Biomedical subjects

Dorothée Caille

Publications and source records attributed to Dorothée Caille.

11 recordsLinked to original sources

PubMed2006

Connexin36 and pancreatic beta-cell functions.

Most cell types are functionally coupled by connexin (Cx) channels, i.e. exchange cytoplasmic ions and small metabolites through gap junction domains of their membrane. This form of direct cell-to-cell communication occurs in all existing animals, whatever their position in the phylogenetic scale, and up to humans. Pancreatic beta-cells are no exception, and normally cross-talk with their neighbors via channels made of Cx36. These exchanges importantly contribute to coordinate and synchronize the function of individual cells within pancreatic islets, particularly in the context of glucose-induced insulin secretion. Compelling evidence now indicates that Cx36-mediated coupling, and/or the Cx36 protein per se, play significant regulatory roles in various beta-cell functions, ranging from the biosynthesis, storage and release of insulin. Recent preliminary data further suggest that the protein may also be implicated in the balance of beta-cell growth versus necrosis and apoptosis, and in the regulated expression of specific genes. Here, we review this evidence, discuss the possible involvement of Cx36 in the pathophysiology of diabetes, and evaluate the relevance of this connexin in the therapeutic approaches to the disease.

Animals↗

PubMed2006

Rhamnolipids are virulence factors that promote early infiltration of primary human airway epithelia by Pseudomonas aeruginosa.

The opportunistic bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis and immunocompromised individuals. Bacterial adherence to the basolateral domain of the host cells and internalization are thought to participate in P. aeruginosa pathogenicity. However, the mechanism by which the pathogen initially modulates the paracellular permeability of polarized respiratory epithelia remains to be understood. To investigate this mechanism, we have searched for virulence factors secreted by P. aeruginosa that affect the structure of human airway epithelium in the early stages of infection. We have found that only bacterial strains secreting rhamnolipids were efficient in modulating the barrier function of an in vitro-reconstituted human respiratory epithelium, irrespective of their release of elastase and lipopolysaccharide. In contrast to previous reports, we document that P. aeruginosa was not internalized by epithelial cells. We further report that purified rhamnolipids, applied on the surfaces of the epithelia, were sufficient to functionally disrupt the epithelia and to promote the paracellular invasion of rhamnolipid-deficient P. aeruginosa. The mechanism involves the incorporation of rhamnolipids within the host cell membrane, leading to tight-junction alterations. The study provides direct evidence for a hitherto unknown mechanism whereby the junction-dependent barrier of the respiratory epithelium is selectively altered by rhamnolipids.

Adult↗

PubMed2005

Involvement of gap junctional communication in secretion.

Glands were the first type of tissues in which the permissive role of gap junctions in the cell-to-cell transfer of membrane-impermeant molecules was shown. During the 40 years that have followed this seminal finding, gap junctions have been documented in all types of multicellular secretory systems, whether of the exocrine, endocrine or pheromonal nature. Also, compelling evidence now indicates that gap junction-mediated coupling, and/or the connexin proteins per se, play significant regulatory roles in various aspects of gland functions, ranging from the biosynthesis, storage and release of a variety of secretory products, to the control of the growth and differentiation of secretory cells, and to the regulation of gland morphogenesis. This review summarizes this evidence in the light of recent reports.

Animals↗

PubMed2005

Quality control of fibrinogen secretion in the molecular pathogenesis of congenital afibrinogenemia.

Congenital afibrinogenemia is a rare bleeding disorder characterized by the absence in circulation of fibrinogen, a hexamer composed of two sets of three polypeptides (Aalpha, Bbeta and gamma). Each polypeptide is encoded by a distinct gene, FGA, FGB and FGG, all three clustered in a region of 50 kb on 4q31. A subset of afibrinogenemia mutations has been shown to specifically impair fibrinogen secretion, but the underlying molecular mechanisms remained to be elucidated. Here, we show that truncation of the seven most C-terminal residues (R455-Q461) of the Bbeta chain specifically inhibits fibrinogen secretion. Expression of additional mutants and structural modelling suggests that neither the last six residues nor R455 is crucial per se for secretion, but prevent protein misfolding by protecting hydrophobic residues in the betaC core. Immunofluorescence and immuno-electron microscopy studies indicate that secretion-impaired mutants are retained in a pre-Golgi compartment. In addition, expression of Bbeta, gamma and angiopoietin-2 chimeric molecules demonstrated that the betaC domain prevents the secretion of single chains and complexes, whereas the gammaC domain allows their secretion. Our data provide new insight into the mechanisms accounting for the quality control of fibrinogen secretion and confirm that mutant fibrinogen retention is one of the pathological mechanisms responsible for congenital afibrinogenemia.

Afibrinogenemia↗

PubMed2005

Expression and secretion of alpha1-proteinase inhibitor are regulated by proinflammatory cytokines in human pancreatic islet cells.

