Dynamic changes in the cervical epithelial tight junction complex and differentiation occur during cervical ripening and parturition

Cervical epithelia have numerous functions that include proliferation, differentiation, maintenance of fluid balance, protection from environmental hazards, and paracellular transport of solutes via tight junctions (TJs). Epithelial functions must be tightly regulated during pregnancy and parturition as the cervix undergoes extensive growth and remodeling. This study evaluated TJ proteins, as well as markers of epithelial cell differentiation in normal and cervical ripening defective mice to gain insights into how the permeability barrier is regulated during pregnancy and parturition. Although numerous TJ proteins are expressed in the nonpregnant cervix, claudins 1 and 2 are temporally regulated in pregnancy. Claudin 1 mRNA expression is increased, whereas claudin 2 expression declines. The cellular localization of claudin 1 shifts at the end of pregnancy (gestation d 18.75) to the plasma membrane in a lattice pattern, consistent with TJs in the apical cells. The timing of claudin 1-enriched TJs coincides with initiation of terminal differentiation of cervical squamous epithelia as evidenced by the increased expression of genes by differentiated epithelia late on gestation d 18. The cervical ripening defective steroid 5alpha-reductase type 1 deficient mouse, which has an elevated local progesterone concentration, also has aberrant claudin 1 and 2 expressions, fails to form claudin 1-enriched TJs, and lacks normal expression of genes involved in epithelial terminal differentiation. These data suggest that changes in permeability barrier properties during cervical ripening are, in part, negatively regulated by progesterone, and that dynamic changes in barrier properties of the cervix occur during pregnancy and parturition.     

Expression and phylogeny of claudins in vertebrate primordia

Claudins, the major transmembrane proteins of tight junctions, are members of the tetraspanin superfamily of proteins that mediate cellular adhesion and migration. Their functional importance is demonstrated by mutations in claudin genes that eliminate tight junctions in myelin and the testis, abolish Mg(2+) resorption in the kidney, and cause autosomal recessive deafness. Here we report that two paralogs among 15 claudin genes in the zebrafish, Danio rerio, are expressed in the otic and lateral-line placodes at their earliest stages of development. Related claudins in amphibians and mammals are expressed in a similar manner in vertebrate primordia such as sensory placodes, branchial arches, and limb buds. We also show that the claudin gene family may have expanded along the chordate stem lineage from urochordates to gnathostomes, in parallel with the elaboration of vertebrate characters. We propose that tight junctions not only form barriers in mature epithelia, but also participate in vertebrate morphogenesis.     

Changes in expression and distribution of claudin 2, 5 and 8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn's disease

BACKGROUND: Epithelial barrier function is impaired in Crohn's disease. AIM: To define the underlying cellular mechanisms with special attention to tight junctions. METHODS: Biopsy specimens from the sigmoid colon of patients with mild to moderately active or inactive Crohn's disease were studied in Ussing chambers, and barrier function was determined by impedance analysis and conductance scanning. Tight junction structure was analysed by freeze fracture electron microscopy, and tight junction proteins were investigated immunohistochemically by confocal laser scanning microscopy and quantified in immunoblots. Epithelial apoptosis was analysed in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and 4',6-diamidino-2-phenylindole staining. RESULTS: Patients with active Crohn's disease showed an impaired intestinal barrier function as indicated by a distinct reduction in epithelial resistance. As distribution of conductivity was even, focal epithelial lesions (eg, microerosions) did not contribute to barrier dysfunction. Instead, freeze fracture electron microscopy analysis showed reduced and discontinuous tight junction strands. Occludin and the sealing tight junction proteins claudin 5 and claudin 8 were downregulated and redistributed off the tight junction, whereas the pore-forming tight junctions protein claudin 2 was strongly upregulated, which constitute the molecular basis of tight junction changes. Other claudins were unchanged (claudins 1, 4 and 7) or not detectable in sigmoid colon (claudins 11, 12, 14, 15 and 16). Claudin 2 upregulation was less pronounced in active Crohn's disease compared with active ulcerative colitis and was inducible by tumour necrosis factor alpha. As a second source of impaired barrier function, epithelial apoptosis was distinctly increased in active Crohn's disease (mean (SD) 5.2 (0.5)% v 1.9 (0.2)% in control). By contrast, barrier function, tight junction proteins and apoptosis were unaffected in Crohn's disease in remission. CONCLUSION: Upregulation of pore-forming claudin 2 and downregulation and redistribution of sealing claudins 5 and 8 lead to altered tight junction structure and pronounced barrier dysfunction already in mild to moderately active Crohn's disease.     

