pH perturbation was enhanced solely in TJ-forming cells. This was supported by simulations with increased ion permeability in the paracellular pathway. Following administration of Clostridium perfringens enterotoxin as a specific TJ inhibitor to the MDCK cells, our method detected TJ breakdown at a 13× lower concentration than a conventional trans-epithelial electrical resistance assay. Thus, the semiconductor-based active pH sensing could offer an alternative to current in vitro assays for TJs in pathological, toxicological, and pharmaceutical studies.

Abnormal Golgi pH Homeostasis in Cancer Cells Impairs Apical Targeting of Carcinoembryonic Antigen by Inhibiting Its Glycosyl-Phosphatidylinositol Anchor-Mediated Association with Lipid Rafts.

AIMS: Carcinoembryonic antigen (CEACAM5, CEA) is a known tumor marker for colorectal cancer that localizes in a polarized manner to the apical surface in normal colon epithelial cells whereas in cancer cells it is present at both the apical and basolateral surfaces of the cells. Since the Golgi apparatus sorts and transports most proteins to these cell surface domains, we set out here to investigate whether any of the factors commonly associated with tumorigenesis, including hypoxia, generation of reactive oxygen species (ROS), altered redox homeostasis, or an altered Golgi pH, are responsible for mistargeting of CEA to the basolateral surface in cancer cells. RESULTS: Using polarized nontumorigenic Madin-Darby canine kidney (MDCK) cells and CaCo-2 colorectal cancer cells as targets, we show that apical delivery of CEA is not affected by hypoxia, ROS, nor changes in the Golgi redox state. Instead, we find that an elevated Golgi pH induces basolateral targeting of CEA and increases its TX-100 solubility, indicating impaired association of CEA with lipid rafts. Moreover, disruption of lipid rafts by methyl-ß-cyclodextrin induced accumulation of the CEA protein at the basolateral surface in MDCK cells. Experiments with the glycosylphosphatidylinositol (GPI)-anchorless CEA mutant and CEA-specific GPI-anchored enhanced green fluorescent protein (EGFP-GPI) fusion protein revealed that the GPI-anchor was critical for the pH-dependent apical delivery of the CEA in MDCK cells. Innovation and Conclusion: The findings indicate that an abnormal Golgi pH homeostasis in cancer cells is an important factor that causes mistargeting of CEA to the basolateral surface of cancer cells via inhibiting its GPI-anchor-mediated association with lipid rafts.

Regulation of epithelial migration by epithelial cell adhesion molecule requires its Claudin-7 interaction domain.

Epithelial cell adhesion molecule (EpCAM) is a glycoprotein on the surface of epithelial cells that is essential for intestinal epithelial integrity and expressed at high levels in many epithelial derived cancers and circulating tumor cells. Here we show the effect of EpCAM levels on migration of Madin-Darby-Canine Kidney (MDCK) epithelial cells. MDCK cells depleted of EpCAM show increased activation of extracellular signal-regulated kinase (ERK) and of myosin, and increased cell spreading and epithelial sheet migration into a gap. In contrast, over-expression of EpCAM inhibits ERK and myosin activation, and slows epithelial sheet migration. Loss of EpCAM is rescued by EpCAM-YFP mutated in the extracellular domain required for cis-dimerization whereas EpCAM-YFP with a mutation that inhibits Claudin-7 interaction cannot rescue increased ERK, myosin activation, and increased migration in EpCAM-depleted cells. In summary, these results indicate that interaction of EpCAM and Claudin-7 at the cell surface negatively regulates epithelial migration by inhibiting ERK and actomyosin contractility.

Treponema denticola Induces Epithelial Barrier Dysfunction in Polarized Epithelial Cells.

Treponema denticola, an anaerobic spirochete found mainly in the oral cavity, is associated with periodontal disease and has a variety of virulence factors. Although in vitro studies have shown that T. denticola is able to penetrate epithelial cell monolayers, its effect on the epithelial barrier junction is not known. Human gingival epithelial cells are closely associated with adjacent membranes, forming barriers in the presence of tight junction proteins, including zonula occludens-1 (ZO-1), claudin-1, and occludin. Tight junction proteins are also expressed by Madin-Darby canine kidney (MDCK) cells in culture. In this study, the MDCK cell profile was investigated following infection with T. denticola (ATCC 35405) wild-type, as well as with its dentilisin-deficient mutant, K1. Basolateral exposure of MDCK cell monolayers to T. denticola at a multiplicity of infection (MOI) of 104 resulted in a decrease in transepithelial electrical resistance (TER). Transepithelial electrical resistance in MDCK cell monolayers also decreased following apical exposure to T. denticola (MOI=104), although this took longer with basolateral exposure. The effect on the TER was time-dependent and required the presence of live bacteria. Meanwhile, MDCK cell viability showed a decrease with either basolateral or apical exposure. Immunofluorescence analysis demonstrated decreases in the amounts of immunoreactive ZO-1 and claudin-1 in association with disruption of cell-cell junctions in MDCK cells exposed apically or basolaterally to T. denticola. Western blot analysis demonstrated degradation of ZO-1 and claudin-1 in culture lysates derived from T. denticola-exposed MDCK cells, suggesting a bacteria-induced protease capable of cleaving these tight junction proteins.