An siRNA screen identifies transmembrane 7 superfamily member 3 (TM7SF3), a seven transmembrane orphan receptor, as an inhibitor of cytokine-induced death of pancreatic beta cells.

AIMS/HYPOTHESIS: Pro-inflammatory cytokines induce death of pancreatic beta cells, leading to the development of type 1 diabetes. We sought to identify novel players and the underlying mechanisms involved in this process. METHODS: A high-throughput screen of 3,850 mouse small interfering RNAs (siRNAs) was performed in cytokine-treated MIN6 beta cells. Cells were transfected with the different siRNAs and then treated with a combination of TNFα, IL-1ß and IFNγ. Cellular apoptosis (caspase-3/7 activity), and changes in cellular reducing power and cell morphology were monitored. The resulting data were analysed and the corresponding z scores calculated. RESULTS: Several gene families were identified as promoting cytokine-induced beta cell apoptosis, the most prominent being those encoding ubiquitin ligases and serine/threonine kinases. Conversely, deubiquitinating enzymes appeared to reduce apoptosis, while protein phosphatases were mainly associated with lowering cellular reducing power. The screen suggested with high confidence the involvement of several novel genes in cytokine-induced beta cell death, including Camkk2, Epn3, Foxp3 and Tm7sf3, which encodes an orphan seven transmembrane receptor. siRNAs to Tm7sf3 promoted cytokine-induced death of MIN6 cells and human pancreatic islets, and abrogated insulin secretion in these cells. These findings implicate transmembrane 7 superfamily member 3 as a potential new player in the inhibition of cytokine-induced death and in the promotion of insulin secretion from pancreatic beta cells. CONCLUSIONS/INTERPRETATION: The signalling pathways and novel genes that we identified in this screen and that mediate beta cell death offer new possible targets for therapeutic intervention in diabetes and its adverse complications.

pp60(c-src) and related tyrosine kinases: a role in the assembly and reorganization of matrix adhesions.

Activation of tyrosine kinases during integrin-mediated cell-matrix adhesion is involved both in the regulation of focal contact assembly and in the initiation of signaling processes at the cell-matrix adhesive interface. In order to determine the role of pp60(c-src) and related kinases in these processes, we have compared the dynamic reorganization of phosphotyrosine, vinculin, focal adhesion kinase and tensin in cells with altered expression of Src-family kinases. Both null cells for pp60(c-src) and triple knockout cells for pp60(c-src), pp59(fyn), and pp62(c-yes) exhibited decreased phosphotyrosine levels in focal contacts when compared with wild-type cells. pp60(c-src)-null cells also exhibited faster assembly of cell-matrix adhesions and a more exuberant recruitment of FAK to these sites. Tensin, which normally segregates into fibrillar adhesions was localized in large focal contacts in the two mutant cell lines, suggesting involvement of pp60(c-src) in the segregation of focal contacts and fibrillar adhesions. Moreover, treatment of wild-type cells with tyrphostin AG1007, which inhibits both pp60(c-src) and FAK activity, induced accumulation of tensin in peripheral focal adhesions. These findings demonstrate that Src family kinases, and pp60(c-src) in particular, have a central role in regulating protein dynamics at cell-matrix interfaces, both during early stages of interaction and in mature focal contacts.

Effect of H-7 on cultured human trabecular meshwork cells.

PURPOSE: To determine the effect of the serine-threonine kinase inhibitor H-7, which blocks actomyosin contractility and increases outflow facility in live monkeys, on morphology, cytoskeleton, and cellular adhesions of human trabecular meshwork (HTM) cells in culture. METHODS: Cultured HTM cells were videographically recorded and evaluated before and after exposure to H-7 at different concentrations. The subcellular distribution of the actin-based cytoskeleton and associated anchor proteins including vinculin, paxillin, and beta-catenin, as well as phosphotyrosine-containing proteins were evaluated by fluorescence immunocytochemistry and digital fluorescence microscopy. RESULTS: H-7 induced pronounced but reversible HTM cell thickening toward the cell center and deterioration of the actin cytoskeletal network. Cell-extracellular matrix (ECM) and cell-cell adhesions were also affected, but the beta-catenin-rich, vinculin-containing adherens junctions were clearly more resistant than focal contacts. Phosphotyrosine labeling in focal contacts was highly sensitive to H-7. CONCLUSIONS: H-7 induces alterations in cell shape, actin cytoskeleton, and associated focal adhesions in cultured HTM cells, which may be responsible for the effects of H-7 on outflow facility in live monkey eyes.