AIMS/HYPOTHESIS: Alpha1-proteinase inhibitor (alpha1-PI) has been considered a key player in inflammatory processes. In humans, the main production site of alpha1-PI is the liver, but other tissues, including pancreatic islets, also synthesise this molecule. The aims of this study were to assess the islet cell types that produce alpha1-PI, to determine whether alpha1-PI is actually secreted by islet cells, and to assess how its production and/or secretion are regulated. METHODS: Expression of alpha1-PI in human islet cells was assessed by immunofluorescence, electron microscopy and western blotting. Release of alpha1-PI was analysed by reverse haemolytic plaque assay and ELISA. The effects of cytokines on alpha1-PI synthesis and secretion were tested. RESULTS: Immunofluorescence showed that alpha and delta cells do express alpha1-PI, whereas beta cells do not. By electron microscopy, we demonstrated a colocalisation of alpha1-PI with glucagon and somatostatin within secretory granules. Immunolabelling also revealed localisation of alpha1-PI within the Golgi apparatus, related vesicles and lysosomal structures. The expression of alpha1-PI in islet cells was also demonstrated by western blotting and ELISA of protein extracts. ELISA and reverse haemolytic plaque assay showed that alpha1-PI is secreted into the culture medium. Treatment of islet cells with IL-1beta and oncostatin M for 4 days increased the production and release of alpha1-PI. CONCLUSIONS/INTERPRETATION: Our results demonstrate that alpha1-PI is expressed by the alpha and delta cells of human islets, and that proinflammatory cytokines enhance the production and release of this inhibitor.

1-Methyl-3-isobutylxanthine↗

PubMed2005

Different mechanisms preclude mutant CLDN14 proteins from forming tight junctions in vitro.

Mutations in claudin 14 (CLDN14) cause nonsyndromic DFNB29 deafness in humans. The analysis of a murine model indicated that this phenotype is associated with degeneration of hair cells, possibly due to cation overload. However, the mechanism linking these alterations to CLDN14 mutations is unknown. To investigate this mechanism, we compared the ability of wild-type and missense mutant CLDN14 to form tight junctions. Ectopic expression in L mouse fibroblasts (LM cells) of wild-type CLDN14 protein induced the formation of tight junctions, while both the c.254T>A (p.V85D) mutant, previously identified in a Pakistani family, and the c.301 G>A (p.G101R) mutant, identified in this study through the screen of 183 Spanish and Greek patients affected with sporadic nonsyndromic deafness, failed to form such junctions. However, the two mutant proteins differed in their ability to localize at the plasma membrane. We further identified hitherto undescribed exons of CLDN14 that are utilized in alternative spliced transcripts. We demonstrated that different mutations of CLDN14 impaired by different mechanisms the ability of the protein to form tight junctions. Our results indicate that the ability of CLDN14 to be recruited to these junctions is crucial for the hearing process.

Alternative Splicing↗

PubMed2005

Loss of connexin36 channels alters beta-cell coupling, islet synchronization of glucose-induced Ca2+ and insulin oscillations, and basal insulin release.

Normal insulin secretion requires the coordinated functioning of beta-cells within pancreatic islets. This coordination depends on a communications network that involves the interaction of beta-cells with extracellular signals and neighboring cells. In particular, adjacent beta-cells are coupled via channels made of connexin36 (Cx36). To assess the function of this protein, we investigated islets of transgenic mice in which the Cx36 gene was disrupted by homologous recombination. We observed that compared with wild-type and heterozygous littermates that expressed Cx36 and behaved as nontransgenic controls, mice homozygous for the Cx36 deletion (Cx36(-/-)) featured beta-cells devoid of gap junctions and failing to exchange microinjected Lucifer yellow. During glucose stimulation, islets of Cx36(-/-) mice did not display the regular oscillations of intracellular calcium concentrations ([Ca(2+)](i)) seen in controls due to the loss of cell-to-cell synchronization of [Ca(2+)](i) changes. The same islets did not release insulin in a pulsatile fashion, even though the overall output of the hormone in response to glucose stimulation was normal. However, under nonstimulatory conditions, islets lacking Cx36 showed increased basal release of insulin. These data show that Cx36-dependent signaling is essential for the proper functioning of beta-cells, particularly for the pulsatility of [Ca(2+)](i) and insulin secretion during glucose stimulation.

Animals↗

PubMed2004

Disruption of the cingulin gene does not prevent tight junction formation but alters gene expression.

