Expressions of Tight Junction Proteins Occludin and Claudin-1 Are under the Circadian Control in the Mouse Large Intestine: Implications in Intestinal Permeability and Susceptibility to Colitis

Background & Aims

The circadian clock drives daily rhythms in behavior and physiology. A recent study suggests that intestinal permeability is also under control of the circadian clock. However, the precise mechanisms remain largely unknown. Because intestinal permeability depends on tight junction (TJ) that regulates the epithelial paracellular pathway, this study investigated whether the circadian clock regulates the expression levels of TJ proteins in the intestine.

Methods

The expression levels of TJ proteins in the large intestinal epithelium and colonic permeability were analyzed every 4, 6, or 12 hours between wild-type mice and mice with a mutation of a key clock gene Period2 (Per2; mPer2^m/m). In addition, the susceptibility to dextran sodium sulfate (DSS)-induced colitis was compared between wild-type mice and mPer2^m/m mice.

Results

The mRNA and protein expression levels of Occludin and Claudin-1 exhibited daily variations in the colonic epithelium in wild-type mice, whereas they were constitutively high in mPer2^m/m mice. Colonic permeability in wild-type mice exhibited daily variations, which was inversely associated with the expression levels of Occludin and Claudin-1 proteins, whereas it was constitutively low in mPer2^m/m mice. mPer2^m/m mice were more resistant to the colonic injury induced by DSS than wild-type mice.

Conclusions

Occludin and Claudin-1 expressions in the large intestine are under the circadian control, which is associated with temporal regulation of colonic permeability and also susceptibility to colitis.     

Translation of Vascular Endothelial Growth Factor mRNA by Internal Ribosome Entry: Implications for Translation under Hypoxia

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic growth factor that promotes compensatory angiogenesis in circumstances of oxygen shortage. The requirement for translational regulation of VEGF is imposed by the cumbersome structure of the 5′ untranslated region (5′UTR), which is incompatible with efficient translation by ribosomal scanning, and by the physiologic requirement for maximal VEGF production under conditions of hypoxia, where overall protein synthesis is compromised. Using bicistronic reporter gene constructs, we show that the 1,014-bp 5′UTR of VEGF contains a functional internal ribosome entry site (IRES). Efficient cap-independent translation is maintained under hypoxia, thereby securing efficient production of VEGF even under unfavorable stress conditions. To identify sequences within the 5′UTR required for maximal IRES activity, deletion mutants were analyzed. Elimination of the majority (851 nucleotides) of internal 5′UTR sequences not only maintained full IRES activity but also generated a significantly more potent IRES. Activity of the 163-bp long “improved” IRES element was abrogated, however, following substitution of a few bases near the 5′ terminus as well as substitutions close to the translation start codon. Both the full-length 5′UTR and its truncated version function as translational enhancers in the context of a monocistronic mRNA.     

Ricin A-Chain and Ricin A-Chain Immunotoxins Rapidly Damage Human Endothelial Cells: Implications for Vascular Leak Syndrome

The results of Phase I/II clinical trials indicate that ricin A-chain-containing immunotoxins cause vascular leak syndrome, characterized by hypoalbuminemia with resultant weight gain and edema. Vascular leak syndrome may be a dose-limiting factor during treatment with ricin A-chain-containing immunotoxins. In this report, we determined the effect of ricin A-chain and ricin A-chain-containing immunotoxins on human umbilical vein endothelial cells with the aim of developing an in vitro model to study vascular leak syndrome. The major findings of our study are: (1) Human umbilical vein endothelial cells undergo rapid and dramatic changes in morphology after treatment with ricin A-chain and ricin A-chain-containing immunotoxins. These changes include rounding of the cells and, eventually, the formation of gaps between them. (2) The permeability of human umbilical vein endothelial cell monolayers to passage of molecules increases after exposure to ricin A-chain or ricin A-chain-containing immunotoxins and this is consistent with the morphologic changes. (3) Human umbilical vein endothelial cells bind ¹²⁵ I-rRTA in a dose-dependent manner but binding is not specific. (4) Human umbilical vein endothelial cells are moderately more sensitive to ricin A-chain-induced inhibition of protein synthesis and proliferation than simian virus-transformed mouse endothelial cells. (5) The morphologic changes are observed 1 h after exposure to the toxins, whereas inhibition of protein synthesis is not detectable until 4 h after a similar exposure. The in vitro model represents a first step in dissecting the complex events which occur in cancer patients who develop vascular leak syndrome after treatment with ricin A-chain-containing immunotoxins.     

Diurnal Variation of Tight Junction Integrity Associates Inversely with Matrix Metalloproteinase Expression in Xenopus laevis Corneal Epithelium: Implications for Circadian Regulation of Homeostatic Surface Cell Desquamation

Background and Objectives

The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs) are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium.

Methodology/Principal Findings

Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2), membrane type 1-MMP (MT1-MMP) and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohistochemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime.

Conclusions/Significance

MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and activation, tight junction protein cleavage, and subsequent surface cell desquamation and renewal may be orchestrated by nocturnal circadian signals.     

