Specific Tight Junction Proteins Research Articles

Specific Modulation of Airway Epithelial Tight Junctions by Apical Application of an Occludin Peptide

Tight junctions are directly involved in regulating the passage of ions and macromolecules (gate functions) in epithelial and endothelial cells. The modulation of these gate functions to transiently regulate the paracellular permeability of large solutes and ions could increase the delivery of pharmacological agents or gene transfer vectors. To reduce the inflammatory responses caused by tight junction-regulating agents, alternative strategies directly targeting specific tight junction proteins could prove to be less toxic to airway epithelia. The apical delivery of peptides corresponding to the first extracellular loop of occludin to transiently modulate apical paracellular flux has been demonstrated in intestinal epithelia. We hypothesized that apical application of these occludin peptides could similarly modulate tight junction permeability in airway epithelia. Thus, we investigated the effects of apically applied occludin peptide on the paracellular permeability of molecular tracers and viral vectors in well differentiated human airway epithelial cells. The effects of occludin peptide on cellular toxicity, tight junction protein expression and localization, and membrane integrity were also assessed. Our data showed that apically applied occludin peptide significantly reduced transepithelial resistance in airway epithelia and altered tight junction permeability in a concentration-dependent manner. These alterations enhanced the paracellular flux of dextrans as well as gene transfer vectors. The occludin peptide redistributed occludin but did not alter the expression or distribution of ZO-1, claudin-1, or claudin-4. These data suggest that specific targeting of occludin could be a better-suited alternative strategy for tight junction modulation in airway epithelial cells compared with current agents that modulate tight junctions.

AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins

Antrum mucosal protein (AMP)-18 and a synthetic peptide of amino acids 77-97 have mitogenic and motogenic properties for epithelial cells. The possibility that AMP-18 is also protective was evaluated in the colonic mucosa of mice and monolayer cultures of human colonic epithelial Caco-2/bbe (C2) cells. Administration of AMP peptide to mice with dextran sulfate sodium (DSS)-induced colonic injury delayed the onset of bloody diarrhea and reduced weight loss. Treatment of C2 cells with AMP peptide protected monolayers against decreases in transepithelial electrical resistance induced by the oxidant monochloramine, indomethacin, or DSS. A molecular mechanism for these barrier-protective effects was sought by asking whether AMP peptide acted on specific tight junction (TJ) proteins. Immunoblots of detergent-insoluble fractions of C2 cells treated with AMP peptide exhibited increased accumulation of specific TJ proteins. Occludin immunoreactivity was also increased in detergent-insoluble fractions obtained from colonic mucosal cells of mice injected with AMP peptide. Observations using laser scanning confocal (CF) microscopy supported the capacity of AMP peptide to enhance accumulation of occludin and zonula occludens-1 in TJ domains of C2 cell monolayers and together with immunoblot analysis showed that the peptide protected against loss of these TJ proteins following oxidant injury. AMP peptide also protected against a fall in TER during disruption of actin filaments by cytochalasin D and stabilized perijunctional actin during oxidant injury when assessed by CF. These findings suggest that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin.

HIV-1 Tat protein alters tight junction protein expression and distribution in cultured brain endothelial cells.

Disruption of the blood-brain barrier (BBB) is widely believed to be the main route of human immunodeficiency virus (HIV) entry into the central nervous system (CNS). Although mechanisms of this process are not fully understood, alterations of tight junction protein expression can contribute, at least in part, to this phenomenon. Tight junctions are critical structural and functional elements of cerebral microvascular endothelial cells and the BBB. The aim of the present study was to examine the effects of HIV-1 Tat protein on expression of tight junction proteins. Primary cultures of brain microvascular endothelial cells (BMEC) were employed in these experiments. A 24-hr exposure of BMEC to Tat(1-72) resulted in a decrease of claudin-1, claudin-5, and zonula occludens (ZO)-2 expression, whereas total levels of occludin and ZO-1 remained unchanged. In addition, a short (3-hr) exposure of BMEC to Tat(1-72) induced cellular redistribution of claudin-5 immunoreactivity. Tat(1-72)-induced alterations of claudin-5 expression also were confirmed in vivo where Tat(1-72) was injected into the right hippocampus of mice. These findings indicate that HIV-1 Tat protein can markedly affect expression and distribution of specific tight junction proteins in brain endothelium. Alterations of only distinct tight junction proteins suggest a finely tuned effect of Tat(1-72) on the BBB. Because tight junction proteins are critical for the barrier function of the BBB, such alterations can lead to disturbances of the BBB integrity and contribute to HIV trafficking into the brain.

Age-related changes in rat cerebral occludin and zonula occludens-1 (ZO-1)

The endothelial or epithelial tight junctions create a rate-limiting barrier to diffusion of solutes. A major determinant of the barrier function is the density of tight junction proteins. Since aging is associated with significant alterations in the blood–brain barrier (BBB) it is possible that specific tight junction proteins may be altered in the cerebrum of aging rats. To test this hypothesis, Western and Northern blot analysis were carried out to measure the steady-state level of occludin and zonula occludens-one (ZO-1) proteins and their mRNA in cerebral tissue of 3-, 12- and 24-month-old rats. The cerebral occludin content in 24-month-old rats (732.5±99.9 arbitrary units) was significantly reduced compared to 12-month-old rats (1043.4±131.8) or 3-month-old rats (1021.4±62.8), P<0.01. The cerebral ZO-1 protein content in 24-month-old rats (161.7±8.1 arbitrary units) and 12-month-old rats (144.3±35.9) were not significantly reduced compared to 4-month-old rats (189.0±27.2). The occludin mRNA content relative to G3PDH mRNA was 1.11±0.05, 1.11±0.07 and 1.00±0.05 in 3-, 12- and 24-month-old rats, respectively. The differences did not achieve statistical significance. The ZO-1 mRNA content of cerebral tissue relative to G3PDH mRNA was significantly increased in 24-month-old rats compared to 3-month-old rats (1.280±0.030 vs. 0.956±0.038), P<0.001. It is concluded that aging in rats may alter the molecular anatomy of the BBB by altering the content of select structural proteins of tight junctions.

Diabetes-related changes in rat cerebral occludin and zonula occludens-1 (ZO-1) expression.

The endothelial or epithelial tight junctions create a barrier to diffusion of solutes. Since experimental diabetes mellitus is associated with considerable alterations in the blood-brain barrier (BBB), it is possible that specific tight junction proteins may be altered in diabetes. To test this hypothesis, Western and Northern blot analysis were carried out to measure the steady-state level of occludin and zonula occludens-one (ZO-1) proteins and mRNA levels in cerebral tissue of streptozotocin-induced diabetic rats and the results were compared to insulin treated diabetic rats and vehicle injected control rats. The cerebral occludin content in diabetic rats (115.4 +/- 18.6 arbitrary units) was significantly reduced compared to insulin-treated diabetic rats (649.1 +/- 141.2) or control rats (552.9 +/- 82.9), p < 0.001. The ZO-1 content of cerebral tissue from diabetic rats (1,240.6 +/- 199.7 arbitrary units) was not significantly altered compared to controls (1,310.8 +/- 256.9). The cerebral occludin mRNA content relative to G3PDH mRNA was 1.35 +/- 0.07 and 1.34 +/- 0.19 in control and diabetic rats respectively. The cerebral ZO-1 mRNA content relative to G3PDH mRNA in diabetic and control rats was 1.135 +/- 0.123 and 0.956 +/- 0.038 respectively. These differences did not achieve statistical significance. It is concluded that diabetes alters the molecular anatomy of the tight junctions in cerebral tissue by altering the content of select structural proteins.