紧密连接蛋白claudins和胃癌

Claudins是构成上皮细胞和血管内皮细胞紧密连接的一类跨膜蛋白家族,维持紧密连接特有的栅栏和屏障功能.Claudins广泛分布于正常组织和不同肿瘤组织,且存在差异性的表达.近年,在胃癌前病变和胃癌中也发现几种claudins蛋白表达异常,并与预后有关.通过对claudins在胃癌中异常分布和表达的研究,人们能更好得理解胃癌的发生发展机制,从而有利于胃癌的诊断和治疗.     

The role of claudins in determining paracellular charge selectivity

Tight junctions create regulated barriers in the paracellular space between epithelial cells, including those of the airway and alveolus. Junctions vary widely throughout the body in their electrical resistance and, to some extent, in their ionic charge selectivity. Paracellular differences complement transcellular transport to define overall water, ion, and solute movements. A large family of transmembrane proteins called claudins has recently been implicated in creating the variable properties of the junction. Here we highlight the evidence that claudins are functional components of the barrier. Supportive data include evidence from deletion of selected claudins in mice, human genetic diseases of claudins 14 and 16, and direct experimental tests of the hypothesis that the extracellular charged residues on claudins influence the passage of ions through the junction. Alterations in claudin expression profiles may contribute to epithelial lung dysfunction during infection and inflammation. Much work remains to be done in the molecular characterization of tight junctions in the lung in normal physiology and during pathologic processes.     

Changes in the Expression and Distribution of Claudins,Increased Epithelial Apoptosis,and a Mannan-Binding Lectin-Associated Immune Response Lead to Barrier Dysfunction in Dextran Sodium Sulfate-Induced Rat Colitis

Background/Aims

This animal study aimed to define the underlying cellular mechanisms of intestinal barrier dysfunction.

Methods

Rats were fed 4% with dextran sodium sulfate (DSS) to induce experimental colitis. We analyzed the sugars in 24-hour urine output by high pressure liquid chromatography. The expression of claudins, mannan-binding lectin (MBL), and MBL-associated serine proteases 2 (MASP-2) were detected in the colonic mucosa by immunohistochemistry; and apoptotic cells in the colonic epithelium were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method assay.

Results

The lactulose and sucralose excretion levels in the urine of rats with DSS-induced colitis were significantly higher than those in the control rats. Mannitol excretion was lower and lactulose/mannitol ratios and sucralose/mannitol ratios were significantly increased compared with those in the control group (p<0.05). Compared with the controls, the expression of sealing claudins (claudin 3, claudin 5, and claudin 8) was significantly decreased, but that of claudin 1 was increased. The expression of pore-forming claudin 2 was upregulated and claudin 7 was downregulated in DSS-induced colitis. The epithelial apoptotic ratio was 2.8%±1.2% in controls and was significantly increased to 7.2%±1.2% in DSS-induced colitis. The expression of MBL and MASP-2 in the intestinal mucosa showed intense staining in controls, whereas there was weak staining in the rats with colitis.

Conclusions

There was increased intestinal permeability in DSS-induced colitis. Changes in the expression and distribution of claudins, increased epithelial apoptosis, and the MASP-2-induced immune response impaired the intestinal epithelium and contributed to high intestinal permeability.     