Effect of latrunculin-A on morphology and actin-associated adhesions of cultured human trabecular meshwork cells.

PURPOSE: Determine the effects of the actin cytoskeleton disrupting compound latrunculin-A (LAT-A) on morphology, cytoskeleton, and cellular adhesions of cultured human trabecular meshwork (HTM) cells. METHODS: HTM cells were cultured to high confluence with endothelial-like morphology and treated with LAT-A at different doses and duration. Topography of living cells was evaluated by videomicroscopy. Distribution and organization of the actin-based cytoskeleton, vinculin- and paxillin-containing focal contacts, and beta-catenin-rich intercellular adhesions were determined by immunofluorescence and digital microscopy. RESULTS: LAT-A induced pronounced but highly reversible rounding of HTM cells, intercellular separation, and disruption of actin filaments. beta-catenin-rich intercellular adherens junctions were particularly sensitive to LAT-A. Vinculin- and paxillin-containing focal contacts were only partially affected and appeared to be more resistant to the drug than the intercellular interactions. CONCLUSIONS: The increase in outflow facility in the living primate eye induced by LAT-A may be due to the disorganization and disruption of the actin cytoskeleton and its associated cellular adhesions in the trabecular meshwork.

Disruption of microtubules in living cells by tyrphostin AG-1714.

Tyrphostin AG-1714 and several related molecules with the general structure of nitro-benzene malononitrile (BMN) disrupt microtubules in a large variety of cultured cells. This process can be inhibited by the stabilization of microtubules with taxol or by pretreatment of the cells with pervanadate, which inhibits tyrosine phosphatases and increases the overall levels of phosphotyrosine in cells. Unlike other microtubule-disrupting drugs such as nocodazole or colchicine, tyrphostin AG-1714 does not interfere with microtubule polymerization or stability in vitro, suggesting that the effect of this tyrphostin on microtubules is indirect. These results imply an involvement of protein tyrosine phosphorylation in the regulation of overall microtubule dynamics. Tyrphostins of AG-1714 type could thus be powerful tools for the identification of such microtubule regulatory pathways.

Latrunculin-A increases outflow facility in the monkey.

PURPOSE: To determine the effect of Latrunculin (LAT)-A, a macrolide that binds to G-actin, which leads to the disassembly of actin filaments, on shape, junctions, and the cytoskeleton of cultured bovine aortic endothelial cells (BAECs) and on outflow facility in living monkeys. METHODS: Latrunculin-A dose-time-response relationships in BAECs were determined by immunofluorescence and phase contrast light microscopy, facility by two-level constant pressure anterior chamber perfusion. RESULTS: In BAECs, LAT-A caused dose- and incubation time- dependent destruction of actin bundles, cell separation, and cell loss. Cell-cell adhesions were more sensitive than focal contacts. Recovery was also dose- and time-dependent. In monkeys, exchange intracameral infusion and topical application of LAT-A induced dose- and time-dependent several-fold facility increases. The facility increase was completely reversed within several hours after drug removal. However, for at least 24 hours after a single topical LAT-A dose, perfusion with drug-free solution caused an accelerated increase in facility beyond that attributed to normal resistance washout. CONCLUSIONS: Pharmacological disorganization of the actin cytoskeleton in the trabecular meshwork by specific actin inhibitors like LAT-A may be a useful antiglaucoma strategy.

H-7 disrupts the actin cytoskeleton and increases outflow facility.

