Decreased interaction between ZO-1 and occludin is involved in alteration of tight junctions in transplanted epiphora submandibular glands

Tight junctions (TJs) in salivary epithelium play an important role in regulating saliva secretion. Autologous transplantation of submandibular glands (SMGs) is an effective method to treat severe dry eye syndrome. However, epiphora occurs in some patients 6 months after transplantation. We previously found that the acinar TJs are enlarged in rabbit SMGs after long-term transplantation, but the exact TJ components involved in the epiphora are still unknown. Here, we found that the mRNA and protein expression of ZO-1 and occludin were increased in the transplanted SMGs obtained from epiphora patients, while other TJs were unchanged. The intensity of ZO-1 and occludin at the apicolateral membranes as well as occludin in the cytoplasm were increased in epiphora SMGs, but the interaction between ZO-1 and occludin was decreased as evidenced by both co-immunoprecipitation assay and co-immunofluorescence staining. Mechanically, the expression of casein kinase 2α (CK2α) and CK2β, which was reported to affect occludin modification and the interaction of occludin with ZO-1 in previous literatures, were increased in epiphora glands. Moreover, activation of muscarinic acetylcholine receptor (mAChR) by carbachol directly decreased the interaction between ZO-1 and occludin and increased the acinar TJ width in the freshly isolated human SMGs, whereas these effects were abolished by pretreatment with CK2 inhibitor. Taken together, our findings suggest that decreased interaction between ZO-1 and occludin might contribute to the epiphora occurred in the transplanted SMGs, and mAChR together with the intracellular molecule CK2 might be responsible for the alteration of TJs in epiphora glands.     

Src homology 2 (SH2) domain containing protein tyrosine phosphatase-1 (SHP-1) dephosphorylates VEGF Receptor-2 and attenuates endothelial DNA synthesis, but not migration*

Background

Sustained agonist-promoted ubiquitination of β-arrestin has been correlated with increased stability of the GPCR – β-arrestin complex. Moreover, abrogation of β-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation.

Results

Herein we report that agonist activation of M₁ mAChRs produces a sustained β-arrestin ubiquitination but no stable co-localization with β-arrestin. In contrast, sustained ubiquitination of β-arrestin by activation of M₂ mAChRs does result in stable co-localization between the M₂ mAChR and β-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of β-arrestin following agonist treatment, we sought to determine the effects of β-arrestin ubiquitination on M₁ and M₂ mAChR down-regulation. A constitutively ubiquitinated β-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of β-arrestin 2 (YFP-β-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M₁ and M₂ mAChRs, with the effect substantially higher on the M₂ mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the β-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M₂ mAChR was not affected by expression of β-arrestin lysine mutants lacking putative ubiquitination sites, β-arrestin 2^{K18R, K107R, K108R, K207R, K296R}, while down-regulation and stable co-localiztion of the receptor with this β-arrestin lysine mutant were significantly reduced. Interestingly, expression of β-arrestin 2^{K18R, K107R, K108R, K207R, K296R} increased the agonist-promoted down-regulation of the M₁ mAChR but did not result in a stable co-localiztion of the receptor with this β-arrestin lysine mutant.

Conclusion

These findings indicate that ubiquitination of β-arrestin has a distinct role in the differential trafficking and degradation of M₁ and M₂ mAChRs.     

Ca<Superscript>2+</Superscript>-CaMKKβ pathway is required for adiponectin-induced secretion in rat submandibular gland

