coculture-analysis-extracellular-protein-interactions-affecting

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

Glutamic acid decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes and is discharged from injured islet beta cells. GAD65 may also be released by transplanted islets undergoing immunological rejection. To test hypotheses regarding the utility of GAD65 as a biomarker for transplant rejection or diabetes-associated islet damage and also regarding the timing and instigators of GAD65 release in humans or animal models, a sensitive assay capable of measuring GAD65 in serum or plasma will be necessary. Ideally, this assay would also be resistant to interference by anti-GAD65 autoantibodies.

A highly sensitive immunoassay resistant to autoantibody interference for detection of the diabetes-associated autoantigen glutamic acid decarboxylase 65 in blood and other biological samples.

Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5 dpc in the blood vessels of the tunica vasculosa, while it is first detected in both the corneal epithelium and lens between 13.5 and 14.5 dpc. In the corneal epithelium, JAM-A levels remain appreciable throughout life, while JAM-A immunostaining becomes stronger in the lens as the animals age. Both the cornea and lens of mice lacking an intact JAM-A gene are transparent until at least a year of age, although the cells of the JAM-A null corneal epithelium are irregularly shaped. In wild-type mice, JAM-A protein is found at the leading edge of repairing corneal epithelial wounds, however, corneal epithelial wound repair was qualitatively normal in JAM-A null animals. In summary, JAM-A is expressed in the corneal epithelium where it appears to regulate cell shape.

Deletion of JAM-A causes morphological defects in the corneal epithelium.

The enzyme glutamate decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes. The mechanism whereby autoreactivity to GAD65, an intracellular protein, is triggered is unknown, and it is possible that immunoreactive GAD65 is released by injured pancreatic islet beta-cells. There is a great need for methods by which to detect and monitor ongoing islet injury. If GAD65 were released and, furthermore, were able to reach the circulation, it could function as a marker of beta-cell injury. Here, a novel GAD65 plasma immunoassay is used to test the hypotheses that beta-cell injury induces GAD65 discharge in vivo and that discharged GAD65 reaches the bloodstream. Plasma GAD65 levels were determined in rats treated with alloxan, and with diabetogenic and low, subdiabetogenic doses of streptozotocin. beta-Cell injury resulted in GAD65 release into the circulation in a dose-dependent manner, and low-dose streptozotocin resulted in a more gradual increase in plasma GAD65 levels than did diabetogenic doses. Plasma GAD65 levels were reduced in rats that had undergone partial pancreatectomy and remained undetectable in mice. Together, these data demonstrate that GAD65 can be released into the circulation by injured beta-cells. Autoantigen shedding may contribute to the pathogenesis of islet autoimmunity in the multiple low-dose streptozocin model and perhaps, more generally, in other forms of autoimmune diabetes. These results demonstrate that, as is true with other tissues, islet injury, at least in some circumstances, can be monitored by use of discharged, circulating proteins. GAD65 is the first such confirmed protein marker of islet injury.

Release of glutamate decarboxylase-65 into the circulation by injured pancreatic islet beta-cells.

The metastatic potential of cancer cells is directly attributed to their ability to invade through the extracellular matrix. The mechanisms regulating this cellular invasiveness are poorly understood. Here, we show that junctional adhesion molecule A (JAM-A), a tight junction protein, is a key negative regulator of cell migration and invasion. JAM-A is robustly expressed in normal human mammary epithelium, and its expression is down-regulated in metastatic breast cancer tumors. In breast cancer cell lines, an inverse relationship between JAM-A expression and the ability of these cells to migrate on a collagen matrix was observed, which correlates with the known ability of these cells to metastasize. The T47D and MCF-7 cells, which migrate least, are found to express high levels of JAM-A, whereas the more migratory MDA-MB-468 cells have lower levels of JAM-A on the cell surface. MDA-MB-231 cells, which are highly migratory, express the least amount of JAM-A. Overexpression of JAM-A in MDA-MB-231 cells inhibited both migration and invasion through collagen gels. Furthermore, knockdown of JAM-A using short interfering RNAs enhanced the invasiveness of MDA-MB-231 cells as well as T47D cells. The ability of JAM-A to attenuate cell invasion correlated with the formation of increased numbers of focal adhesions and the formation of functional tight junctions. These results show for the first time that an immunoglobulin superfamily cell adhesion protein expressed at tight junctions could serve as a key negative regulator of breast cancer cell invasion and possibly metastasis. Furthermore, loss of JAM-A could be used as a biomarker for aggressive breast cancer.

