Cross-Talk between Adherens Junctions and Desmosomes Depends on Plakoglobin

Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immediately obvious why formation of an adherens junction is a prerequisite for the formation of a desmosome. The adherens junction is composed of a transmembrane classical cadherin (E-cadherin and/or P-cadherin in squamous epithelial cells) linked to either β-catenin or plakoglobin, which is linked to α-catenin, which is linked to the actin cytoskeleton. The desmosome is composed of transmembrane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the intermediate filament cytoskeleton, presumably through plakoglobin and desmoplakin. To begin to study the role of adherens junctions in the assembly of desmosomes, we produced an epithelial cell line that does not express classical cadherins and hence is unable to organize desmosomes, even though it retains the requisite desmosomal components. Transfection of E-cadherin and/or P-cadherin into this cell line did not restore the ability to organize desmosomes; however, overexpression of plakoglobin, along with E-cadherin, did permit desmosome organization. These data suggest that plakoglobin, which is the only known common component to both adherens junctions and desmosomes, must be linked to E-cadherin in the adherens junction before the cell can begin to assemble desmosomal components at regions of cell–cell contact. Although adherens junctions can form in the absence of plakoglobin, making use only of β-catenin, such junctions cannot support the formation of desmosomes. Thus, we speculate that plakoglobin plays a signaling role in desmosome organization

Lis1 is essential for cortical microtubule organization and desmosome stability in the epidermis

Desmoplakin recruits the centrosomal protein Lis1 to the epidermal cell cortex, where it regulates cortical microtubule organization and desmosome stability

Pre-maturation with cAMP modulators in conjunction with EGF-like peptides during in vitro maturation enhances mouse oocyte developmental competence

Recent studies have independently shown that cyclic adenosine 3'5'-monophosphate (cAMP) modulation prior to in vitro maturation (IVM) and epidermal growth factor (EGF)-like peptide supplementation during IVM improve subsequent oocyte developmental outcomes. This study investigated the effects of an IVM system that incorporates these two concepts. Cumulus-oocyte complexes (COCs) were collected from pre-pubertal mice either 46 hr post-equine chorionic gonadotropin (eCG) (IVM) or post-eCG + post-human chorionic gonadotropin (hCG) stimulation (in vivo maturation; IVV). IVM COCs were treated with the cAMP modulators forskolin and IBMX for 1, 2, or 4 hr (pre-IVM phase) prior to IVM. COCs then underwent IVM with the EGF-like peptides amphiregulin or epiregulin, or with the common IVM stimulants follicle-stimulating hormone (FSH) or EGF. A pre-IVM phase increased the size of the subsequent blastocysts' inner-cell-mass compared to standard IVM, regardless of IVM treatment (P < 0.05). Unlike FSH or EGF, amphiregulin or epiregulin significantly increased blastocyst quality (trophectoderm and total cell numbers) and/or yield (P < 0.01) compared to standard IVM, and were the only treatments that produced blastocysts comparable to IVV-derived blastocysts. Forskolin acutely up-regulated EGF-like peptide mRNA expression after a 2-hr pre-IVM phase (P < 0.001), although EGF receptor and ERK1/2 activities were not significantly different than control. IVV-like levels of EGF-like peptide mRNA expression during IVM were maintained only by supplementing with EGF-like peptides and EGF, since expression levels induced by FSH were significantly lower in vitro than during IVV. However, EGF receptor and ERK1/2 phosphorylation levels were not significantly different across treatment groups. In conclusion, a pre-IVM phase in conjunction with IVM in the presence of EGF-like peptides endows high oocyte developmental competence, as evidenced by increased embryo yield and/or quality relative to FSH and EGF.D. Richani, X. Wang, H. Zeng, J.E.J. Smitz, J.G. Thompson, and R.B. Gilchris

α-Catenin-Vinculin Interaction Functions to Organize the Apical Junctional Complex in Epithelial Cells

αE-catenin, a cadherin-associated protein, is required for tight junction (TJ) organization, but its role is poorly understood. We transfected an αE-catenin–deficient colon carcinoma line with a series of αE-catenin mutant constructs. The results showed that the amino acid 326–509 domain of this catenin was required to organize TJs, and its COOH-terminal domain was not essential for this process. The 326–509 internal domain was found to bind vinculin. When an NH2-terminal αE-catenin fragment, which is by itself unable to organize the TJ, was fused with the vinculin tail, this chimeric molecule could induce TJ assembly in the αE-catenin–deficient cells. In vinculin-null F9 cells, their apical junctional organization was impaired, and this phenotype was rescued by reexpression of vinculin. These results indicate that the αE-catenin-vinculin interaction plays a role in the assembly of the apical junctional complex in epithelia

Regulation of sheep oocyte maturation using cAMP modulators

Physical removal of mammalian cumulus-oocyte complexes (COCs) from ovarian follicles results in spontaneous resumption of meiosis, largely because of a decrease in cAMP concentrations, causing asynchrony between cytoplasmic and nuclear maturation and decreased oocyte developmental competence. The aim of this study was to modulate cAMP concentrations within ovine COCs to delay spontaneous nuclear maturation and improve developmental competence. Abattoir-derived sheep COCs were cultured for 2 hours (pre-IVM) in 100 μM forskolin (FSK) plus 500 μM 3-isobutyl-1-methylxanthine (IBMX). Pre-IVM (100 μM FSK and 500 μM IBMX) culture increased COC cAMP concentrations 10-fold compared with controls (P < 0.05). With regard to nuclear maturation, with FSK and IBMX and/or with FSH and cilostamide delayed completion of meiosis (metaphase II) by 3 to 4 hours compared with standard IVM (FSH-stimulated induction of meiosis). In this study, pre-IVM (with FSK and IBMX) followed by IVM (with FSH and cilostamide), increased ovine COC cAMP concentrations and delayed, but did not inhibit, completion of nuclear maturation. This did not affect embryo development rates, but increased total cell number of blastocysts compared with IVM with FSH alone (103 ± 6 vs. 66 ± 4 cells, respectively; mean ± SEM; P < 0.05). We inferred that regulation of ovine oocyte cAMP concentrations during IVM improved embryo quality compared with embryos produced by standard IVM methods.Ryan D. Rose, Robert B. Gilchrist, Jennifer M. Kelly, Jeremy G. Thompson, Melanie L. Sutton-McDowal

