Regulation Of Tight Junction Proteins Research Articles

Exogenous GDF9 but not Activin A, BMP15 or TGFβ alters tight junction protein transcript abundance in zebrafish ovarian follicles

The tight junction (TJ) complex plays an important role in regulating paracellular permeability and provides mechanical stability in vertebrate epithelia and endothelia. In zebrafish ovarian follicles, TJ complexes in the follicular envelope degenerate as the follicles develop towards maturation. In the current study, transcript abundance of claudins ( cldn d, g, h, 1, and 12) and occludins ( ocln, and ocln b) were assessed in mid-vitellogenic follicles in response to treatment with exogenous growth factors that are reported to be involved in zebrafish follicle development (i.e. Activin A, BMP15, GDF9 and TGFβ). Exogenous GDF9 reduced the transcript abundance of cldn g, ocln and ocln b in mid-vitellogenic follicles, whereas Activin A, BMP15, and TGFβ had no effect. Subsequent studies with GDF9 revealed that this factor did not alter TJ protein transcript abundance in pre-vitellogenic follicles but did increase the abundance of ocln b in fully grown (maturing) follicles. GDF9 was also seen to increase the abundance of StAR mRNA in all but primary stage follicles. These data suggest a role for GDF9 in the regulation of TJ integrity in zebrafish ovarian follicles, perhaps in the facilitation of ovulation, and support a previously postulated role for GDF9 in zebrafish ovarian follicle development. In addition, data also support the idea that endocrine factors play an important role in the regulation of TJ proteins during ovarian follicle development.

Up-regulation of caveolin-1 and blood–brain barrier breakdown are attenuated by N-acetylcysteine in thiamine deficiency

Wernicke's encephalopathy is a cerebral metabolic disorder caused by thiamine (vitamin B1) deficiency (TD). Neuropathologic consequences of TD include region-selective neuronal cell loss and blood–brain barrier (BBB) breakdown. Caveolin-1 is involved in the regulation of tight junction proteins and BBB permeability, and is modulated by oxidative stress, a feature of vulnerable brain regions in TD. We hypothesized that TD-related oxidative stress alters BBB integrity via induction of the caveolin-1 pathway. TD was induced in C57BL6 mice by treatment with a thiamine-deficient diet and administration of the thiamine antagonist pyrithiamine, in the absence or presence of the antioxidant N-acetylcysteine (NAC). A significant and focal increase in both caveolin-1 gene and protein expression was detected in the thalamus of thiamine-deficient mice, concomitant with IgG extravasation. Reduction of oxidative stress by NAC, as shown by normalization of reduced glutathione levels and attenuation of endothelial heme oxygenase-1 and nitric oxide synthase expression, resulted in prevention of the up-regulation of caveolin-1 in TD. Normalization of caveolin-1 levels by NAC was accompanied by a reduction in BBB breakdown, indicated by decreased IgG extravasation, normalization of occludin levels and prevention of matrix metalloproteinase-9 up-regulation. These findings demonstrate a role for caveolin-1 in TD pathogenesis, and suggest that oxidative stress contributes to BBB alterations in TD via modulation of this pathway.

Reduction of endothelial tight junction proteins is related to cerebral aneurysm formation in rats

The formation of cerebral aneurysms is associated with endothelial damage and macrophage migration. Hypothesizing that the opening of tight junctions due to the disappearance of the tight junction proteins occludin and zona occludens-1 (ZO-1) in damaged endothelia allows macrophage migration, leading to cerebral aneurysm formation, we investigated the role of tight junction proteins. The vascular wall of female rats subjected to hypertension, oophorectomy (OVX), and hemodynamic stress to induce cerebral aneurysms was evaluated morphologically, immunohistochemically, and by quantitative RT-PCR. We also assessed the regulation of tight junction proteins in human brain endothelial cells (HBECs). In the very early stage before aneurysm formation, the expression of occludin and ZO-1 was reduced in injured endothelial cell junctions exhibiting gaps. In the course of aneurysmal progression their reduction progressed and was correlated with macrophage migration. In hypertension along with OVX rats we observed an increase in angiotensin II and the degradation molecules matrix metalloproteinase-9 (MMP-9), nicotinamide-adenine dinucleotide phosphate oxidases and monocyte chemoattractant protein-1. The mineralocorticoid receptor blocker eplerenone increased occludin and ZO-1 expression; this was associated with a reduction in angiotensin II and the degradation molecules and resulted in the inhibition of macrophage exudation and aneurysm formation. In HBECs, occludin and ZO-1 downregulation by angiotensin II and estrogen deficiency was reversed by eplerenone, the MMP inhibitor SB3CT, and apocynin. Our results suggest that macrophage migration is associated with the reduction in tight junction proteins induced by the degradation molecules. In rats, the destruction of tight junctions may facilitate macrophage migration and cerebral-aneurysm formation.