Acute regulation of tight junction ion selectivity in human airway epithelia

Electrolyte transport through and between airway epithelial cells controls the quantity and composition of the overlying liquid. Many studies have shown acute regulation of transcellular ion transport in airway epithelia. However, whether ion transport through tight junctions can also be acutely regulated is poorly understood both in airway and other epithelia. To investigate the paracellular pathway, we used primary cultures of differentiated human airway epithelia and assessed expression of claudins, the primary determinants of paracellular permeability, and measured transepithelial electrical properties, ion fluxes, and La³⁺ movement. Like many other tissues, airway epithelia expressed multiple claudins. Moreover, different cell types in the epithelium expressed the same pattern of claudins. To evaluate tight junction regulation, we examined the response to histamine, an acute regulator of airway function. Histamine stimulated a rapid and transient increase in the paracellular Na⁺ conductance, with a smaller increase in Cl⁻ conductance. The increase was mediated by histamine H₁ receptors and depended on an increase in intracellular Ca²⁺ concentration. These results suggest that ion flow through the paracellular pathway can be acutely regulated. Such regulation could facilitate coupling of the passive flow of counter ions to active transcellular transport, thereby controlling net transepithelial salt and water transport.     

Cyclic AMP and acidic fibroblast growth factor have opposing effects on tight and adherens junctions in microvascular endothelial cells in vitro

Endothelial adherens junctions (AJ) and tight junctions (TJ) are important determinants of vascular permeability and cell morphology. Here, we investigate their regulation, in primary human placental microvascular endothelial cell (HPMEC) cultures, by either aFGF plus heparin (ECGS) or elevated cAMP. The proliferation of HPMEC was weakly stimulated by ECGS, while cAMP was inhibitory. ECGS had little effect on transendothelial resistance (TER), but increased macromolecular permeability, whereas cAMP induced a twofold increase in TER and reduced macromolecular permeability. Ultrastructurally, ECGS-treated HPMEC exhibited an "activated" phenotype typified by proliferating cells, with poorly organized cell-cell junctions, whereas cAMP-treated cells appeared quiescent and markedly flattened with extended paracellular junctions, resembling endothelium in situ. The expression and localization of junctional molecules, F-actin, and junctional phosphotyrosine were examined by confocal microscopy and immunoblotting. Junctional molecules in ECGS-treated cells were less organized at lateral membranes than in control cells, whereas in cAMP-treated cells, they were highly localized at continuous contacts. These differences correlated with the intensity of junctional phosphotyrosine, being lowest with cAMP treatment. In the AJ of ECGS-treated and control cells, beta-catenin predominated but in cAMP-treated cells, gamma-catenin/plakoglobin was enriched. In addition, cAMP upregulated junctional expression of VE-cadherin and PECAM-1 and increased the levels of the TJ molecules occludin and ZO-1. The expression levels of junctional components, and their tyrosine phosphorylation, play an important role in dynamic regulation of endothelial cell-cell junctions.     

Peripheral myelin protein 22 is a constituent of intercellular junctions in epithelia

Alterations in peripheral myelin protein 22 (PMP22) gene expression are associated with a host of heritable demyelinating peripheral neuropathies, yet the function of the protein remains unknown. PMP22 expression is highest in myelinating Schwann cells of peripheral nerves; however, significant levels of PMP22 mRNAs can be detected in a variety of non-neural tissue, including epithelia. To date, PMP22 protein expression and localization in non-neural tissues have not been studied in detail. In adult rat liver and intestine, and cultured epithelial cells, we detected PMP22-like immunoreactivity associated with markers of the tight junctional complex, including zonula occludens 1 (ZO-1) and occludin. Upon disruption of intercellular contacts, PMP22 was internalized into vesicles that were immunoreactive for both anti-occludin and anti-PMP22 antibodies. Nonionic detergent extraction of cultured epithelial cells did not solubilize PMP22, as the majority of the protein remained in the detergent insoluble fraction, as did ZO-1 and occludin. We also observed the targeting of exogenous myc-tagged PMP22 to apical cell junctions in polarized epithelia and to anti-ZO-1 antibody immunoreactive cell contacts of L fibroblasts. These studies support a role for PMP22 at intercellular junctions of epithelia and may indicate a similar function in myelinating Schwann cells. Furthermore, our findings could provide an explanation for certain phenotypes of PMP22 neuropathy mice that cannot be accounted for by dysmyelination.     