Adiponectin functions as a promoter of saliva secretion in rat submandibular gland via activation of adenosine monophosphate-activated protein kinase (AMPK) and increased paracellular permeability. Ca²⁺ mobilization is the primary signal for fluid secretion in salivary acinar cells. However, whether intracellular Ca²⁺ mobilization is involved in adiponectin-induced salivary secretion is unknown. Here, we found that full-length adiponectin (fAd) increased intracellular Ca²⁺ and saliva secretion in submandibular glands. Pre-perfusion with ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) combined with thapsigargin (TG), an endoplasmic reticulum Ca²⁺-ATPase inhibitor, abolished fAd-induced salivary secretion, AMPK phosphorylation, and enlarged tight junction (TJ) width. Furthermore, in cultured SMG-C6 cells, co-pretreatment with EGTA and TG suppressed fAd-decreased transepithelial electrical resistance and increased 4-kDa FITC-dextran flux responses. Moreover, fAd increased phosphorylation of calcium/calmodulin-dependent protein kinase (CaMKKβ), a major kinase that is activated by elevated levels of intracellular Ca²⁺, but not liver kinase B1 phosphorylation. Pre-perfusion of the isolated gland with STO-609, an inhibitor of CaMKKβ, abolished fAd-induced salivary secretion, AMPK activation, and enlarged TJ width. CaMKKβ shRNA suppressed, whereas CaMKKβ re-expression rescued fAd-increased paracellular permeability. Taken together, these results indicate that adiponectin induced Ca²⁺ modulation in rat submandibular gland acinar cells. Ca²⁺-CaMKKβ pathway is required for adiponectin-induced secretion through mediating AMPK activation and increase in paracellular permeability in rat submandibular glands.     

Proteomic analysis of human transplanted submandibular gland in patients with epiphora after transplantation

Submandibular gland autotransplantation is effective for treating severe dry eye syndrome. However, more than 40% of patients show epiphora within 3-6 months after treatment. The mechanism underlying the hypersecretion in epiphora remains to be elucidated for developing novel interventions. Since salivary gland secretion is dependent on a variety of proteins, we analyzed the changes in protein expression in transplanted glands of epiphora patients with 2-D gel electrophoresis and electrospray ionization quadrupole/time-of-flight mass spectrometry and evaluated their possible roles in epiphora. There were 23 proteins that showed altered expression in the glands of epiphora patients, 15 being up-expressed and 8 being down-expressed. The expression of secretory proteins was decreased in these glands, including alpha-amylase, cystatin S, SA, and SN. In contrast, cytoskeletal proteins were all up-regulated, including actin and vimentin. Immunofluorescence revealed that the intensity ratio of F-actin in apical and lateral cytoplasm to total F-actin in acini was decreased in the glands of epiphora patients. Carbachol stimulation induced a similar redistribution of F-actin in the control glands. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was increased in both carbachol-stimulated and epiphora glands. Preincubation of submandibular glands with ERK1/2 inhibitors PD98059 or U0126 inhibited carbachol-induced F-actin redistribution. These results indicated that differentially expressed proteins participated in the hypersecretion of transplanted submandibular glands and the redistribution of F-actin might be involved in this hypersecretion in an ERK1/2-dependent manner.     

VIP and noncholinergic vasodilatation in rabbit submandibular gland

The effect of parasympathetic nerve activation on rabbit submandibular gland (SMG) blood flow and saliva secretion were studied before and after systemic administration of atropine or hexamethonium. The parasympathetic fibers were stimulated electrically (2 and 15 Hz, 10 V, 1 msec) at the plexus around the submandibular salivary duct or at the chorda lingual nerve. In untreated animals, stimulation of parasympathetic fibers caused a frequency-dependent increase of salivary secretion and blood flow in the SMG. Atropine treatment completely abolished saliva secretion at 2 Hz and 15 Hz and the increase in SMG blood flow during stimulation at 2 Hz. Although atropine significantly reduced the vasodilatory response at 15 Hz, the highest blood flow measured under such circumstances was still about 2.5 times the prestimulation value. After hexamethonium administration no blood flow increase or saliva secretion was seen upon chorda lingual stimulation. The concentration of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the venous effluent of the SMG increased during nerve stimulation. Atropine significantly reduced, and hexamethonium abolished this VIP-output elicited by parasympathetic nerve stimulation. Local infusion of VIP, peptide histidine isoleucine (PHI) and substance P all caused atropine-resistant vasodilation but no salivation. The present data suggest that VIP and possibly PHI play a role in the atropine-resistant vasodilatation in rabbit submandibular gland elicited by parasympathetic nerve stimulation. The contribution of sensory mediators such as substance P released by stimulation of afferent nerves in the chorda lingual nerve to the salivary and vasodilatory responses seems to be of minor importance in the rabbit submandibular gland.     

