Epithelial transport and barrier function in occludin-deficient mice

Background and Aims

This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model.

Methods

Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (R^e, epithelial resistance). Conductance scanning differentiated transcellular (G^c) and tight junctional conductance (G^tj). The pH-stat technique quantified gastric acid secretion.

Results

In occludin+/+ mice, R^e was 23±5 Ω cm² in jejunum, 66±5 Ω cm² in distal colon and 33±6 Ω cm² in gastric corpus and was not altered in heterozygotic occludin+/− or homozygotic occludin−/− mice. Additionally, [³H]mannitol fluxes were unaltered. In the control colon, G^c and G^tj were 7.6±1.0 and 0.3±0.1 mS/cm² and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin−/− mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control R^t was 5.8±0.8 kΩ cm² in urinary bladder and 33±6 Ω cm² in stomach and not altered in occludin−/− mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin−/− mice.

Conclusion

Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.     

Epithelial barrier function: assembly and structural features of the cornified cell envelope

Terminally differentiating stratified squamous epithelial cells assemble a specialized protective barrier structure on their periphery termed the cornified cell envelope (CE). It is composed of numerous structural proteins that become cross-linked by several transglutaminase enzymes into an insoluble macromolecular assembly. Several proteins are involved in the initial stages of CE assembly, but only certain proteins from a choice of more than 20 different proteins are used in the final stages of CE reinforcement, apparently to meet tissue-specific requirements. In addition, a variable selection of proteins may be upregulated in response to genetic defects of one of the CE proteins or tissue injury, in an effort to maintain an effective barrier. Additionally, in the epidermis and hair fiber cuticle, a layer of lipids is covalently attached to the proteins, which provides essential water barrier properties. Here we describe our current understanding of CE structure, a possible mechanism of its assembly, and various disorders that cause a defective barrier.     

Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms

The phorbol ester phorbol12-myristate 13-acetate (PMA) inhibits Cl secretion(short-circuit current, I_sc) and decreasesbarrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes inthis response, we compared PMA with two non-phorbol activators of PKC(bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozymeselectivity profiles. PMA sequentially inhibited cAMP-stimulatedI_sc and decreased TER, as measured byvoltage-current clamp. By subcellular fractionation and Western blot,PMA (100 nM) induced sequential membrane translocation of the novelPKC followed by the conventional PKC and activated both isozymesby in vitro kinase assay. PKC was activated by PMA but did nottranslocate. By immunofluorescence, PKC redistributed to thebasolateral domain in response to PMA, whereas PKC moved apically.Inhibition of I_sc by PMA was prevented by theconventional and novel PKC inhibitor Gö-6850 (5 µM) but not theconventional isoform inhibitor Gö-6976 (5 µM) or the PKCinhibitor rottlerin (10 µM), implicating PKC in inhibition ofCl secretion. In contrast, both Gö-6976 andGö-6850 prevented the decline of TER, suggesting involvement ofPKC. Bryostatin-1 (100 nM) translocated PKC and PKC andinhibited cAMP-elicited I_sc. However, unlikePMA, bryostatin-1 downregulated PKC protein, and the decrease in TERwas only transient. Carbachol (100 µM) translocated only PKC andinhibited I_sc with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1and carbachol inhibition of I_sc. We concludethat basolateral translocation of PKC inhibits Clsecretion, while apical translocation of PKC decreases TER. Thesedata suggest that epithelial transport and barrier function can bemodulated by distinct PKC isoforms.

     

Luminal hyperosmolarity decreases Na transport and impairs barrier function of sheep rumen epithelium

The effects of luminal hyperosmolarity on Na and Cl transport were studied in rumen epithelium of sheep. An increase of luminal osmotic pressure with mannitol (350 and 450 mosm/l) caused a significant increase of tissue conductance, G _T, which is linearly correlated with flux rates of ⁵¹Cr-EDTA and indicates an increase of passive permeability. Studies with microelectrodes revealed, that an increase of the osmotic pressure caused a significant increase of the conductance of the shunt pathway from 1.23±0.10 (control) to 1.92±0.14 mS cm⁻² (450 mosm/l) without a change of fractional resistance. Hyperosmolarity significantly increased J _sm and reduced J _net Na. The effect of hyperosmolarity on J _ms Na is explained by two independent and opposed effects: increase of passive permeability and inhibition of the Na⁺/H⁺ exchanger. Hypertonic buffer solution induced a decrease of the intracellular pH (pH_i) of isolated ruminal cells, which is consistent with an inhibition of Na⁺/H⁺ exchange, probably isoform NHE-3, because NHE-3-mRNA was detectable in rumen epithelium. These data are in contrast to previous reports and reveal a disturbed Na transport and an impaired barrier function of the rumen epithelium, which predisposes translocation of rumen endotoxins and penetration of bacteria.     