Effect of inactivated schistosome ova on intestinal epithelial tight junctions during tinitrobenzene sulfonic acid-induced colitis

Objective To observe the possible effect of inactivated schistosome ova on the expression of intestinal epithelial tight junctions ZO-1 and Occludin gene in mouse colitis induced by tinitrobenzene sulfonic acid (TNBS) and its mechanism. Methods Fifty mice were divided into control group (group A, n= 10),TNBS plus normal saline(NS) group(group B, n= 20) and TNBS plus inactivated schistosome ova group(group C, n= 20). Group C was exposed to 10 000 freeze-killed schistosome ova by intraperitoneal injection at day 14 and day 3 before colitis induction. Meanwhile,group B was exposed to 1 ml NS by intraperitoneal injection. The mice in group B and C were challenged with 3 mg TNBS to induce colitis. All mice were killed 7-day after colitis induction and assessed with following variables including mortality, pathological change with HE staining of colon.The transcription levels of ZO-1 and Oceludin in colon tissues were examined using Real-time PCR.The expression and distribution of ZO-1 and Occludin proteins were detected by Western blotting and immunohistochemistry. Results In comparison with group B inactivated schistosome ova most effectively reduced the mortality (30% vs 15 %) and histopathologic severity of TNBS-induced colitis (4.21±0.40 vs 1.74±0.10). The transcription levels of ZO-1 and Occludin in group B were decreased compared with those in group A and group C (P<0.01). When compared with group B,group C showed a significant elevation of the alteration of ZO-1, Occludin proteins expression and localization. Conclusion The results clearly show that schistosome ova treatment reduced the severity of experimental colitis through the regulation of tight junction proteins. Key words: Inflammatory bowel diseases; Schistosorna; Tight junctions protein; Modles, animal

Nitric oxide and airway epithelial barrier function: Regulation of tight junction proteins and epithelial permeability

Acute airway inflammation is associated with enhanced production of nitric oxide (NO ) and altered airway epithelial barrier function, suggesting a role of NO or its metabolites in epithelial permeability. While high concentrations of S-nitrosothiols disrupted transepithelial resistance (TER) and increased permeability in 16HBE14o− cells, no significant barrier disruption was observed by NONOates, in spite of altered distribution and expression of some TJ proteins. Barrier disruption of mouse tracheal epithelial (MTE) cell monolayers in response to inflammatory cytokines was independent of NOS2, based on similar effects in MTE cells from NOS2−/− mice and a lack of effect of the NOS2-inhibitor 1400W. Cell pre-incubation with LPS protected MTE cells from TER loss and increased permeability by H 2O 2, which was independent of NOS2. However, NOS2 was found to contribute to epithelial wound repair and TER recovery after mechanical injury. Overall, our results demonstrate that epithelial NOS2 is not responsible for epithelial barrier dysfunction during inflammation, but may contribute to restoration of epithelial integrity.

I-42 Regulation of a hepatitis C virus co-receptor claudin-1 in hepatocytes

The polarization of hepatocytes involves formation of functionally distinct sinusoidal (basolateral) and bile canalicular (apical) plasma membrane domains that are separated by tight junctions. In murine livers, integral tight junction proteins claudin-1, -2, -3, -5, -7, -8, -12, -14 which can form tight junction strands are detected together with occludin, JAM, CAR and tricellulin, and claudin-1, -2, -3 are expressed in the bile canaliculus region of hepatocytes. In the livers (mouse, rat, human), claudin-1 and -3 are expressed in the whole liver lobule, whereas claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes. It is reported that downregulation of claudin-1 serves as a potential marker for a poor prognosis in HCC. More recently, it is found that the claudin-1 is a hepatitis C virus co-receptor with CD81. Thus, the study of regulation of claudin-1 in hepatocytes is very important for analysis of pathogenesis of hepatitis C virus infection. We have been studied for the regulation of tight junction proteins including claudin-1 in hepatocytes in vivo and in vitro. In this lecture, we present the regulation in expression and function of hepatic claudin-1 by growth factors (EGF, TGF-beta), cytokines (IL-1beta, oncostatin M) and partial hepatectomy via distinct signal transduction pathways.