The Reversible Increase in Tight Junction Permeability Induced by Capsaicin Is Mediated via Cofilin-Actin Cytoskeletal Dynamics and Decreased Level of Occludin

Previous results demonstrated that capsaicin induces the reversible tight junctions (TJ) opening via cofilin activation. The present study investigated the mechanisms underlying the reversible TJ opening and compared the effect to the irreversible opening induced by actin inhibitors. Capsaicin treatment induced the F-actin alteration unique to capsaicin compared to actin-interacting agents such as latrunculin A, which opens TJ irreversibly. Along with TJ opening, capsaicin decreased the level of F-actin at bicellular junctions but increased it at tricellular junctions accompanied with its concentration on the apical side of the lateral membrane. No change in TJ protein localization was observed upon exposure to capsaicin, but the amount of occludin was decreased significantly. In addition, cosedimentation analyses suggested a decrease in the interactions forming TJ, thereby weakening TJ tightness. Introduction of cofilin, LIMK and occludin into the cell monolayers confirmed their contribution to the transepithelial electrical resistance decrease. Finally, exposure of monolayers to capsaicin augmented the paracellular passage of both charged and uncharged compounds, as well as of insulin, indicating that capsaicin can be employed to modulate epithelial permeability. Our results demonstrate that capsaicin induces TJ opening through a unique mechanism, and suggest that it is a new type of paracellular permeability enhancer.     

Phenylephrine protects autotransplanted rabbit submandibular gland from apoptosis

Submandibular gland (SMG) autotransplantation is an effective treatment for severe keratoconjunctivitis sicca. Our previous studies have shown that phenylephrine attenuates structural injury and promotes cell proliferation in autotransplanted rabbit SMG. However, the mechanism by which phenylephrine reduces the injury has not been fully evaluated. In this study, we investigate the ability of phenylephrine to inhibit apoptosis in autotransplanted rabbit SMG. We observed that apoptosis occurred in the early phase of SMG transplantation and that phenylephrine treatment protected transplanted SMG from apoptosis. Furthermore, we found that phenylephrine could significantly upregulate the expression of Bcl-2, downregulate the expression of Bax, and inhibit the activation of both caspase-3 and p38 mitogen-activated protein kinase in autotransplanted SMG. Therefore, the cytoprotective effects of phenylephrine on autotransplanted SMG may be a novel clinical strategy for autotransplanted SMG protection during the early postoperative stage of transplantation.     

Regulation of the MDCK Cell Tight Junction

The sodium flux across individual tight junctions (TJ) of low-resistance MDCK cell monolayers grown on glass coverslips was determined as a measure of paracellular permeability. Increases in perfusate glucose concentration from 5 to 25 mm decreased tight junction Na permeability. This permeability decrease was not specific as nonmetabolizable analogues of glucose caused similar diminutions in TJ Na permeability. Stimulation of protein kinase A increased TJ Na permeability, and inhibition of protein kinase A decreased TJ Na permeability. Transepithelial electrical resistance of monolayers grown on permeable supports did not change as predicted from the observed alterations in TJ Na permeability of monolayers grown on glass coverslips. Fluorescent labeling of cell F-actin showed that increased F-actin in the perijunctional ring correlated with higher TJ Na permeability. Although a low dose of cytochalasin D did not change TJ Na permeability, it disrupted the cytoskeleton and blocked the decrease in TJ Na permeability caused by glucose. Cytochalasin D failed to block the effects of protein kinase A stimulation or inhibition on TJ Na permeability. We conclude that tight junction sodium permeability is regulated both by protein kinase A activity and by other processes involving the actin cytoskeleton. Received: 17 June 1997/Revised: 28 August 1997     

Agonist mediated internalization of M2 mAChR is β-arrestin-dependent

Background

Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is β-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of β-arrestins have reported that agonist-promoted internalization of M₂ mAChRs is a β-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous β-arrestin that may have existed in these earlier studies, we examined agonist-promoted internalization of the M₂ mAChR in mouse embryonic fibroblasts (MEFs) derived from β-arrestin knockout mice that lack expression of either one or both isoforms of β-arrestin (β-arrestin 1 and 2).