The acyl-CoA binding protein is required for normal epidermal barrier function in mice

The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP(+/+) and ACBP(-/-) mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP(-/-) mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP(-/-) mice display a ～50% increased transepidermal water loss compared with ACBP(+/+) mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function.     

Maternal obesity induces gut inflammation and impairs gut epithelial barrier function in nonobese diabetic mice

Impairment of gut epithelial barrier function is a key predisposing factor for inflammatory bowel disease, type 1 diabetes (T1D) and related autoimmune diseases. We hypothesized that maternal obesity induces gut inflammation and impairs epithelial barrier function in the offspring of nonobese diabetic (NOD) mice. Four-week-old female NOD/ShiLtJ mice were fed with a control diet (CON; 10% energy from fat) or a high-fat diet (HFD; 60% energy from fat) for 8 weeks to induce obesity and then mated. During pregnancy and lactation, mice were maintained in their respective diets. After weaning, all offspring were fed the CON diet. At 16 weeks of age, female offspring were subjected to in vivo intestinal permeability test, and then ileum was sampled for biochemical analyses. Inflammasome mediators, activated caspase-1 and mature forms of interleukin (IL)-1β and IL-18 were enhanced in offspring of obese mothers, which was associated with elevated serum tumor necrosis factor α level and inflammatory mediators. Consistently, abundance of oxidative stress markers including catalase, peroxiredoxin-4 and superoxide dismutase 1 was heightened in offspring ileum (P<.05). Furthermore, offspring from obese mothers had a higher intestinal permeability. Morphologically, maternal obesity reduced villi/crypt ratio in the ileum of offspring gut. In conclusion, maternal obesity induced inflammation and impaired gut barrier function in offspring of NOD mice. The enhanced gut permeability in HFD offspring might predispose them to the development of T1D and other gut permeability-associated diseases.     

Epithelial PBLD attenuates intestinal inflammatory response and improves intestinal barrier function by inhibiting NF-κB signaling

Intestinal barrier function defects and dysregulation of intestinal immune responses are two key contributory factors in the pathogenesis of ulcerative colitis (UC). Phenazine biosynthesis-like domain-containing protein (PBLD) was recently identified as a tumor suppressor in gastric cancer, hepatocellular carcinoma, and breast cancer; however, its role in UC remains unclear. Therefore, we analyzed colonic tissue samples from patients with UC and constructed specific intestinal epithelial PBLD-deficient (PBLD^IEC−/−) mice to investigate the role of this protein in UC pathogenesis. We found that epithelial PBLD was decreased in patients with UC and was correlated with levels of tight junction (TJ) and inflammatory proteins. PBLD^IEC−/− mice were more susceptible to dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid-induced colitis compared with wild-type (WT) mice. In DSS-induced colitis, PBLD^IEC−/− mice had impaired intestinal barrier function and greater immune cell infiltration in colonic tissue than WT mice. Furthermore, TJ proteins were markedly reduced in PBLD^IEC−/− mice compared with WT mice with colitis. Nuclear factor (NF)-κB activation was markedly elevated and resulted in higher expression levels of downstream effectors (C–C motif chemokine ligand 20, interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α) in colonic epithelial cells isolated from PBLD^IEC−/− mice than WT mice with colitis. PBLD overexpression in intestinal epithelial cells (IECs) consistently inhibited TNF-α/interferon-γ-induced intestinal barrier disruption and TNF-α-induced inflammatory responses via the suppression of NF-κB. In addition, IKK inhibition (IKK-16) rescued excessive inflammatory responses induced by TNF-α in PBLD knockdown FHC cells. Co-immunoprecipitation assays showed that PBLD may interact with IKKα and IKKβ, thus inhibiting NF-κB signaling, decreasing inflammatory mediator production, attenuating colonic inflammation, and improving intestinal barrier function. Modulating PBLD expression may provide a novel approach for treatment in patients with UC.Subject terms: Ulcerative colitis, Chronic inflammation     

Blood-brain barrier and neuroendocrine function

Blood-brain barrier and neuroendocrine function     

Epithelial V-like antigen regulates permeability of the blood-CSF barrier

Epithelial V-like antigen (EVA), a CD3-binding immunoglobulin-like protein, regulates embryonic thymic development. Here we demonstrate that EVA is expressed in choroid plexus from mature immune competent and lymphocyte-deficient (RAG−/−) mice. Choroid plexus epithelial cells from RAG−/− mice demonstrated reduced junctional integrity and enhanced permeability that was associated with decreased expression of E-cadherin and EVA mRNA as compared to wild-type mice. Following iv infusion of an anti-CD3 antibody (145-2C11) that also binds EVA, expression of E-cadherin and EVA mRNA approached levels seen in wild-type mice. Immuno-fluorescent staining for cadherin also revealed decreased expression in untreated RAG−/− mice that could be increased by 145-2C11 treatment. Expression of mouse EVA in HEK-293 cells followed by challenge with 145-2C11 resulted in increased cytosolic calcium that was not seen in control cells. These results suggest that EVA expressed in choroid plexus cells may regulate the permeability of the blood-CSF barrier.     