Hepatocyte Nuclear Factor 4 Provokes Expression of Epithelial Marker Genes, Acting As a Morphogen in Dedifferentiated Hepatoma Cells

Abstract. We have recently shown that stable expression of an epitope-tagged cDNA of the hepatocyte- enriched transcription factor, hepatocyte nuclear factor (HNF)4, in dedifferentiated rat hepatoma H5 cells is sufficient to provoke reexpression of a set of hepatocyte marker genes. Here, we demonstrate that the effects of HNF4 expression extend to the reestablishment of differentiated epithelial cell morphology and simple epithelial polarity. The acquisition of epithelial morphology occurs in two steps. First, expression of HNF4 results in reexpression of cytokeratin proteins and partial reestablishment of E-cadherin production. Only the transfectants are competent to respond to the synthetic glucocorticoid dexamethasone, which induces the second step of morphogenesis, including formation of the junctional complex and expression of a polarized cell phenotype. Cell fusion experiments revealed that the transfectant cells, which show only partial restoration of E-cadherin expression, produce an extinguisher that is capable of acting in trans to downregulate the E-cadherin gene of well-differentiated hepatoma cells. Bypass of this repression by stable expression of E-cadherin in H5 cells is sufficient to establish some epithelial cell characteristics, implying that the morphogenic potential of HNF4 in hepatic cells acts via activation of the E-cadherin gene. Thus, HNF4 seems to integrate the genetic programs of liver-specific gene expression and epithelial morphogenesis

Pharmacological Analyses of Protein Kinases Regulating Egg Maturation in Marine Nemertean Worms: A Review and Comparison with Mammalian Eggs

For development to proceed normally, animal eggs must undergo a maturation process that ultimately depends on phosphorylations of key regulatory proteins. To analyze the kinases that mediate these phosphorylations, eggs of marine nemertean worms have been treated with pharmacological modulators of intracellular signaling pathways and subsequently probed with immunoblots employing phospho-specific antibodies. This article both reviews such analyses and compares them with those conducted on mammals, while focusing on how egg maturation in nemerteans is affected by signaling pathways involving cAMP, mitogen-activated protein kinases, Src-family kinases, protein kinase C isotypes, AMP-activated kinase, and the Cdc2 kinase of maturation-promoting factor

Desmosome structure, composition and function

AbstractDesmosomes are intercellular junctions of epithelia and cardiac muscle. They resist mechanical stress because they adopt a strongly adhesive state in which they are said to be hyper-adhesive and which distinguishes them from other intercellular junctions; desmosomes are specialised for strong adhesion and their failure can result in diseases of the skin and heart. They are also dynamic structures whose adhesiveness can switch between high and low affinity adhesive states during processes such as embryonic development and wound healing, the switching being signalled by protein kinase C. Desmosomes may also act as signalling centres, regulating the availability of signalling molecules and thereby participating in fundamental processes such as cell proliferation, differentiation and morphogenesis. Here we consider the structure, composition and function of desmosomes, and their role in embryonic development and disease

The Pathogenic Potential of Campylobacter concisus Strains Associated with Chronic Intestinal Diseases

Campylobacter concisus has garnered increasing attention due to its association with intestinal disease, thus, the pathogenic potential of strains isolated from different intestinal diseases was investigated. A method to isolate C. concisus was developed and the ability of eight strains from chronic and acute intestinal diseases to adhere to and invade intestinal epithelial cells was determined. Features associated with bacterial invasion were investigated using comparative genomic analyses and the effect of C. concisus on host protein expression was examined using proteomics. Our isolation method from intestinal biopsies resulted in the isolation of three C. concisus strains from children with Crohn's disease or chronic gastroenteritis. Four C. concisus strains from patients with chronic intestinal diseases can attach to and invade host cells using mechanisms such as chemoattraction to mucin, aggregation, flagellum-mediated attachment, “membrane ruffling”, cell penetration and damage. C. concisus strains isolated from patients with chronic intestinal diseases have significantly higher invasive potential than those from acute intestinal diseases. Investigation of the cause of this increased pathogenic potential revealed a plasmid to be responsible. 78 and 47 proteins were upregulated and downregulated in cells infected with C. concisus, respectively. Functional analysis of these proteins showed that C. concisus infection regulated processes related to interleukin-12 production, proteasome activation and NF-κB activation. Infection with all eight C. concisus strains resulted in host cells producing high levels of interleukin-12, however, only strains capable of invading host cells resulted in interferon-γ production as confirmed by ELISA. These findings considerably support the emergence of C. concisus as an intestinal pathogen, but more significantly, provide novel insights into the host immune response and an explanation for the heterogeneity observed in the outcome of C. concisus infection. Moreover, response to infection with invasive strains has substantial similarities to that observed in the inflamed mucosa of Crohn's disease patients