Results

In wild type MEF cells transiently expressing M₂ mAChRs, 40% of surface M₂ mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M₂ mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF β-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either β-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M₂ mAChRs led to a stable co-localization with GFP-tagged β-arrestin within endocytic structures in multiple cell lines; the compartment to which β-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M₂ mAChRs was moderately rescued in MEF β-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (β-arrestin 2 ΔLIELD), AP-2 (β-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of β-arrestin 1 (319–418) completely abrogated agonist-promoted internalization of M₂ mAChRs in wild type MEF cells.

Conclusion

In summary, this study demonstrates that agonist-promoted internalization of M₂ mAChRs is β-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with β-arrestin in early endosomal vesicles.     

Expression and characteristics of vanilloid receptor 1 in the rabbit submandibular gland

Vanilloid receptor 1 (VR1) is a polymodal receptor originally found in sensory neurons of the central nervous system. Recent evidence indicates that VR1 is also expressed in non-neuronal tissues. We report here endogenous expression of VR1 in rabbit submandibular gland (SMG) and its possible role in regulating saliva secretion based on: (i) the expression of VR1 mRNA and protein detected in SMG; (ii) VR1 was mainly localized in the basolateral membrane of duct cells and the cytoplasm of acinar cells and also in cytoplasm of primary cultured neonatal rabbit SMG cells; (iii) stimulation of neonatal rabbit SMG cells with capsaicin induced a significant increase in intracellular calcium, and capsazepine, a VR1 antagonist, abolished this increase; (iv) infusion of capsaicin via the external carotid artery to isolated SMG increased saliva secretion of the gland. These findings indicated that VR1 was expressed in SMG and appeared to play an important role in regulating saliva secretion.     

Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex

Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABalphaC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function.     

Rotavirus-induced structural and functional alterations in tight junctions of polarized intestinal Caco-2 cell monolayers

We provide here new insights into rotavirus (RRV) pathogenicity by showing that RRV infection promotes structural and functional injuries localized at the tight junctions (TJ) in the cell-cell junctional complex of cultured polarized human intestinal Caco-2 cells forming monolayers. RRV infection resulted in a progressive increase in the paracellular permeability to [(3)H]mannitol as a function of the time postinfection. We observed a disorganization of the TJ-associated protein occludin as a function of the time postinfection, whereas distribution of the zonula adherens associated E-cadherin was not affected. These structural and functional RRV-induced TJ injuries were not accompanied by alteration in cell and monolayer integrity, as assessed by the lack of change in transepithelial membrane resistance and lactate dehydrogenase release. Finally, using the stabilizer of actin filaments Jasplakinolide, we demonstrated that the RRV-induced structural and functional alterations in TJ are independent of the RRV-induced apical F-actin rearrangements.     

Partitioning of paracellular conductance along the ileal crypt-villus axis: A hypothesis based on structural analysis with detailed consideration of tight junction structure-function relationships