Adrenomedullin treatment reduces intestinal inflammation and maintains epithelial barrier function in mice administered dextran sulphate sodium

Hyperactivation and hyperpermeability of the intestinal epithelium is a hallmark of IBD. AM has been shown to reduce the severity of colitis in the acetic acid and TNBS-induced colitis model, however the mechanism of the therapeutic effect of AM against the colitis has not been clarified. Here, we show that the protective capability of AM is associated with suppression of inflammation and maintenance of the intestinal epithelial barrier function. In the DSS-induced colitis model, intra-rectal AM-treated mice showed a reduction in loss of body weight and severity of colitis. AM-treatment suppressed phosphorylation of STAT1 and STAT3 in the colonic epithelium, and altered the cytokine balance in the intestinal T cells, with lower levels of IFN-γ and TNF-α but higher levels of TGF-β. Expression of the epithelial intercellular junctions such as tight and adherence junctions were sustained in the AM-treated mice. In contrast, the epithelial junctions were down-regulated in the control mice, leading to loss of epithelial barrier integrity and enhanced permeability. Collectively, these data indicate a broad spectrum of AM-induced effects with respect to protection against DSS-induced colitis, and suggest a potential therapeutic value of this treatment for IBD.     

Nutrient transport and the blood-brain barrier in developing animals

Structural alterations in the development of the blood-brain barrier (BBB) can be seen in capillary profiles from the rat cortex. The neonatal luminal membrane is amplified with irregular folds, a possible adaptation to reduced cerebral blood flow rates. By 21 days the capillaries have resolved to a smooth-surfaced, adult-like appearance. Developmental alterations in the basement membrane, tight junctions, capillary seams, Golgi, pinocytotic vesicles, and cytoplasmic thickness are observed. Two studies have addressed developmental modulations in BBB polarity; both indicate that brain-to-blood transport mechanisms that were inoperative in the early neonatal rat become functional in weanlings. Six of the seven major independent BBB nutrient transport systems that regulate plasma-to-brain uptake have been kinetically characterized in the newborn rabbit, and comparisons have been made in the weanling (28-day-old) rabbit. All of these saturable transport systems are operative at birth, which suggests that the immature rabbit has a mature BBB with respect to regulation of nutrients. Purine base permeability, affinity, and uptake velocities are virtually unchanged during postnatal development. Subtle alterations in amino acid and amine transport were suggested by the lower-affinity (high-capacity) transport mechanisms characterized in the newborn as compared to the 28-day-old BBB. Under conditions of elevated plasma levels (typical of the neonate), these higher-capacity mechanisms would facilitate a relative increase in metabolite influx to the developing brain. Significant differences in kinetics were also observed for the monocarboxylic acid and hexose transport systems in the absence of developmental changes in permeability times surface area products. A low-affinity, high-capacity monocarboxylic acid transport system operates at birth. It supplies the developing brain with increased quantities of ketone bodies, but is seen as a high-affinity, low-capacity mechanism in the 28-day-old rabbit. Concomitantly, the higher-affinity glucose carrier defined in newborn rabbits modulates, and by 28 days becomes a lower-affinity, high-capacity mechanism capable of delivering about 2 mumol X min-1 X g-1 of glucose to the (anesthetized) brain.     

Epithelial water transport in a balanced gradient system.

The relationship between epithelial fluid transport, standing osmotic gradients, and standing hydrostatic pressure gradients has been investigated using a perturbation expansion of the governing equations. The assumptions used in the expansion are: (a) the volume of lateral intercellular space per unit volume of epithelium is small; (b) the membrane osmotic permeability is much larger than the solute permeability. We find that the rate of fluid reabsorption is set by the rate of active solute transport across lateral membranes. The fluid that crosses the lateral membranes and enters the intercellular cleft is driven longitudinally by small gradients in hydrostatic pressure. The small hydrostatic pressure in the intercellular space is capable of causing significant transmembrane fluid movement, however, the transmembrane effect is countered by the presence of a small standing osmotic gradient. Longitudinal hydrostatic and osmotic gradients balance such that their combined effect on transmembrane fluid flow is zero, whereas longitudinal flow is driven by the hydrostatic gradient. Because of this balance, standing gradients within intercellular clefts are effectively uncoupled from the rate of fluid reabsorption, which is driven by small, localized osmotic gradients within the cells. Water enters the cells across apical membranes and leaves across the lateral intercellular membranes. Fluid that enters the intercellular clefts can, in principle, exit either the basal end or be secreted from the apical end through tight junctions. Fluid flow through tight junctions is shown to depend on a dimensionless parameter, which scales the resistance to solute flow of the entire cleft relative to that of the junction. Estimates of the value of this parameter suggest that an electrically leaky epithelium may be effectively a tight epithelium in regard to fluid flow.