Cingulin, a component of vertebrate tight junctions, contains a head domain that controls its junctional recruitment and protein interactions. To determine whether lack of junctional cingulin affects tight-junction organization and function, we examined the phenotype of embryoid bodies derived from embryonic stem cells carrying one or two alleles of cingulin with a targeted deletion of the exon coding for most of the predicted head domain. In homozygous (-/-) embryoid bodies, no full-length cingulin was detected by immunoblotting and no junctional labeling was detected by immunofluorescence. In hetero- and homozygous (+/- and -/-) embryoid bodies, immunoblotting revealed a Triton-soluble, truncated form of cingulin, increased levels of the tight junction proteins ZO-2, occludin, claudin-6 and Lfc, and decreased levels of ZO-1. The +/- and -/- embryoid bodies contained epithelial cells with normal tight junctions, as determined by freeze-fracture and transmission electron microscopy, and a biotin permeability assay. The localization of ZO-1, occludin and claudin-6 appeared normal in mutant epithelial cells, indicating that cingulin is not required for their junctional recruitment. Real-time quantitative reverse-transcription PCR (real-time qRT-PCR) showed that differentiation of embryonic stem cells into embryoid bodies was associated with up-regulation of mRNAs for several tight junction proteins. Microarray analysis and real-time qRT-PCR showed that cingulin mutation caused a further increase in the transcript levels of occludin, claudin-2, claudin-6 and claudin-7, which were probably due to an increase in expression of GATA-6, GATA-4 and HNF-4alpha, transcription factors implicated in endodermal differentiation. Thus, lack of junctional cingulin does not prevent tight-junction formation, but gene expression and tight junction protein levels are altered by the cingulin mutation.

Animals↗

PubMed2004

Replacement by a lacZ reporter gene assigns mouse connexin36, 45 and 43 to distinct cell types in pancreatic islets.

Transcripts of three connexin isoforms (Cx36, Cx43 and Cx45) have been reported in rodent pancreatic islets, but the precise distribution of the cognate proteins is still unknown. We determined expression of Cx36 in a cell-autonomous manner using mice with a targeted replacement of the Cx36 coding region by a lacZ reporter gene. For cell-autonomous monitoring of Cx43 expression, we used the Cre/loxP system: Mice carrying the Cx43 coding region flanked by loxP sites (floxed) also carried an embedded lacZ gene that is activated after Cre-mediated recombination in cells with transcriptional activity of the Cx43 gene. Deletion of the Cx43 coding region in beta-cells did not result in the activation of the embedded lacZ reporter gene. Instead, Cx43 expression was found in endothelial cells of the islets of Langerhans in mice with endothelium-specific deletion. Ubiquitous deletion of Cx43 led to a similar endothelial lacZ expression, but again, activity of the reporter gene was not detected in beta-cells. Mice with targeted replacement of the Cx45 coding region by lacZ showed a vascular expression similar to Cx43. The data show that native insulin-producing cells express a connexin isoform (Cx36) which differs from those (Cx43 and Cx45) expressed by vascular islet cells.

Animals↗

PubMed2003

Lentivirus-mediated transduction of connexin cDNAs shows level- and isoform-specific alterations in insulin secretion of primary pancreatic beta-cells.

We have generated novel lentiviral vectors to integrate various connexin cDNAs into primary, non-dividing cells. We have used these vectors to test whether proper control of insulin secretion depends on a specific connexin isoform and/or on its level of expression. We have observed that transduced connexin32, connexin36 and connexin43 were expressed by primary adult beta-cells at membrane interfaces, were packed into typical gap junction plaques and formed functional channels that allowed a variable coupling, depending on the type and level of connexin expressed. The infected cells spontaneously reaggregated into three-dimensional pseudo-islet organs that could be maintained in culture. We have found that pseudo-islets made by cells transduced with either GFP- or connexin43-expressing lentivirus released insulin in response to various secretagogues similarly to controls. By contrast, pseudo-islets made by cells expressing connexin32, a connexin exogenous to pancreatic islets, or over-expressing connexin36, the endogenous islet connexin, featured a marked decrease in the secretory response to glucose. The data show: (1) that lentiviral vectors allow stable modulation of various connexin in primary, non-proliferating cells; (2) that specific connexin isoforms affect insulin secretion differently; and (3) that adequate levels of coupling via connexin36 channels are required for proper beta-cell function.

Animals↗

PubMed2002

Connexins and secretion.

Connexin channels clustered at gap junctions are obligatory attributes of all macroscopic endocrine and exocrine glands investigated so far and also connect most types of cells which produce secretory products in other tissues. Increasing evidence indicates that connexins, and the cell-to-cell communications that these proteins permit, contribute to control the growth of secretory cells, their expression of specific genes and their differentiated function, including their characteristic ability to biosynthetize and release secretory products in a regulated manner. Since the previous reviews which have been published on this topic, several lines of evidence have been added in support of multiple regulatory roles of gland connexins. Here, we review this novel evidence, point to the many questions which are still open and discuss some interesting perspectives of the field.

Animals↗