Alcohol increases the permeability of airway epithelial tight junctions in Beas-2B and NHBE cells

Background: Tight junctions form a continuous belt‐like structure between cells and act to regulate paracellular signaling. Protein kinase C (PKC) has been shown to regulate tight junction assembly and disassembly and is activated by alcohol. Previous research has shown that alcohol increases the permeability of tight junctions in lung alveolar cells. However, little is known about alcohol’s effect on tight junctions in epithelium of the conducting airways. We hypothesized that long‐term alcohol exposure reduces zonula occluden‐1 (ZO‐1) and claudin‐1 localization at the cell membrane and increases permeability through a PKC‐dependent mechanism. Methods: To test this hypothesis, we exposed normal human bronchial epithelial (NHBE) cells, cells from a human bronchial epithelial transformed cell line (Beas‐2B), and Beas‐2B expressing a PKCα dominant negative (DN) to alcohol (20, 50, and 100 mM) for up to 48 hours. Immunofluorescence was used to assess changes in ZO‐1, claudin‐1, claudin‐5, and claudin‐7 localization. Electric cell–substrate impedance sensing was used to measure the permeability of tight junctions between monolayers of NHBE, Beas‐2B, and DN cells. Results: Alcohol increased tight junction permeability in a concentration‐dependent manner and decreased ZO‐1, claudin‐1, claudin‐5, and claudin‐7 localization at the cell membrane. To determine a possible signaling mechanism, we measured the activity of PKC isoforms (alpha, delta, epsilon, and zeta). PKCα activity significantly increased in Beas‐2B cells from 1 to 6 hours of 100 mM alcohol exposure, while PKCζ activity significantly decreased at 1 hour and increased at 3 hours. Inhibiting PKCα with Gö‐6976 prevented the alcohol‐induced protein changes in both ZO‐1 and claudin‐1 at the cell membrane. PKCα DN Beas‐2B cells were resistant to alcohol‐induced protein alterations. Conclusions: These results suggest that alcohol disrupts ZO‐1, claudin‐1, claudin‐5, and claudin‐7 through the activation of PKCα, leading to an alcohol‐induced “leakiness” in bronchial epithelial cells. Such alcohol‐induced airway‐leak state likely contributes to the impaired airway host defenses associated with acute and chronic alcohol ingestion.     

Tight and adherens junctions in the ovine uterus: differential regulation by pregnancy and progesterone

In species with noninvasive implantation by conceptus trophectoderm, fetal/maternal communications occur across the endometrial epithelia. The present studies identified changes in junctional complexes in the ovine endometrium that regulate paracellular trafficking of water, ions, and other molecules, and the secretory capacity of the uterine epithelia. Distinct temporal and spatial alterations in occludin, tight junction protein 2, and claudin 1-4 proteins were observed in the endometrium of cyclic and early pregnant ewes. Dynamic changes in tight junction formation were characterized by an abundance of tight junction proteins on d 10 of the estrous cycle and pregnancy that substantially decreased by d 12. Early progesterone administration advanced conceptus development on d 9 and 12 that was associated with loss of tight-junction-associated proteins. Pregnancy increased tight-junction-associated proteins between d 14-16. Cadherin 1 and beta-catenin, which form adherens junctions, were abundant in the endometrial glands, but decreased after d 10 of pregnancy in the luminal epithelium and then increased by d 16 with the onset of implantation. Results support the ideas that progesterone elicits transient decreases in tight and adherens junctions in the endometrial luminal epithelium between d 10-12 that increases selective serum and tissue fluid transudation to enhance blastocyst elongation, which is subsequently followed by an increase in tight and adherens junctions between d 14-16 that may be required for attachment and adherence of the trophectoderm for implantation. The continuous presence of tight and adherens junctions in the uterine glands would allow for vectorial secretion of trophic substances required for conceptus elongation and survival.     