OBJECTIVES: To determine the effects of the serine-threonine kinase inhibitor H-7 on (1) cell junctions and the attached actin-based cytoskeleton in cultured bovine aortic endothelial cells, and (2) outflow facility in living monkeys. METHODS: Bovine aortic endothelial cells were cultured by standard techniques. The architecture and distribution of actin filaments, vinculin, and beta-catenin in bovine aortic endothelial cells were studied by immunolabeling before and after exposure to H-7 at various concentrations and durations. Outflow facility (perfusion) and intraocular pressure (Goldmann tonometer) were determined before and after the intracameral or topical administration of H-7 or a vehicle. RESULTS: In bovine aortic endothelial cells, exposure to H-7 produced a reversible time- and concentration-dependent disruption of actin microfilaments and an alteration in the organization of cell-cell and cell-matrix adhesions. In monkeys, intracameral and topical administration of H-7 dose dependently and reversibly doubled facility, and topical H-7 reduced intraocular pressures. CONCLUSION: H-7 increases outflow facility in monkeys, probably by inhibiting cell contractility, cytoskeletal support, and cell-cell adhesions in the trabecular meshwork.

Focal adhesion formation by F9 embryonal carcinoma cells after vinculin gene disruption.

The assembly of focal adhesions was investigated in F9 embryonal carcinoma cells in which the expression of vinculin was eliminated by a targeted disruption of the vinculin gene. Vinculin-deficient F9 cells were capable of adhering to fibronectin-coated surfaces, though they displayed a reduced spreading compared to the parental cells. Transmission electron microscopy as well as interference reflection microscopy of live cells showed that vinculin-null F9 cells formed focal adhesions that were indistinguishable from those of the control cells. Fluorescent labeling for actin, talin, alpha-actinin, paxillin and phosphotyrosinated components indicated that the organization of all these focal contact-associated components was essentially identical in the vinculin-containing and vinculin-null cells. However, quantitative, digitized microscopy indicated that the intensity of fluorescence labeling in focal adhesions for alpha-actinin, talin and paxillin was significantly higher in cells lacking vinculin. The results suggest that there are multiple molecular mechanisms for the formation of focal adhesions in the absence of vinculin.

Mapping of adherens junction components using microscopic resonance energy transfer imaging.

Quantitative microscopic imaging of resonance energy transfer (RET) was applied for immunological high resolution proximity mapping of several cytoskeletal components of cell adhesions. To conduct this analysis, a microscopic system was developed, consisting of a highly stable field illuminator, computer-controlled filter wheels for rapid multiple-color imaging and a sensitive, high resolution CCD camera, enabling quantitative data recording and processing. Using this system, we have investigated the spatial inter-relationships and organization of four adhesion-associated proteins, namely vinculin, talin, alpha-actinin and actin. Cultured chick lens cells were double labeled for each of the junctional molecules, using fluorescein- and rhodamine-conjugated antibodies or phalloidin. RET images were acquired with fluorescein excitation and rhodamine emission filter setting, corrected for fluorescein and rhodamine fluorescence, and normalized to the fluorescein image. The results pointed to high local densities of vinculin, talin and F-actin in focal adhesions, manifested by mean RET values of 15%, 12% and 10%, respectively. On the other hand, relatively low values (less than 1%) were observed following double immunofluorescence labeling of the same cells for alpha-actinin. Double indirect labeling for pairs of these four proteins (using fluorophore-conjugated antibodies or phalloidin) resulted in RET values of 5% or lower, except for the pair alpha-actinin and actin, which yielded significantly higher values (13-15%). These results suggest that despite their overlapping staining patterns, at the level of resolution of the light microscope, the plaque proteins vinculin and talin are not homogeneously interspersed at the molecular level but form segregated clusters. alpha-Actinin, on the other hand, does not appear to form such clusters but, rather, closely interacts with actin. We discuss here the conceptual and applicative aspects of RET measurements and the implications of the results on the subcellular molecular organization of adherens-type junctions.

Augmentation of adherens junction formation in mesenchymal cells by co-expression of N-CAM or short-term stimulation of tyrosine-phosphorylation.

