TGF-{beta}-induced MiR-491-5p expression promotes Par-3 degradation in rat proximal tubular epithelial cells

Par-3 is a component of Par complex, which is critical for the integrity of tight junction. We previously reported that TGF-β down-regulated Par-3 expression in rat proximal tubular epithelial cells, but the underlying mechanism remains unknown. In the present study, we demonstrated by a luciferase reporter assay that miR-491-5p down-regulated the luciferase activity through a binding site in the 3' UTR of Par-3. Overexpression of miR-491-5p dramatically decreased the expression of endogenous Par-3, disrupted tight junction, and resulted in decreased transepithelial resistance. Moreover, miR-491-5p expression was induced by TGF-β1 through the MEK/p38 MAPK pathway. Importantly, miR-491-5p levels were increased significantly in a rat model of obstructive nephropathy, in parallel with decreased Par-3 levels. Taken together, we conclude that up-regulation of miR-491-5p contributes to TGF-β-regulated Par-3 expression. Our study uncovered a novel mechanism by which TGF-β disrupts cell junction.     

Angiopoietin-2 Stimulation of Endothelial Cells Induces αvβ3 Integrin Internalization and Degradation

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit β3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, αvβ3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser⁹¹⁰ upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from αvβ3 integrin. The αvβ3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/αvβ3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.     

Modulation of the epithelial barrier by dexamethasone and prolactin in cultured Madin-Darby canine kidney (MDCK) cells

Glucocorticoids and prolactin (PRL) have a direct effect on the formation and maintenance of tight junctions (TJs) in cultured endothelial and mammary gland epithelial cells. In this work, we investigated the effect of a synthetic glucocorticoid dexamethasone (DEX) and PRL on the paracellular barrier function in MDCK renal epithelial cells. DEX (4 microM)+PRL (2 microg/ml) and DEX alone increased significantly the transepithelial electrical resistance after chronic treatment (4 days) of confluent MDCK monolayers or after 24 h treatment of subconfluent monolayers. Immunoblotting and immunocytochemistry revealed no changes in the expression and distribution of TJ-associated proteins occludin, ZO-1 and claudin-1 in confluent monolayers after hormone addition. However, a marked increase in junctional content for occludin and ZO-1 with no changes in their total expression was observed in subconfluent MDCK monolayers 24 h exposed to DEX or DEX+PRL. No change in cell proliferation/growth was detected at subconfluent conditions following hormone treatment. An increase in the total number of viable cells was observed only in confluent MDCK monolayers after exposure to DEX+PRL suggesting that the main effect of these hormones on already established barrier may be associated with the inhibition of cell death. In conclusion, our data suggest that these hormones (specially dexamethasone) have an effect on TJ structure and function only during the formation of MDCK epithelial barrier by probably modulating the localization, stability or assembly of TJ proteins to membrane sites of intercellular contact.     

Tight-junction protein ZO-1 isoforms (α+ and α−) show differential extractability and epidermal-growth-factor-induced tyrosine phosphorylation in A431 cells

Summary ZO-1, a cytoplasmic plaque protein of tight junctions, exists in vivo as two major isoforms which are defined by the presence or absence of an 80 amino acid domain termed . The ZO-1 (+) isoform is expressed in most epithelial cells while ZO-1(–) isoform expression is restricted to endothelial cells and some highly specialized epithelial cells, suggesting that the isoforms serve different functions. We had previously demonstrated that both ZO-1 isoforms are expressed in A 431 cells and are tyrosine phosphorylated in response to epidermal-growth-factor treatment. In the present study, we found that the (-) isoform of ZO-1 was more tightly associated with the cytoskeleton than was the (+) isoform, based on extraction in nonionic detergent. In addition, the ZO-1(-) was preferentially tyrosine phosphorylated in response to epidermal-growth-factor treatment. However, both isoforms became more tightly associated with the cytoskeleton after A 431 cells were exposed to epidermal growth factor. Immunofluorescence analysis of A 431 cells with isoform-specific antibodies demonstrated that functional differences in ZO-1 isoform behavior were not due to differences in their subcellular locations. The coincident localization of these isoforms does not rule out different affinities for interacting proteins related to the presence or absence of the domain, and it is these interactions that are likely to explain functional differences between the isoforms.     

Thr(207) of claudin-5 is involved in size-selective loosening of the endothelial barrier by cyclic AMP

We have recently shown that cyclic AMP (cAMP) increases claudin-5 immunoreactivity along cell boundaries and could promote phosphorylation of claudin-5 on threonine residues in porcine blood-brain barrier (BBB) endothelial cells via a protein kinase A (PKA)-dependent pathway (Exp. Cell Res. 290 [2003] 275). Along this line, we identified a putative phosphorylation site for PKA at Thr(207) in the intracytoplasmic carboxyl terminal domain of claudin-5. To clarify the biological significance of this site in regulation of endothelial barrier functions, we established rat lung endothelial (RLE) cells expressing doxycycline (Dox)-inducible wild-type claudin-5 and a mutant with a substitution of Ala for Thr(207) (CL5T207A). We show that induction of wild-type claudin-5 is sufficient to reconstitute the paracellular barrier against inulin (5 kDa), but not mannitol (182 Da), in leaky RLE cells. By contrast, the barrier against both molecules was induced in the mutant cells. We also demonstrate that, upon cAMP treatment, Thr(207) of claudin-5 is involved in enhancement of claudin-5 immunoreactive signals along cell borders, rapid reduction in transendothelial electrical resistance (TER), and loosening of the claudin-5-based endothelial barrier against mannitol, but not inulin. cAMP decreased the claudin-5-based endothelial barrier, strongly suggesting that other tight-junction molecule(s) are required to elevate endothelial barrier functions in response to cAMP.     