Summary Current models of intestinal transport suggest cells which absorb ions are located on the villus while secretory cells are located in the crypt and putatively have paracellular pathways which are highly conductive to Na⁺. One approach to assess possible variation in small intestinal paracellular conductance along the crypt-villus axis is to morphometrically analyze the structural aspects of crypt and villus tight junctions (TJs) which relate to paracellular resistance. Such detailed analysis of junctional structure in this heterogeneous epithelium would permit one to compare intestinal TJ structure-function relationships with those in a structurally simpler epithelium such as that of toad urinary bladder. This comparison would also be of considerable interest since previous similar comparisons have failed to consider in detail the geometric dissimilarity between these two epithelia. We applied light, electron microscopic, and freezefracture morphometric techniques to guinea pig ileal mucosa to quantitatively assess, for both crypts and villi, linear TJ density, relative surface contributions, and TJ strand counts. Mean linear TJ densities were 76.8 m/cm² for crypt cells and 21.8 m/cm² for villus absorptive cells. Mean TJ strand counts were 4.45 for undifferentiated crypt cell TJs and 6.03 for villus absorptive cell TJs. The villus constituted 87% and the crypt 13% of total surface. We utilized these data to predict paracellular conductance of cryptsvs. villi based on equations derived from those of Claude (P. Claude,J. Membrane Biol. 39:219–232, 1978). Such analysis predicts that 73% of ileal paracellular conductance is attributable to the crypt. Furthermore, we obtained literature values for paracellular resistance in mammalian ileum and toad urinary bladder and for toad bladder TJ structure and linear density and constructed a relationship which would allow us to more accurately compare TJ structure-function correlates between these two epithelia. Such a comparison, which considers both surface amplification and TJ structure and distribution in these epithelia, shows that one would predictin vitro measured values for paracellular resistance should be approximately two orders of magnitude less in mammalian ileum than in toad urinary bladder. This predicted discrepancy (115-fold) correlates well with the observed difference (100-fold). These findings suggest that highly similar TJ structure-function relationships apply to these geometrically dissimilar tissues and that, in mammalian ileum, the crypt compartment may be responsible for the majority of net ileal paracellular conductance. We speculate that high crypt linear TJ density and low crypt TJ strand counts may serve as the structural basis of massive paracellular Na⁺ movement which is coupled to active Cl^– secretion and appears to originate from the crypt following exposure to intestinal secretagogues.     

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.     

Dual mode of glucagon receptor internalization: role of PKCα, GRKs and β-arrestins

Glucagon levels are elevated in diabetes and some liver diseases. Increased glucagon secretion leads to abnormal stimulation of glucagon receptors (GRs) and consequent elevated glucose production in the liver. Blocking glucagon receptor signaling has been proposed as a potential treatment option for diabetes and other conditions associated with hyperglycemia. Elucidating mechanisms of GR desensitization and downregulation may help identify new drug targets besides GR itself. The present study explores the mechanisms of GR internalization and the role of PKCα, GPCR kinases (GRKs) and β-arrestins therein. We have reported previously that PKCα mediates GR phosphorylation and desensitization. While the PKC agonist, PMA, did not affect GR internalization when tested alone, it increased glucagon-mediated GR internalization by 25–40% in GR-expressing HEK-293 cells (HEK-GR cells). In both primary hepatocytes and HEK-GR cells, glucagon treatment recruited PKCα to the plasma membrane where it colocalized with GR. We also observed that overexpression of GRK2, GRK3, or GRK5 enhanced GR internalization. In addition, we found that GR utilizes both clathrin- and caveolin-mediated endocytosis in HEK-GR cells. Glucagon triggered translocation of both β-arrestin1 and β-arrestin2 from the cytosol to the perimembrane region, and overexpression of β-arrestin1 and β-arrestin2 increased GR internalization. Furthermore, both β-arrestin1 and β-arrestin2 colocalized with GR and with Cav-1, suggesting the possible involvement of these arrestins in GR internalization.     

Mechanism of IFN-gamma-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane

Disruption of epithelial barrier by proinflammatory cytokines such as IFN-gamma represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN-gamma increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-gamma-induced endocytosis of epithelial TJ proteins. IFN-gamma treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-gamma dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-gamma exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-gamma treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-gamma induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs.     