Differentiation Associated Changes in Gene Expression Profiles of Interstitial Cystitis and Control Urothelial Cells

We evaluated gene expression profiles after inducing differentiation in cultured interstitial cystitis and control urothelial cells. Bladder biopsies were taken from patients with interstitial cystitis and controls, that is women undergoing surgery for stress incontinence. Primary cultures were grown in keratinocyte growth medium with supplements. To induce differentiation in some plates the medium was changed to Dulbecco's modified Eagle's minimal essential medium-F12 (Media Tech, Herndon, Virginia) with supplements. RNA was analyzed with Affymetrix(R) chips. Three patients with nonulcerative interstitial cystitis were compared with 3 controls. After inducing differentiation 302 genes with a described function were altered at least 3-fold in interstitial cystitis and control cells (p <0.01). Functions of the 162 up-regulated genes included cell adhesion (eg claudins, occludin and cingulin), urothelial differentiation, the retinoic acid pathway and keratinocyte differentiation (eg skin cornified envelope components). The 140 down-regulated transcripts included genes associated with basal urothelium (eg p63, integrins beta4, alpha5 and alpha6, basonuclin 1 and extracellular matrix components), vimentin, metallothioneins, and members of the Wnt and Notch pathways. When comparing interstitial cystitis control cells after differentiation, only 7 genes with a described function were altered at least 3-fold (p <0.01). PI3, SERPINB4, CYP2C8, EFEMP2 and SEPP1 were decreased, and AKR1C2 and MKNK1 were increased in interstitial cystitis cases. Differentiation associated changes occurred in interstitial cystitis and control cells. Comparing interstitial cystitis vs control cases revealed few differences. This study may have included patients with interstitial cystitis and minimal urothelial deficiency, and/or we may have selected cells that were most robust in culture. Also, the abnormal urothelium in interstitial cystitis cases may be due to post-translational changes and/or to the bladder environment.

Regulation of testicular tight junctions by gonadotrophins in the adult Djungarian hamster in vivo

This study aimed to assess the effect of gonadotrophin suppression and FSH replacement on testicular tight junction dynamics and blood-testis barrier (BTB) organisation in vivo, utilising the seasonal breeding Djungarian hamster. Confocal immunohistology was used to assess the cellular organisation of tight junction proteins and real-time PCR to quantify tight junction mRNA. The effect of tight junction protein organisation on the BTB permeability was also investigated using a biotin-linked tracer. Tight junction protein (claudin-3, junctional adhesion molecule (JAM)-A and occludin) localisation was present but disorganised after gonadotrophin suppression, while mRNA levels (claudin-11, claudin-3 and occludin) were significantly (two- to threefold) increased. By contrast, both protein localisation and mRNA levels for the adaptor protein zona occludens-1 decreased after gonadotrophin suppression. FSH replacement induced a rapid reorganisation of tight junction protein localisation. The functionality of the BTB (as inferred by biotin tracer permeation) was found to be strongly associated with the organisation and localisation of claudin-11. Surprisingly, JAM-A was also recognised on spermatogonia, suggesting an additional novel role for this protein in trans-epithelial migration of germ cells across the BTB. It is concluded that gonadotrophin regulation of tight junction proteins forming the BTB occurs primarily at the level of protein organisation and not gene transcription in this species, and that immunolocalisation of the organised tight junction protein claudin-11 correlates with BTB functionality.

Juxtacrine activation of EGFR regulates claudin expression and increases transepithelial resistance

Heparin-binding (HB)-EGF, a ligand for EGF receptors, is synthesized as a membrane-anchored precursor that is potentially capable of juxtacrine activation of EGF receptors. However, the physiological importance of such juxtacrine signaling remains poorly described, due to frequent inability to distinguish effects mediated by membrane-anchored HB-EGF vs. mature "secreted HB-EGF." In our studies, using stable expression of a noncleavable, membrane-anchored rat HB-EGF isoform (MDCK(rat5aa) cells) in Madin-Darby canine kidney (MDCK) II cells, we observed a significant increase in transepithelial resistance (TER). Similar significant increases in TER were observed on stable expression of an analogous, noncleavable, membrane-anchored human HB-EGF construct (MDCK(human5aa) cells). The presence of noncleavable, membrane-anchored HB-EGF led to alterations in the expression of selected claudin family members, including a marked decrease in claudin-2 in MDCK(rat5aa) cells compared with the control MDCK cells. Reexpression of claudin-2 in MDCK(rat5aa) cells largely prevented the increases in TER. Ion substitution studies indicated decreased paracellular ionic permeability of Na(+) in MDCK(rat5aa) cells, further indicating that the altered claudin-2 expression mediated the increased TER seen in these cells. In a Ca(2+)-switch model, increased phosphorylation of EGF receptor and Akt was observed in MDCK(rat5aa) cells compared with the control MDCK cells, and inhibition of these pathways inhibited TER changes specifically in MDCK(rat5aa) cells. Therefore, we hypothesize that juxtacrine activation of EGFR by membrane-anchored HB-EGF may play an important role in the regulation of tight junction proteins and TER.