Identification of Proteins Associating with Glycosylphosphatidylinositol- Anchored T-Cadherin on the Surface of Vascular Endothelial Cells: Role for Grp78/BiP in T-Cadherin-Dependent Cell Survival

There is scant knowledge regarding how cell surface lipid-anchored T-cadherin (T-cad) transmits signals through the plasma membrane to its intracellular targets. This study aimed to identify membrane proteins colocalizing with atypical glycosylphosphatidylinositol (GPI)-anchored T-cad on the surface of endothelial cells and to evaluate their role as signaling adaptors for T-cad. Application of coimmunoprecipitation from endothelial cells expressing c-myc-tagged T-cad and high-performance liquid chromatography revealed putative association of T-cad with the following proteins: glucose-related protein GRP78, GABA-A receptor α1 subunit, integrin β₃, and two hypothetical proteins, LOC124245 and FLJ32070. Association of Grp78 and integrin β₃ with T-cad on the cell surface was confirmed by surface biotinylation and reciprocal immunoprecipitation and by confocal microscopy. Use of anti-Grp78 blocking antibodies, Grp78 small interfering RNA, and coexpression of constitutively active Akt demonstrated an essential role for surface Grp78 in T-cad-dependent survival signal transduction via Akt in endothelial cells. The findings herein are relevant in the context of both the identification of transmembrane signaling partners for GPI-anchored T-cad as well as the demonstration of a novel mechanism whereby Grp78 can influence endothelial cell survival as a cell surface signaling receptor rather than an intracellular chaperone.T-cadherin (T-cad, or H-cadherin or cadherin-13) is an atypical member of the cadherin superfamily of adhesion molecules. The “classical” cadherins are transmembrane receptors that mediate homophilic Ca²⁺-dependent adhesion between the cells of solid tissues (2). The extracellular domain organization of T-cad is similar to that of classical cadherins, but it lacks transmembrane and cytosolic domains and is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (57). T-cad was shown to mediate weak homophilic cell aggregation in suspensions of transfected cells (45, 57). However, there is a large amount of data suggesting that, in contrast to classical cadherins, atypical T-cad does not primarily function in the maintenance of intercellular adhesion; T-cad is not concentrated at sites of cell-cell contacts, is expressed on the luminal but not the baso-lateral surface of polarized transfected cells, and locates in lipid raft domains of the plasma membrane (29, 42, 43). In the embryonic nervous system T-cad functions as a negative guidance cue regulating motor axon outgrowth and innervation of skeletal muscle (15). Many studies in the cancer field have demonstrated a relationship between T-cad expression levels in tumor cells and tumor progression, although its influence on cell behavior varies in different cancer types, either inhibiting invasion and growth or correlating with a high proliferative and invasive potential (46, 52).In the cardiovascular system T-cad is highly expressed on endothelial cells (ECs), smooth muscle cells, and cardiomyocytes. Its expression level is increased in atherosclerotic lesions from the human aorta (22), in experimental restenosis during neointima formation after balloon catheterization of rat carotid artery (30), and in ECs from tumor vasculature (59). Together, these data suggest that upregulation or/and ligation of T-cad molecules on vascular cells might importantly contribute to progression of vascular pathologies associated with vascular tissue remodelling and stress, such as atherosclerosis, restenosis, and neovascularization of atherosclerotic lesions or tumors. This hypothesis is supported by studies showing that overexpression and/or homophilic ligation of T-cad in ECs stimulates proliferation, migration, and survival under conditions of oxidative stress and promotes angiogenesis in vitro and in vivo (21, 23, 26, 40).Signaling mechanisms underlying T-cad effects on cell growth and motility are poorly studied. We have identified some target signaling pathways activated in cultured vascular ECs by surface T-cad. Changes in cell phenotype during T-cad ligation-induced migration depend on activation of RhoA and Rac GTPases (41). Overexpression and/or ligation of T-cad induces increases in Akt and GSKβ3 phosphorylation levels and activation of β-catenin, and all these effects are blocked by phosphatidylinositol 3-kinase (PI3-kinase) inhibitors (26).There is scant knowledge regarding how cell surface lipid-anchored T-cad transmits signals through the plasma membrane to its intracellular targets. The absence of transmembrane and cytoplasmic domains implies the existence of transmembrane “adaptors” that interact with T-cad on the outer surface of the plasma membrane. Data in the current literature do not allow prediction of membrane associations of T-cad, which from the structural point of view shares homology with two distinct protein groups, namely, cadherins and GPI-anchored proteins. Absence of the cytoplasmic domain excludes any possibility of direct interactions between T-cad and molecular mechanisms utilized by classical cadherins, such as catenins. Recently we have demonstrated that T-cad overexpression results in stimulation of β-catenin signaling in ECs (25). However, this effect is the consequence of Akt/GSK3β pathway activation rather than the result of a direct physical interaction with β-catenin. A requirement for integrin-linked kinase (ILK) upstream of Akt/GSKβ3/β-catenin modulation by T-cad has recently been shown (25). However, ILK is located intracellularly and thus cannot function as a primary molecular adaptor for surface T-cad.The presence of a lipid GPI anchor on the C terminus of the T-cad molecule suggests that T-cad may utilize some of the signaling mechanisms that depend on its localization within lipid rafts, cholesterol- and sphingolipid-rich domains of the plasma membrane that act as signal transduction platforms compartmentalizing, clustering, and facilitating interactions between various lipid-anchored signaling molecules (49). However, the group of GPI proteins is highly heterogeneous both structurally and functionally and includes membrane-associated enzymes, adhesion receptors, differentiation markers, protozoan coat components, and other miscellaneous glycoproteins. Likewise, downstream raft-associated signaling is also diverse, including small GTPases, Src kinases, lipid second messengers, and many others (20). Moreover, molecular interactions within lipid rafts have been shown to be determined by many factors, such as the presence of receptor ligands, their precise membrane localization (the leading edge versus overall surface distribution), and lipid composition (ganglioside GM1-enriched versus GM3-enriched lipid rafts), among others (7).This study aimed to identify membrane proteins colocalizing with atypical GPI-anchored T-cad on the surface of cultured ECs and to evaluate the role of identified molecules as adaptors transmitting signals from cell surface T-cad to its intracellular targets. We have identified several candidate proteins with potential functions as membrane adaptors for T-cad, namely, glucose-related protein Grp78/BiP, GABA-A receptor α1 subunit, integrin β₃, and two hypothetical proteins, LOC124245 and FLJ32070. We demonstrate that the interaction between T-cad and surface Grp78 is necessary for T-cad-dependent activation of prosurvival signaling in ECs.     