Adherens-type junctions (AJ) are specialized intercellular contacts, mediated by cadherins and characterized by the association with actin filaments through a vinculin- and catenin-rich submembrane plaque. We describe here two mechanisms which potentiate AJ formation in mesenchymal cells. These include the augmentation of AJ by the co-expression of another adhesion molecule, namely NCAM, and the stimulation of tyrosine phosphorylation. These effects were obtained in NIH-3T3 cells, which, under normal conditions, have poor cadherin- and vinculin-containing intercellular junctions. The transfection of these cells with cDNA encoding the 140kD NCAM resulted in the extensive formation of cadherin- and vinculin-rich AJ, demonstrating a cooperativity between the two junctional systems. AJ could also be induced in 3T3, and in CEF and COS cells, upon a brief exposure to H2O2/vanadate, which elevates cellular levels of phosphotyrosine due to inhibition of tyrosine-specific phosphatases. This induction was, however, transient since prolonged exposure to H2O2/vanadate resulted in an overall destruction of AJ and detachment of cells from each other and from the extracellular matrix. AJ formation appears, therefore, to be modulated by a variety of factors including the level of expression of its intrinsic components, the cooperative effect of other adhesion molecules, and by tyrosine-phosphorylation.

Cytoskeletal involvement in the modulation of cell-cell junctions by the protein kinase inhibitor H-7.

The protein kinase inhibitor H-7 has been shown to block junction dissociation induced by low extracellular calcium in Madin Darby canine kidney epithelial cells (S. Citi, J. Cell Biol. (1992) 117, 169-178). To understand the basis of this effect, we have examined how H-7 affects the organization of junctions and the actin cytoskeleton in different types of epithelial cells in culture. Immunofluorescence microscopy showed that H-7 confers Ca2+ independence on cultured epithelial lens cells, which lack tight junctions and desmosomes but have microfilament-associated adherens junctions. In these cells, H-7 did not protect N-cadherin from trypsin digestion at low extracellular calcium, suggesting that H-7 does not stabilize the 'active' cadherin conformation. In cultured Madin Darby canine kidney cells, H-7 partially prevented the fall in transepithelial resistance induced by cytochalasin D, either alone or in conjunction with calcium chelators. Double-immunofluorescence microscopy showed that H-7 inhibits both the fragmentation of labeling for the tight junction protein cingulin and the condensation of actin into cytoplasmic foci induced by cytochalasin D. Taken together, these observations indicate that H-7 inhibits junction dissociation by affecting the contractility of the adherens junction-associated microfilaments following treatment with calcium chelators or cytochalasin D.

Effect of protein kinase inhibitor H-7 on the contractility, integrity, and membrane anchorage of the microfilament system.

Addition of protein kinase inhibitor H-7 leads to major changes in cell structure and dynamics. In previous studies [Citi, 1992: J. Cell Biol. 117:169-178] it was demonstrated that intercellular junctions in H-7-treated epithelial cells become calcium independent. To elucidate the mechanism responsible for this effect we have examined the morphology, dynamics, and cytoskeletal organization of various cultured cells following H-7-treatment. We show here that drug treated cells display an enhanced protrusive activity. Focal contact-attached stress fibers and the associated myosin, vinculin, and talin deteriorated in such cells while actin, vinculin, and N-cadherin associated with cell-cell junctions were retained. Furthermore, we demonstrate that even before these cytoskeletal changes become apparent, H-7 suppresses cellular contractility. Thus, short pretreatment with H-7 leads to strong inhibition of the ATP-induced contraction of saponin permeabilized cells. Comparison of H-7 effects with those of other kinase inhibitors revealed that H-7-induced changes in cell shape, protrusional activity, and actin cytoskeleton structure are very similar to those induced by selective inhibitor of myosin light chain kinase, KT5926. Specific inhibitors of protein kinase C (Ro31-8220 and GF109203X), on the other hand, did not induce similar alterations. These results suggest that the primary effect of H-7 on cell morphology, motility, and junctional interactions may be attributed to the inhibition of actomyosin contraction. This effect may have multiple effects on cell behavior, including general reduction in cellular contractility, destruction of stress fibers, and an increase in lamellipodial activity. It is proposed that this reduction in tension also leads to the apparent stability of cell-cell junctions in low-calcium medium.

The effect of tyrosine-specific protein phosphorylation on the assembly of adherens-type junctions.