When the levee breaks: The etiologic role of epithelial barrier function defects in ileitis

When the levee breaks: The etiologic role of epithelial barrier function defects in ileitis

Bryostatin-1 enhances barrier function in T84 epithelia through PKC-dependent regulation of tight junction proteins.

Protein kinase C (PKC) is known to regulate epithelial barrier function. However, the effect of specific PKC isozymes, and their mechanism of action, are largely unknown. We determined that the nonphorbol ester PKC agonist bryostatin-1 increased transepithelial electrical resistance (TER), a marker of barrier function, in confluent T84 epithelia. Bryostatin-1, which has been shown to selectively activate PKC-alpha, -epsilon, and -delta (34), was associated with a shift in the subcellular distribution of the tight junction proteins claudin-1 and ZO-2 from a detergent-soluble fraction into a detergent-insoluble fraction. Bryostatin-1 also led to the appearance of a higher-molecular-weight form of occludin previously shown to correspond to protein phosphorylation. These changes were attenuated by the conventional and novel PKC inhibitor Gö-6850 but not the conventional PKC inhibitor Gö-6976 or the PKC-delta inhibitor röttlerin, implicating a novel isozyme, likely PKC-epsilon. The results suggest that enhanced epithelial barrier function induced by bryostatin-1 involves a PKC-epsilon-dependent signaling pathway leading to recruitment of claudin-1 and ZO-2, and phosphorylation of occludin, into the tight junctional complex.

BLADDER EPITHELIAL CELLS FROM PATIENTS WITH INTERSTITIAL CYSTITIS PRODUCE AN INHIBITOR OF HEPARIN-BINDING EPIDERMAL GROWTH FACTOR-LIKE GROWTH FACTOR PRODUCTION

The etiology of interstitial cystitis is unknown. We previously identified an interstitial cystitis urine factor, antiproliferative factor, that inhibits proliferation of bladder epithelial cells in vitro and complex changes in epithelial growth factor levels, including profound decreases in heparin-binding epidermal growth factor-like growth factor (HB-EGF). Bladder and renal pelvic catheterization of patients with interstitial cystitis indicated that the antiproliferative factor is made and/or activated in the distal ureter or bladder. Therefore, we determined whether bladder epithelial cells from interstitial cystitis cases produced the antiproliferative factor and whether purified antiproliferative factor could alter production of growth factors known to be abnormal in interstitial cystitis. Antiproliferative factor activity was determined by 3H-thymidine incorporation into primary bladder epithelial cells. The antiproliferative factor was purified by size fractionation followed by sequential chromatography involving ion exchange, hydrophobic interaction and high performance liquid chromatography. HB-EGF, epidermal growth factor, insulin-like growth factor and insulin-like growth factor binding protein 3 levels were determined by enzyme-linked immunosorbent assay. Bladder epithelial cells from patients with interstitial cystitis produced a single antiproliferative factor with the same purification profile as that purified from interstitial cystitis urine. Purified antiproliferative factor specifically inhibited HB-EGF production by bladder epithelial cells in vitro, and the effect of interstitial cystitis urine or purified antiproliferative factor on bladder cell proliferation was inhibited by recombinant human HB-EGF in a dose dependent manner. Similar to urine HB-EGF, serum HB-EGF was also significantly lower in interstitial cystitis cases than in controls. Bladder epithelial abnormalities in interstitial cystitis may be caused by a negative autocrine growth factor that inhibits cell proliferation by down-regulating HB-EGF production. Furthermore, decreased levels of urine and serum HB-EGF indicate that interstitial cystitis may be a urinary tract manifestation of a systemic disorder.

Regulation of tight junction proteins in human subjects with inflammatory bowel disease

Regulation of tight junction proteins in human subjects with inflammatory bowel disease