Upregulation of Myelin Gene Expression by a Physically-Modified Saline via Phosphatidylinositol 3-Kinase-Mediated Activation of CREB: Implications for Multiple Sclerosis

An increase in central nervous system (CNS) remyelination and a decrease in CNS inflammation are important steps to halt the progression of multiple sclerosis (MS). RNS60 is a bioactive aqueous solution generated by subjecting normal saline to Taylor–Couette–Poiseuille flow under elevated oxygen pressure. Recently we have demonstrated that RNS60 exhibits anti-inflammatory properties. Here, we describe promyelinating property of RNS60. RNS60, but not normal saline (NS), RNS10.3 (TCP-modified saline without excess oxygen) or PNS60 (saline containing excess oxygen without TCP modification), stimulated the expression of myelin-specific genes and proteins (myelin basic protein, MBP; myelin oligodendrocyte glycoprotein, MOG and proteolipid protein, PLP) in primary mouse oligodendroglia and mixed glial cells. While investigating the mechanisms, we found that RNS60 treatment induced the activation of cAMP response element binding protein (CREB) in oligodendrocytes, ultimately leading to the recruitment of CREB to the promoters of myelin-specific genes. Furthermore, activation of type 1A p110β/α, but not type 1B p110γ, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated activation of CREB and upregulation of myelin genes by LY294002 (a specific inhibitor of PI-3 kinase) suggest that RNS60 upregulates the activation of CREB and the expression of myelin-specific molecules in oligodendrocytes via activation of PI3 kinase. These results highlight a novel promyelinating property of RNS60, which may be of benefit for MS and other demyelinating disorders.     

Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind the 3′ end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific, is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3′ end of histone mRNA.     

Activation of human bronchial epithelial cells by inflammatory cytokines IL‐27 and TNF‐α: Implications for immunopathophysiology of airway inflammation

Interleukin (IL)‐27 is a member of IL‐6/IL‐12 family cytokines produced by antigen‐presenting cells in immune responses. IL‐27 can drive the commitment of naive T cells to a T helper type 1 (Th1) phenotype and inhibit inflammation in later phases of infection. Human bronchial epithelial cells have been shown to express IL‐27 receptor complex. In this study, we investigated the in vitro effects of IL‐27, alone or in combination with inflammatory cytokine tumor necrosis factor (TNF)‐α on the pro‐inflammatory activation of human primary bronchial epithelial cells and the underlying intracellular signaling mechanisms. IL‐27 was found to enhance intercellular adhesion molecule 1 (ICAM‐1) expression on the surface of human bronchial epithelial cells, and a synergistic effect was observed in the combined treatment of IL‐27 and TNF‐α on the expression of ICAM‐1. Although IL‐27 did not alter the basal IL‐6 secretion from bronchial epithelial cells, it could significantly augment TNF‐α‐induced IL‐6 release. These synergistic effects on the up‐regulation of ICAM‐1 and IL‐6 were partially due to the elevated expression of TNF‐α receptor (p55TNFR) induced by IL‐27. Further investigations showed that the elevation of ICAM‐1 and IL‐6 in human bronchial epithelial cells stimulated by IL‐27 and TNF‐α was differentially regulated by phosphatidylinositol 3‐OH kinase (PI3K)‐Akt, p38 mitogen‐activated protein kinase, and nuclear factor‐κB pathways. Our results therefore provide a new insight into the molecular mechanisms involved in airway inflammation. J. Cell. Physiol. 223:788–797, 2010. © 2010 Wiley‐Liss, Inc.