Adherens-type junctions (AJs) are major subcellular targets for tyrosine specific protein phosphorylation [Volberg et al. (1991) Cell Regul., 2, 105-120]. Here we report on the apparent effect of such phosphorylation events on the assembly and integrity of AJs. We show that incubation of MDCK cells with potent inhibitors of tyrosine-specific phosphatases (PTP), namely H2O2 and vanadate, leads to a dramatic increase in AJ-associated phosphotyrosine which was apparent already within 2-5 min of treatment and progressed upon further incubation. Examination of H2O2 vanadate treated cells at later time points indicated that intercellular AJs rapidly deteriorated, concomitantly with a marked increase in the number and size of vinculin and actin containing focal contacts. In parallel, major changes were observed in cell structure and topology, as revealed by electron microscopy. These were manifested by rapid rounding-up of the cells followed by reorganization of the cell monolayer. Other intercellular junctions, including desmosomes and tight junctions, visualized by staining with desmoplakin and ZO-I antibodies, were not significantly affected. To verify that modulation of AJs was indeed related to tyrosine phosphorylation, we have carried out reciprocal experiments in which Rovs Sarcoma virus (RSV) transformed chick lens cells, expressing high levels of pp60src kinase, were treated with inhibitors of tyrosine kinases, (tyrphostins). We show that following such treatment, intercellular AJs which were deteriorated in the transformed cells, were reformed. Based on these observations, we propose that specific tyrosine phosphorylation of AJ components is involved in the downregulation of these cellular contacts.

Modulation of intercellular adherens-type junctions and tyrosine phosphorylation of their components in RSV-transformed cultured chick lens cells.

Transformation of cultured chick lens epithelial cells with a temperature-sensitive mutant of Rous sarcoma virus (tsRSV) leads to radical changes in cell shape and interactions. When cultured at the restrictive temperature (42 degrees C), the transformed cells largely retained epithelial morphology and intercellular adherens junctions (AJ), whereas on switch to the permissive temperature (37 degrees C) they rapidly became fibroblastoid, their AJ deteriorated, and cell adhesion molecules (A-CAM) (N-cadherin) largely disappeared from intercellular contact sites. The microfilament system that was primarily associated with these junctions was markedly rearranged on shift to 37 degrees C and remained associated mainly with cell-substrate focal contacts. These apparent changes in intercellular AJ were not accompanied by significant alterations in the cellular content of several junction-associated molecules, including A-CAM, vinculin, and talin. Immunolabeling with phosphotyrosine-specific antibodies indicated that both cell-substrate and intercellular AJ were the major cellular targets for the pp60v-src tyrosine-specific protein kinase. It was further shown that intercellular AJ components serve as substrates to tyrosine kinases also in nontransformed lens cells, because the addition of a combination of vanadate and H2O2--which are potent inhibitors of protein tyrosine phosphatases--leads to a remarkable accumulation of immunoreactive phosphotyrosine-containing proteins in these junctions. This finding suggests that intercellular junctions are major sites of action of protein tyrosine kinases and that protein tyrosine phosphatases play a major role in the regulation of phosphotyrosine levels in AJ of both normal and RSV-transformed cells.

Broad spectrum pan-cadherin antibodies, reactive with the C-terminal 24 amino acid residues of N-cadherin.

We describe here the preparation and application of antibodies directed against a synthetic, 24 amino acid long, peptide corresponding to the conserved cytoplasmic C terminus of N-cadherin. We demonstrate here that the antibodies to the synthetic peptide react extensively with all known members of the cadherin family and, in addition, recognize novel cadherins in a variety of cells and tissues, suggesting that these antibodies indeed exhibit pan-cadherin reactivity. By Western blot screening of chicken tissues at least 4 different immunoreactive bands were resolved, commonly disclosing 2-3 distinct bands within the same tissue. The pan-cadherin antibodies also displayed a broad interspecies cross reactivity, recognizing cadherins in man, bovine, canine, avian, amphibian and teleost cells. This property renders these antibodies excellent reagents for the cloning and identification of novel cadherins. Immunocytochemical labelling with the pan-cadherin antibodies, at the light- and electron-microscope levels, revealed an extensive reactivity with intercellular adherens junctions in cardiac muscle and in various epithelia. We thus propose that the pan-cadherin antibodies may be used as ubiquitous cadherin probes and serve as markers for adherens junctions.