An activating mutant of Cdc42 that fails to interact with Rho GDP-dissociation inhibitor localizes to the plasma membrane and mediates actin reorganization

Cdc42 is a member of the Rho family of GTPases and plays an important role in the regulation of actin cytoskeletal organization. Activation of Cdc42 and associated signal transduction cascades are dependent upon proper localization of this GTPase. The studies described herein address the hypothesis that Rho GDP-dissociation inhibitor, RhoGDI, plays an essential role in the translocation of Cdc42 to signaling complexes at the plasma membrane and is essential for Cdc42-mediated actin cytoskeletal rearrangements. An activating mutant of Cdc42 that is RhoGDI-binding defective (Cdc42(G12V/R66E)) is characterized and used as a tool to study the functional importance of the Cdc42-RhoGDI interaction. Overexpression of mycCdc42(G12V/R66E) in COS-7 cells results in actin cytoskeletal rearrangements that are indistinguishable from those stimulated by overexpression of mycCdc42(G12V). In addition, the G12V activating mutant of Cdc42 was overexpressed in mesangial cells that are null for RhoGDI expression. MycCdc42(G12V) stimulation of filopodia formation in these cells was indistinguishable from that observed in wild-type mesangial cells. Taken together, the results presented herein indicate that although RhoGDI is a critical regulator of guanine nucleotide binding, cycling of Cdc42 between membranes and the cytosol and cellular transformation, it is not essential for Cdc42-mediated organization of the actin cytoskeleton.     

Cell to cell relationships in the seminiferous epithelium in the mouse embryo

Summary Germ cells and Sertoli cells in embryonic mouse testes (day 14 to 20 of gestation) were examined by sectioning and freeze-fracture. Intercellular cytoplasmic bridges between the germ cells are observed in day 14 and older embryos. Membrane specializations with dense fuzzy material similar to the socalled desmosome-like structures are found between Sertoli cells and germ cells. A cell contact area with dense opposed membranes is also found between adjacent germ cells. Asymmetrical dense fuzzy lining of both Sertoli and germ cell membranes is noted. Pinocytotic pits or caveolae are frequently found in the Sertoli cell membrane. Between adjacent Sertoli cells, gap junctions of various sizes and focal meshworks of the occluding junctions are found. Most of the occluding junctional particles are located in the center of the grooves in the E face, and are similar to those in postnatal and adult Sertoli cell junctions. In addition, on both fractured faces there are ridges and grooves devoid of particles which are continuous with occluding junctions with particles, suggesting an initial stage in the formation of occluding junctions of the Sertoli cells. Particles gathered at the site of desmosome-like structures are present on the P face of the Sertoli cell.This work is supported by the Japanese Ministry of Education     

Secretory hyperactivity and mitochondrial changes in neurosecretory cells of an insect

Summary The neurosecretory cells of the corpus cardiacum of the desert locust Schistocerca gregaria secrete their hormonal products by exocytosis. The insecticide lindane is known to cause release of hyperglycaemic and adipokinetic neurohormones. Electron microscopy of lindane-poisoned corpora cardiaca reveals many exocytotic omega figures. Mitochondria are affected either directly by the poison or by the consequences of secretory hyperactivity. They occur in increased number, divide, acquire dense mitochondrial granules larger than in the controls, and sometimes line up along plasma membranes, mitochondria in two neighbouring cells forming pairs in juxtaposition. Between two such mitochondria the plasma membranes form a junction-like structure. It is suggested that these effects reflect an excessive calcium entry caused by lindane.This work was supported by a Wellcome-Carlsberg Travelling Research Fellowship awarded to T.N. and by a Commonwealth Scholarship for Postgraduate Research awarded to M.S.     

Are there specialized junctions in the pars maculata of the distal tubule?

Summary In the present study the tight junctions at the macula densa were compared to those of the adjacent straight and convoluted segments of the distal tubule using freeze fracturing and thin sectioning techniques. Only insignificant differences were found in the number of strands and the apico-basal depth of the tight junctions in the three distal tubular segments of rat, dog and tree shrew. In experiments with horseradish peroxidase on mice and tree shrews, the tracer did not penetrate the apical junctions in any of the distal tubular segments. Our findings do not support the concept of considerably higher permeability of the tight junctions at the macula densa, as previously reported. Gap junctions were never observed in the distal nephron. The present results suggest that the glomerulo-tubular feedback is more likely to be mediated by transcellular resorption of solutes than by passive diffusion through a leaky paracellular shunt pathway.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System