Attenuation of junctional adhesion molecule-A is a contributing factor for breast cancer cell invasion.

The composition of the beta-cell exocytic machinery is very similar to that of neuronal synapses, and the developmental pathway of beta-cells and neurons substantially overlap. beta-Cells secrete gamma-aminobutyric acid and express proteins that, in the brain, are specific markers of inhibitory synapses. Recently, neuronal coculture experiments have identified three families of synaptic cell-surface molecules (neurexins, neuroligins, and SynCAM) that drive synapse formation in vitro and that control the differentiation of nascent synapses into either excitatory or inhibitory fully mature nerve terminals. The inhibitory synapse-like character of the beta-cells led us to hypothesize that members of these families of synapse-inducing adhesion molecules would be expressed in beta-cells and that the pattern of expression would resemble that associated with neuronal inhibitory synaptogenesis. Here, we describe beta-cell expression of the neuroligins, neurexins, and SynCAM, and show that neuroligin expression affects insulin secretion in INS-1 beta-cells and rat islet cells. Our findings demonstrate that neuroligins and neurexins are expressed outside the central nervous system and help confer an inhibitory synaptic-like phenotype onto the beta-cell surface. Analogous to their role in synaptic neurotransmission, neurexin-neuroligin interactions may play a role in the formation of the submembrane insulin secretory apparatus.

Expression of neurexin, neuroligin, and their cytoplasmic binding partners in the pancreatic beta-cells and the involvement of neuroligin in insulin secretion.

Pancreatic islet beta-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since beta-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in beta-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates beta-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 beta-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits beta3B and mu3B are expressed in beta-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that beta-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to beta-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in beta-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 delta-subunit expression. Our findings suggest that beta-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation.

An AP-3-dependent mechanism drives synaptic-like microvesicle biogenesis in pancreatic islet beta-cells.

Pankreatische Inselzellen Beta enthalten synaptische-wie Microvesicles (branches). Herkunft, Menschenhandel und Rolle der diese branches sind oft missverstanden. In Neuronen ist synaptische Vesikel (SV) Biogenese von zwei verschiedenen Zytosolische Adapter-Proteinkomplexe, einem allgegenw&auml;rtigen AP-2-komplex und der Neuron-spezifische AP-3B Komplex vermittelt. M&auml;use, die fehlende AP-3B-Untereinheiten zeigen GABAergic (hemmende) Neurotransmission beeintr&auml;chtigt und neuronale vesikul&auml;ren GABA-Transporter (VGAT) Inhalt reduziert. Da Beta-Zell-Reifung und exocytotic Funktion zu parallelen scheinen, dass von der hemmenden Synapse wir vorhergesagt, dass AP-3B-assoziierten Vesikel in Beta-Zellen vorhanden w&auml;re. Hier testen wir die Hypothese, dass AP-3B &auml;u&szlig;ert sich in kleinen Inseln und Beta-Zellen SLMV Biogenese vermittelt. Eine sekund&auml;re wollte testen, ob die Ablagerung-Eigenschaften des INS-1 Beta-Zellen Microvesicles identisch mit denen der bona-fide branches von PC12 Zellen isoliert werden. Unsere Ergebnisse zeigen, dass die beiden Neuron-spezifische AP-3 Untereinheiten beta3B und mu3B in Beta-Zellen, zum ersten Mal ausgedr&uuml;ckt werden, die, das diese Proteine gefunden worden sind, au&szlig;erhalb des Nervensystems ausgedr&uuml;ckt werden. Wir fanden, dass die Beta-Zellen branches teilen die gleichen Ablagerung-Eigenschaften wie PC12 branches und SV-Proteine, die speziell auf die AP-3B-assoziierten Vesikel im Gehirn sortiert enthalten. Brefeldin A, eine Droge, die Interferenzen mit AP-3-vermittelter SV Biogenese, hemmt die Lieferung von AP-3 Ladungen an Beta-Zellen branches. im Einklang mit einer Rolle f&uuml;r AP-3 in der Biogenese des INS-1 Zelle VGAT Inhalt wird bei der Hemmung der Expression der AP-3-Delta-Untereinheit, GABAergic SLMV in Beta-Zellen. Unsere Forschungsergebnisse legen nahe, dass Beta-Zellen und Neuronen Teilen Molek&uuml;le und Mechanismen, die wichtig f&uuml;r die Vermittlung der Neuron-spezifische Membran-Menschenhandel-Bahnen, die synaptische Vesikel Bildung zugrunde liegen.