Larazotide acetate promotes tight junction assembly in epithelial cells

Tight junctions (TJ) control paracellular permeability and apical-basolateral polarity of epithelial cells. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. TJ formation is dependent on E-cadherin-mediated cell-cell adhesion and actin rearrangement, and is regulated by the Rho family GTPase and aPKC signaling pathways. Larazotide acetate, an 8-mer peptide and TJ modulator, inhibits TJ disassembly and dysfunction caused by endogenous and exogenous stimuli in intestinal epithelial cells. Here, we examined the effect of larazotide acetate on de novo TJ assembly using 2 different model systems. In MDCK cells, larazotide acetate promoted TJ assembly in a calcium switch assay. Larazotide acetate also promoted actin rearrangement, and junctional distribution of zonula occludens-1 (ZO-1), occludin, claudins, and E-cadherin. Larazotide acetate promoted TJ maturation and decreased paracellular permeability in "leaky" Caco-2 cells. Taken together, our data indicate that larazotide acetate enhances TJ assembly and barrier function by promoting actin rearrangement and redistribution of TJ and AJ proteins.     

β-Arrestin prevents cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt pathways

Our previous studies have shown that β-arrestin 2 plays an anti-apoptotic effect. However, the mechanisms by which β-arrestin contribute to anti-apoptotic role remain unclear. In this study, we show that a deficiency of either β-arrestin 1 or β-arrestin 2 significantly increases serum deprivation (SD)-induced percentage of apoptotic cells. β-arrestin 2 deficient-induced apoptosis was inhibited by transfection with β-arrestin 2 full-length plasmid, revealing that SD-induced apoptosis is dependent on β-arrestin 2. Furthermore, in the absence of either β-arrestin 1 or β-arrestin 2 significantly enhances SD-induced the level of pro-apoptotic proteins, including cleaved caspase-3, extracellular-signal regulated kinase 1/2 (ERK1/2) and p38, members of mitogen-activated protein kinases (MAPKs). In addition, a deficiency of either β-arrestin 1 or β-arrestin 2 inhibits phosphorylation of Akt. The SD-induced changes in cleaved caspase-3, ERK1/2 and p38 MAPKs, Akt, and apoptotic cell numbers could be blocked by double knockout of β-arrestin 1/2. Our study thus demonstrates that β-arrestin inhibits cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt signaling pathways.     

Protamine-induced epithelial barrier disruption involves rearrangement of cytoskeleton and decreased tight junction-associated protein expression in cultured MDCK strains

Natural and synthetic polycationic proteins, such as protamine, have been used to reproduce the tissue injury and changes in epithelial permeability caused by positively charged substances released by polymorphonuclear cells during inflammation. Protamine has diverse and often conflicting effects on epithelial permeability. The effects of this polycation on the distribution and expression of tight junction (TJ)-associated proteins have not yet been investigated. In this work, we examined the influence of protamine on paracellular barrier function and TJ structure using two strains of the epithelial Madin-Darby canine kidney (MDCK) cell line that differed in their TJ properties ("tight" TJ-strain I and "leaky" TJ-strain II). Protamine induced concentration-, time- and strain-dependent alterations in transepithelial electrical resistance (Rt) only when applied to apical or apical+basolateral monolayer surfaces, indicating a polarity of action. In MDCK II cells, protamine (50 microg/ml) caused a significant increase in Rt that returned to control values after 2 h. However, the treatment of this MDCK strain with a higher concentration of protamine (250 microg/ml) significantly decreased the Rt after 30 min. In contrast, treated MDCK I monolayers showed a significant decrease in Rt after apical treatment with protamine at both concentrations. The protamine-induced decrease in Rt was paralleled by an increase in the phenol red basal-to-apical flux in both MDCK strains, suggesting disruption of the paracellular barrier. Marked changes in cytoskeletal F-actin distribution/polymerization and a significant reduction in the junctional expression of the tight junctional proteins occludin and claudin-1 but subtle alterations in ZO-1 were observed following protamine-elicited paracellular barrier disruption. In conclusion, protamine induces alterations in the epithelial barrier function of MDCK monolayers that may involve the cytoskeleton and TJ-associated proteins. The various actions of protamine on epithelial function may reflect different degrees of interaction of protamine with the plasma membrane and different intracellular processes triggered by this polycation.