Cleavage of A-CAM by endogenous proteinases in cultured lens cells and in developing chick embryos.

We describe two truncated forms of A-CAM (N-cadherin) and present evidence suggesting that both forms are proteolytically derived from the intact A-CAM molecule. The first is a membrane-bound fragment of A-CAM displaying an apparent molecular weight of 78 kDa. This polypeptide, containing the C-terminal portion of the protein, may be generated in cultured chicken lens cells, either by a short treatment with trypsin-EGTA, or by endogenous proteinase(s) during incubation in low Ca2+ medium. Immunofluorescent labeling of normal and EGTA-treated cells indicated that the 78-kDa fragment is uniformly distributed over the cell surface. Moreover, staining of developing chick embryos with pairs of antibodies which distinguish the 78-kDa fragment from intact A-CAM indicated that, at early stages of sclerotome dissociation in developing somites, a truncated derivative of the molecule is generated. The second truncated form of A-CAM is a 97-kDa polypeptide which is constitutively released by cultured lens cells into the culture medium in the presence of normal medium. We present evidence that the 97-kDa molecule is proteolytically derived from A-CAM by the action of an endogenous proteinase. We discuss possible mechanisms leading to the formation of these two truncated derivatives and their possible involvement in the physiological modulation of A-CAM-mediated interactions.

Spatial and temporal distribution of the adherens-junction-associated adhesion molecule A-CAM during avian embryogenesis.

A-CAM (adherens-junction-specific cell adhesion molecule) is a calcium-dependent adhesion molecule which is associated with intercellular adherens junctions in various tissues (Volk & Geiger, 1986, J. Cell Biol. 103, 1441-1450 and 1451-1464). In the present report, we have investigated the distribution of A-CAM during avian morphogenesis by immunofluorescence microscopy and immunoblotting. A-CAM appeared at the onset of gastrulation on developing mesodermal and endodermal cells and was then expressed on tissues derived from the three primary germ layers. During embryonic life, A-CAM was constitutively expressed in a number of tissues including the central and peripheral nervous system, myocardium, muscles, notochord, skin and lens whereas it was found transiently in many tissues ranging from the nephritic tubules and the endoderm of visceral arches to ectodermal placodes. In the adult, in addition to the nervous system, A-CAM was restricted to the skin, lens, heart and testis, and exhibited an apparent molecular weight higher than the one found in the embryo. The prevalence and cell-surface modulation of A-CAM could frequently be correlated with morphogenetic events such as mesenchyme condensation into epithelia or cell clusters (e.g. formation of the somitic epithelium, kidney tubules and peripheral ganglia), dissociation of epithelia (e.g. dissociation of the somitic epithelium and segregation of neural crest from the neural tube), separation of cell populations (e.g. fibroblasts and myotubes in the heart) and reorganizations of epithelia (e.g. neurulation). In addition, using electron microscopy, the expression of A-CAM on the surface of aggregating and separating cells could be correlated with the formation and disappearance of adherens junctions. This precisely scheduled control of A-CAM correlated with early morphogenetic events during embryogenesis suggests that this CAM could play a crucial role in these processes.

Molecular interactions in adherens-type contacts.

Adherens junctions are members of a molecularly and structurally heterogeneous family of cell contacts sharing a common association with the microfilament system. Various topics related to the biogenesis of these cellular contacts and the molecular interactions involved in their formation are discussed. The role of vinculin, a cytoplasmic 'plaque' component present in all adherens junctions tested to date and its possible interactions with the other junctional domains have been investigated by both biochemical analyses and studies of molecular dynamics in microinjected living cells. The importance of A-CAM, which apparently functions as a 'junctional receptor' is emphasized and its roles in junction formation in cell cultures and in developing embryos are discussed. In addition, its relationship to other Ca2+-dependent cell adhesion molecules (in particular L-CAM) is considered. The evidence indicating that the level of expression of vinculin-specific mRNA is affected by culture conditions and may be markedly modulated by changes in the adhesiveness of the substratum on which the cells grow is reviewed.