Phosphorylation state regulates the localization of Scribble at adherens junctions and its association with E-cadherin-catenin complexes

Mammalian ortholog of Scribble tumor suppressor has been reported to regulate cadherin-mediated epithelial cell adhesion by stabilizing the coupling of E-cadherin with catenins, but the molecular mechanism involved remains unknown. In this study, we investigated the relationship between the localization of mouse Scribble at cadherin-based adherens junctions (AJs) and its phosphorylation state. Immunofluorescence staining confirmed that Scribble was localized at AJs as well as at the basolateral plasma membrane in epithelial cells. We found that Scribble was detected as two bands by Western blotting analysis and that the band shift to the higher molecular weight was dependent on its phosphorylation at Ser 1601. Triton X-100 treatment extracted Scribble localized on the basolateral membrane but not Scribble localized at AJs in cultured epithelial cells, and the Triton X-100-resistant Scribble was the Ser 1601-unphosphorylated form. Conversely, an in-house-generated antibody that predominantly recognized Ser 1601-phosphorylated Scribble only detected Scribble protein on the lateral plasma membrane. Furthermore, Ser 1601-unphosphorylated Scribble was selectively coprecipitated with E-cadherin–catenin complexes in E-cadherin-expressing mouse L fibroblasts. Taken together, these results suggest that the phosphorylation state of Scribble regulates its complex formation with the E-cadherin–catenin system and may control cadherin-mediated cell–cell adhesion.     

Hymenolepis diminuta: The effects of infection on transepithelial ion transport and tight junctions in rat intestines

In this study, we examine the effect of Hymenolepis diminuta on ion transport in the ileum and on tight junctions in the ileum and colon of rats. We also evaluate the effect of H. diminuta on C-fiber endings in the ileum, the direct habitat of H. diminuta, before and after mechanical stimulation and pharmacological modification by capsaicin (C-fiber irritant).Wistar rats were orally infected with five cysticercoids of H. diminuta. Using a modified Ussing chamber, electrophysiological parameters of the ileum were measured (transepithelial electrical potential difference and transepithelial electrical resistance) as well as the deposition of occludin (a tight junction protein) in the ileum and colon of the rats 8, 16, 25, 35, 40 and 60 days post infection.We observed a significant reduction in transepithelial electrical potential difference in the ileum of rats infected with H. diminuta. In both the ileum and colon of rats infected with H. diminuta we also observed a decrease in occludin deposition, which indicates leakage of tight junctions, correlating with the decrease in transepithelial electrical resistance of these tissues. The application of capsaicin confirmed the hypothesis that H. diminuta in rats affects the C-fiber sensory receptors, causing changes in ion transport in the ileum.The results of the performed electrophysiological and immunohistochemical examinations indicate hymenolepidosis-related changes in the active transport of ions and the passive movement of ions.     

Hydrostatic pressure regulates tight junctions, actin cytoskeleton and transcellular ion transport

In the epithelia and endothelia, tight junctions regulate the movement of several substances through the paracellular pathway, maintaining several gradients between apical and basal compartments including osmolality and hydrostatic pressure. In this study, we show that the change of hydrostatic pressure gradient affected tight junctions as well as actin cytoskeleton, cell height and transcellular ion transport. Hydrostatic pressure gradient from basolateral to apical side increased transepithelial conductance and altered claudin-1 localization within several tens of minutes. These changes were promptly restored by the elimination of hydrostatic pressure gradient. Hydrostatic pressure gradient also induced dynamic changes in the actin structure and cell height. We further found that hydrostatic pressure gradient from basolateral to apical side stimulates transcellular Cl⁻ transport. Our present findings indicate that the epithelial cell structures and functions are regulated by the hydrostatic pressure gradient which is generated and maintained by the epithelia themselves.     

Fatty aldehyde and fatty alcohol metabolism: Review and importance for epidermal structure and function

Normal fatty aldehyde and alcohol metabolism is essential for epidermal differentiation and function. Long-chain aldehydes are produced by catabolism of several lipids including fatty alcohols, sphingolipids, ether glycerolipids, isoprenoid alcohols and certain aliphatic lipids that undergo α- or ω-oxidation. The fatty aldehyde generated by these pathways is chiefly metabolized to fatty acid by fatty aldehyde dehydrogenase (FALDH, alternately known as ALDH3A2), which also functions to oxidize fatty alcohols as a component of the fatty alcohol:NAD oxidoreductase (FAO) enzyme complex. Genetic deficiency of FALDH/FAO in patients with Sjögren–Larsson syndrome (SLS) results in accumulation of fatty aldehydes, fatty alcohols and related lipids (ether glycerolipids, wax esters) in cultured keratinocytes. These biochemical changes are associated with abnormalities in formation of lamellar bodies in the stratum granulosum and impaired delivery of their precursor membranes to the stratum corneum (SC). The defective extracellular SC membranes are responsible for a leaky epidermal water barrier and ichthyosis. Although lamellar bodies appear to be the pathogenic target for abnormal fatty aldehyde/alcohol metabolism in SLS, the precise biochemical mechanisms are yet to be elucidated. Nevertheless, studies in SLS highlight the critical importance of FALDH and normal fatty aldehyde/alcohol metabolism for epidermal function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