Expression of homocellular and heterocellular gap junctions in hamster arterioles and feed arteries

OBJECTIVES: Conduction of vasoconstrictor and vasodilator responses in the microcirculation involves electrical coupling through gap junction channels among cells of the vascular wall. The present study determined whether reported differences in the properties of conduction along the arterioles of the epithelial hamster cheek pouch (CPA) and feed arteries of its retractor skeletal muscle (RFA) result from differences in the expression profile of specific connexin (Cx) isoforms and the gap junctions they comprise. METHODS: Real-time PCR, immunohistochemistry and serial section electron microscopy were used to compare wall morphology and the distribution of gap junctions between respective vessels. RESULTS: Expression of mRNA for Cx37, 40, 43 and 45 was similar between CPA and RFA. In the endothelium, Cx37, 40 and 43 proteins were expressed abundantly between adjacent cells while Cx37 was present in the smooth muscle. In both vessels, endothelial and smooth muscle cell (SMC) layers were well connected by myoendothelial gap junctions (MEGJs), which were found near endothelial cell (EC) gap junctions. CONCLUSIONS: The absence of differential gap junctional expression between CPA and RFA, in spite of documented differences in cellular conduction pathways, supports the hypothesis that conductance of vascular gap junction channels can be differentially modulated in resistance microvessels.     

Localization of connexin26 and connexin32 in putative CO(2)-chemosensitive brainstem regions in rat.

Recent studies have suggested that cell-to-cell coupling, which occurs via gap junctions, may play a role in CO(2) chemoreception. Here, we used immunoblot and immunohistochemical analyses to investigate the presence, distribution, and cellular localization of the gap junction proteins connexin26 (Cx26) and connexin32 (Cx32) in putative CO(2)-chemosensitive brainstem regions in both neonatal and adult rats. Immunoblot analyses revealed that both Cx subtypes were expressed in putative CO(2)-chemosensitive brainstem regions; however, regional differences in expression were observed. Immunohistochemical experiments confirmed Cx expression in each of the putative CO(2)-chemosensitive brainstem regions, and further demonstrated that Cx26 and Cx32 were found in neurons and Cx26 was also found in astrocytes in these regions. Thus, our findings suggest the potential for gap junctional communication in these regions in both neonatal and adult rats. We propose that the gap junction proteins Cx26 and Cx32, at least in part, form the neuroanatomical substrate for this gap junctional communication, which is hypothesized to play a role in central CO(2) chemoreception.     

Catenins in the rat epididymis: their expression and regulation in adulthood and during postnatal development

Tight and adhering junctions are important in maintaining the integrity of the epididymal epithelium and formation of the blood epididymal barrier, which are crucial for sperm maturation and storage. The composition of the catenin-adhering junctional family of proteins and their relationship with tight junctions remain to be established in the epididymis. In the normal adult rat epididymis, immunostaining for three anticatenin antibodies (alpha, beta-, and p120ctn) was noted along the lateral plasma membranes (LPM) between adjacent epithelial cells. Although alpha-catenin and beta-catenin were maximally expressed in the corpus and cauda epididymis, p120 expression was intense and similar in all epididymal regions. Bilateral orchidectomy of adult rats indicated that the expression of p120 at the LPM was not altered compared with that in control animals. On the other hand, staining at the LPM for alpha- and beta-catenin was markedly reduced, concomitant with an increased cytoplasmic reaction in each epididymal region. As the staining pattern for alpha- and beta-catenin returned to that seen in control animals after testosterone supplementation, it is suggested that their localization and targeting to the LPM are regulated by androgens. This is confirmed by postnatal studies in which maximal expression at the LPM for each catenin occurs by d 49, when androgen levels are adult-like. Immunolocalization of zona occludens-1 along with immunoprecipitation of epididymal homogenates of the initial segment/caput region of the epididymis revealed that zona occludens-1 is an integral part of the adhering junctional complex in young rats and coprecipitates with beta-catenin at the level of the apical tight junctions.     