Ein AP-3-abh&auml;ngigen Mechanismus f&auml;hrt synaptische-artigen Microvesicle Biogenese in pankreatischen Inselzellen Beta.

Normal glucose-stimulated insulin secretion is dependent on interactions between neighboring Î² cells. Elucidation of the reasons why this cell-to-cell contact is essential will probably yield critical insights into Î² cell maturation and function. In the central nervous system, transcellular protein interactions (i.e. interactions between proteins on the surfaces of different cells) involving neuroligins are key mediators of synaptic functional development. We previously demonstrated that Î² cells express neuroligin-2 and that insulin secretion is affected by changes in neuroligin-2 expression. Here we show that the effect of neuroligin-2 on insulin secretion is mediated by transcellular interactions. Neuroligin-2 binds with nanomolar affinity to a partner on the Î² cell surface and contributes to the increased insulin secretion brought about by Î² cell-to-Î² cell contact. It does so in a manner seemingly independent of interactions with neurexin, a known binding partner. As in the synapse, transcellular neuroligin-2 interactions enhance the functioning of the submembrane exocytic machinery. Also, as in the synapse, neuroligin-2 clustering is important. Neuroligin-2 in soluble form, rather than presented on a cell surface, decreases insulin secretion by rat islets and MIN-6 cells, most likely by interfering with endogenous neuroligin interactions. Prolonged contact with neuroligin-2-expressing cells increases INS-1 Î² cell proliferation and insulin content. These results extend the known parallels between the synaptic and Î² cell secretory machineries to extracellular interactions. Neuroligin-2 interactions are one of the few transcellular protein interactions thus far identified that directly enhance insulin secretion. Together, these results indicate a significant role for transcellular neuroligin-2 interactions in the establishment of Î² cell function.

Article	Year
A highly sensitive immunoassay resistant to autoantibody interference for detection of the diabetes-associated autoantigen glutamic acid decarboxylase 65 in blood and other biological sample... Diabetes technology & therapeutics\| PubMed ID: 16734550	2006
Deletion of JAM-A causes morphological defects in the corneal epithelium. The international journal of biochemistry & cell biology\| PubMed ID: 17118692	2007
Release of glutamate decarboxylase-65 into the circulation by injured pancreatic islet beta-cells. Endocrinology\| PubMed ID: 17584960	2007
Attenuation of junctional adhesion molecule-A is a contributing factor for breast cancer cell invasion. Cancer research\| PubMed ID: 18381425	2008
Expression of neurexin, neuroligin, and their cytoplasmic binding partners in the pancreatic beta-cells and the involvement of neuroligin in insulin secretion. Endocrinology\| PubMed ID: 18755801	2008
An AP-3-dependent mechanism drives synaptic-like microvesicle biogenesis in pancreatic islet beta-cells. American journal of physiology. Endocrinology and metabolism\| PubMed ID: 20442321	2010
Transcellular neuroligin-2 interactions enhance insulin secretion and are integral to pancreatic Î² cell function. The Journal of biological chemistry\| PubMed ID: 22528485	2012

Arthur T. Suckow

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