The important role of stratum corneum lipids for the cutaneous barrier function

The skin protects the body from unwanted influences from the environment as well as excessive water loss. The barrier function of the skin is located in the stratum corneum (SC). The SC consists of corneocytes embedded in a lipid matrix. This lipid matrix is crucial for the lipid skin barrier function. This paper provides an overview of the reported SC lipid composition and organization mainly focusing on healthy and diseased human skin. In addition, an overview is provided on the data describing the relation between lipid modulations and the impaired skin barrier function. Finally, the use of in vitro lipid models for a better understanding of the relation between the lipid composition, lipid organization and skin lipid barrier is discussed. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

The role of sphingolipid metabolism in cutaneous permeabilitybarrier formation

The epidermal permeability barrier of mammalian skin is localized in the stratum corneum. Corneocytes are embedded in an extracellular, highly ordered lipid matrix of hydrophobic lipids consisting of about 50% ceramides, 25% cholesterol and 15% long and very long chain fatty acids. The most important lipids for the epidermal barrier are ceramides. The scaffold of the lipid matrix is built of acylceramides, containing ω-hydroxylated very long chain fatty acids, acylated at the ω-position with linoleic acid. After glucosylation of the acylceramides at Golgi membranes and secretion, the linoleic acid residues are replaced by glutamate residues originating from proteins exposed on the surface of corneocytes. Removal of their glucosyl residues generates a hydrophobic surface on the corneocytes used as a template for the formation of extracellular lipid layers of the water permeability barrier. Misregulation or defects in the formation of extracellular ceramide structures disturb barrier function. Important anabolic steps are the synthesis of ultra long chain fatty acids, their ω-hydroxylation, and formation of ultra long chain ceramides and glucosylceramides. The main probarrier precursor lipids, glucosylceramides and sphingomyelins, are packed in lamellar bodies together with hydrolytic enzymes such as glucosylceramide-β-glucosidase and acid sphingomyelinase and secreted into the intercelullar space between the stratum corneum and stratum granulosum. Inherited defects in the extracellular hydrolytic processing of the probarrier acylglucosylceramides impair epidermal barrier formation and cause fatal diseases: such as prosaposin deficiency resulting in lack of lysosomal lipid binding and transfer proteins, or the symptomatic clinical picture of the “collodion baby” in the absence of glucocerebrosidase. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

The coxsackie- and adenovirus receptor (CAR) is an in vivo marker for epithelial tight junctions, with a potential role in regulating permeability and tissue homeostasis

The coxsackie- and adenovirus receptor (CAR) is a transmembrane protein belonging to the immunoglobulin superfamily. The function of CAR as a virus receptor has been extensively analyzed, while its physiological role and expression pattern in adult tissues have remained less clear. CAR associates with epithelial tight junctions in vitro and mediates cell-cell adhesion. Using a set of affinity-purified antibodies, we show that CAR is predominantly expressed in epithelial cells lining the body cavities in adult mice, where it specifically co-localizes with the tight junction components ZO-1 and occludin. Notably, CAR could not be detected in endothelial cells of the vasculature, including brain capillaries. CAR expression correlated positively with the maturity of tight junctions and inversely with permeability. With a few exceptions, the two known CAR isoforms were co-expressed in most epithelial cells analyzed. A CAR mutant lacking the intracellular tail over-expressed in transgenic mice was diffusely localized over the plasma membrane, showing the importance of this domain for correct subcellular localization in vivo. We conclude that CAR is localized to epithelial tight junctions in vivo where it may play a role in the regulation of epithelial permeability and tissue homeostasis.     

Sodium-dependent glucose transporter reduces peroxynitrite and cell injury caused by cisplatin in renal tubular epithelial cells

Cisplatin causes nephropathy accompanied by two types of cell death, necrosis and apoptosis, according to its dosage. The mechanisms of necrosis are still unclear. In this study, we examined how high doses of cisplatin induce cell injury and whether a high affinity sodium-dependent glucose transporter (SGLT1) has a cytoprotective function in renal epithelial LLC-PK₁ cells. Cisplatin decreased in transepithelial electrical resistance (TER) and increased in the number of necrotic dead cells in a time dependent manner. Phloridzin, a potent SGLT1 inhibitor, enhanced both TER decrease and increase of necrotic dead cells caused by cisplatin. Cisplatin increased in the intracellular nitric oxide, superoxide anion and peroxynitrite productions. Phloridzin enhanced the peroxynitrite production caused by cisplatin. The intracellular diffusion of ZO-1 and TER decrease caused by cisplatin were inhibited by N-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor. Protein kinase C was not involved in the cisplatin-induced injury. 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato iron (III) and reduced glutathione, peroxynitrite scavengers, inhibited the cisplatin-induced ZO-1 diffusion, TER decrease, and increase of necrotic dead cells. These results suggest that peroxynitrite is a key mediator in the nephrotoxicity caused by high doses of cisplatin. SGLT1 endogenously carries out the cytoprotective function by the reduction of peroxynitrite production.     

Crosstalk of tight junction components with signaling pathways

Tight junctions (TJs) regulate the passage of ions and molecules through the paracellular pathway in epithelial and endothelial cells. TJs are highly dynamic structures whose degree of sealing varies according to external stimuli, physiological and pathological conditions. In this review we analyze how the crosstalk of protein kinase C, protein kinase A, myosin light chain kinase, mitogen-activated protein kinases, phosphoinositide 3-kinase and Rho signaling pathways is involved in TJ regulation triggered by diverse stimuli. We also report how the phosphorylation of the main TJ components, claudins, occludin and ZO proteins, impacts epithelial and endothelial cell function.     

Mutated C-terminal fragments of Clostridium perfringens enterotoxin have increased affinity to claudin-4 and reversibly modulate tight junctions in vitro

Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator.     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.