Sialylated MUC1 mucin expression in normal pancreas, benign pancreatic lesions, and pancreatic ductal adenocarcinoma.

BACKGROUND/AIMS: Pancreatic cancer has the poorest prognosis of all gastrointestinal cancers. Because sialylated mucin influences the biologic behavior of carcinoma cells, we investigated sialylated MUC1 mucin expression in patients with pancreatic ductal adenocarcinoma. METHODOLOGY: The expression of sialylated MUC1 mucin was examined in 55 pancreatic ductal adenocarcinomas, 2 normal pancreas specimens, 3 chronic pancreatitis specimens, 1 ductal hyperplasia of the pancreas, 3 mucinous cystadenomas, and 2 liver metastases from pancreatic ductal adenocarcinoma. Expression was assessed by immunohistochemistry with a new monoclonal antibody (mAb) (MY.1E12). RESULTS: Sialylated MUC1 mucin was expressed in the cancer cell membrane in all the ductal carcinomas. The reaction product was seen at the apical aspect of cells when these were in tubule formation. This pattern was also detected in mucinous cystadenomas. However, it was seen diffusely in the cell membrane in single cancer cells or small clusters of cells without tubule formation and in metastatic liver tumors. Namely, invading or metastatic cancer cells expressed this type of mucin throughout the entire cell membrane. The expression of sialylated MUC1 mucin was not observed in specimens from normal pancreas, chronic pancreatitis, or ductal hyperplasia of the pancreas. In normal pancreas and these lesions, expression of sialylated Mession of sialylated MUC1 was limited to acini and secreted mucin. CONCLUSIONS: Sialylated MUC1 mucin, which is expressed throughout the cancer cell membrane, may be a factor in the metastatic potential of pancreatic ductal adenocarcinoma.     

Expression Pattern, Ethanol-Metabolizing Activities, and Cellular Localization of Alcohol and Aldehyde Dehydrogenases in Human Pancreas: Implications for Pathogenesis of Alcohol-Induced Pancreatic Injury

Background:  Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are major enzymes responsible for metabolism of ethanol. Genetic polymorphisms of ADH1B , ADH1C , and ALDH2 occur among racial populations. The metabolic effect and metabolites contribute to pathogenesis of pancreatic injury. The goal of this study was to determine the functional expressions and cellular localization of ADH and ALDH families in human pancreas.
Methods:  Fifty five surgical specimens of normal pancreas as well as 15 samples each for chronic pancreatitis and pancreatic cancer from archival formalin-fixed paraffin-embedded tissue specimens were investigated. Class-specific antibodies were prepared by affinity chromatographies from rabbit antisera raised against recombinant human ADH1C1, ADH4, ADH5, ADH7, ALDH1A1, ALDH2, and ALDH3A1. The isozyme expression patterns of ADH/ALDH were identified by isoelectric focusing, and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting, and the cellular localizations were detected by immunohistochemistry and histochemistry.
Results:  At 33 mM ethanol, pH 7.5, the activities were significantly different between allelic phenotypes of ADH1B. The activity of ALDH2-inactive phenotypes was slightly lower than ALDH2-active phenotypes at 200 μM acetaldehyde. The protein contents were in the following decreasing order: ALDH1A1, ALDH2, ADH1, and ADH5. ADH1B was detected in the acinar cells and ADH1C in the ductular, islet, and stellate cells. The expression of ADH1C appeared to be increased in the activated pancreatic stellate cells in chronic pancreatitis and pancreatic cancer.
Conclusions:  Alcohol dehydrogenase and ALDH family members are differentially expressed in the various cell types of pancreas. ADH1C may play an important role in modulation of activation of pancreatic stellate cells.