Bruno Constantin

Expression of VEGF, semaphorin SEMA3F, and their common receptors neuropilins NP1 and NP2 in preinvasive bronchial lesions, lung tumours, and cell lines

Two receptors, neuropilin 1 (NP1) and neuropilin 2 (NP2), bind class 3 semaphorins, axon guidance molecules including SEMA3F, the gene for which was isolated from a 3p21.3 deletion in lung cancer. In addition, they bind VEGF (vascular endothelial growth factor), enhancing the effects of VEGF binding to KDR/Flk‐1. Elevated VEGF levels are associated with the loss and cytoplasmic delocalization of SEMA3F in lung cancer, suggesting competition for their NP1 and NP2 receptors. To determine the timing of these events, we compared by immunohistochemistry VEGF, SEMA3F, NP1 and NP2 expression in 50 preneoplastic lesions and 112 lung tumours. In preneoplastic lesions, VEGF increased from low‐grade to high‐grade dysplasia (p = 0.001) whereas SEMA3F levels remained low. NP1 and NP2 levels increased from dysplasia to microinvasive carcinoma (p = 0.0001) and correlated with VEGF expression (p = 0.04 and 0.0002, respectively). Non‐small cell lung carcinoma overexpressed VEGF and NP1 and NP2 significantly more often than neuroendocrine tumours including small cell lung carcinoma. SEMA3F loss or delocalization correlated with advanced tumour stage. Migrating cells overexpressed VEGF, SEMA3F, NP1 and NP2 with cytoplasmic delocalization of NP1 as demonstrated in an in vitro wound assay. These results demonstrate early alteration of the VEGF/SEMA3F/NP pathway in lung cancer progression. Copyright

Semaphorin SEMA3F Localization in Malignant Human Lung and Cell Lines: A Suggested Role in Cell Adhesion and Cell Migration

Semaphorins/collapsins are a family of secreted and membrane-associated proteins involved in nerve growth cone migration. However, some are expressed widely in adult tissues suggesting additional functions. SEMA3F/H.SemaIV was previously isolated from a 3p21.3 homozygous deletion region in human lung cancer. We studied SEMA3F cellular localization using our previously characterized anti-SEMA3F antibody. In normal lung, SEMA3F was found in all epithelial cells at the cytoplasmic membrane and, to a lesser extent, in the cytoplasm. In lung tumors, the localization was predominantly cytoplasmic, and the levels were comparatively reduced. In non-small-cell lung carcinomas, low levels correlated with higher stage. In all tumors, an exclusive cytoplasmic localization of SEMA3F correlated with high levels of vascular endothelial growth factor and was related to the grade and aggressiveness. This suggests that vascular endothelial growth factor might compete with SEMA3F for binding to their common receptors, neuropilin-1 and -2 and might contribute to SEMA3F delocalization and deregulation in lung tumor. In parallel studies, SEMA3F distribution was examined in cell cultures by confocal microscopy. Marked staining was observed in pseudopods and in the leading edge or ruffling membranes of lamellipods or cellular protrusions in motile cells. SEMA3F was also observed at the interface of adjacent interacting cells suggesting a role in cell motility and cell adhesion.

Semaphorin SEMA3F and VEGF have opposing effects on cell attachment and spreading.

SEMA3F, isolated from a 3p21.3 deletion, has antitumor activity in transfected cells, and protein expression correlates with tumor stage and histology. In primary tumors, SEMA3F and VEGF surface staining is inversely correlated. Coupled with SEMA3F at the leading edge of motile cells, we previously suggested that both proteins competitively regulate cell motility and adhesion. We have investigated this using the breast cancer cell line, MCF7. SEMA3F inhibited cell attachment and spreading as evidenced by loss of lamellipodia extensions, membrane ruffling, and cell-cell contacts, with cells eventually rounding-up and detaching. In contrast, VEGF had opposite effects. Although SEMA3F binds NRP2 with 10-fold greater affinity than NRP1, the effects in MCF7 were mediated by NRP1. This was determined by receptor expression and blocking of anti-NRP1 antibodies. Similar effects, but through NRP2, were observed in the C100 breast cancer cell line. Although we were unable to demonstrate changes in total GTP-bound Rac1 or RhoA, we did observe changes in the localization of Rac1-GFP using time lapse microscopy. Following SEMA3F, Rac1 moved to the base of lamellipodia and - with their collapse - to the membrane. These results support the concept that SEMA3F and VEGF have antagonistic actions affecting motility in primary tumor cell.

Regulation of capacitative calcium entries by α1-syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of α1-syntrophin

Calcium mishandling in Duchenne dystrophic muscle suggested that dystrophin, a membrane‐associated cytoskeleton protein, might regulate calcium signaling cascade such as calcium influx pathway. It was previously shown that abnormal calcium entries involve uncontrolled stretch‐activated currents and store‐operated Ca2+ currents supported by TRPC1 channels. Moreover, our recent work demonstrated that reintroduction of minidystrophin in dystrophic myotubes restores normal capacitative calcium entries (CCEs). However, until now, no molecular link between the dystrophin complex and calcium entry channels has been described. This study is the first to show by coimmunoprecipitation assays the molecular association of TRPC1 with dystrophin and α1‐syntrophin in muscle cells. TRPC1 was also associated with α1‐syntrophin in dystrophic muscle cells independently of dystrophin. Furthermore, glutathione S‐transferase (GST) pull‐down assays showed that TRPC1 binds to the α1‐syntrophin PDZ domain. Transfected recombinant α1‐syntrophin formed a complex with TRPC1 channels and restored normal CCEs in dystrophic muscle cells. We suggest that normal regulation of CCEs in skeletal muscle depends on the association between TRPC1 channels and α1‐syntrophin that may anchor the store‐operated channels to the dystrophin‐associated protein complex (DAPC). The loss of this molecular association could participate in the calcium alterations observed in dystrophic muscle cells. This study provides a new model for the regulation of calcium influx by interaction with the scaffold of the DAPC in muscle cells.—Vandebrouck, A., Sabourin, J., Rivet, J., Balghi, H., Sebille, S., Kitzis, A., Raymond, G., Cognard, C., Bourmeyster, N., Constantin, B. Regulation of capacitative calcium entries by α1‐syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of α1‐syntrophin. FASEB J. 21, 608–617 (2007)

Dystrophin complex functions as a scaffold for signalling proteins

Dystrophin is a 427kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force transduction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaffold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle cytoskeleton, and β-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. The adaptor proteins syntrophin contribute to recruit and regulate various signalling proteins such as ion channels, into a macromolecular complex. Although dystrophin and dystroglycan can be directly involved in signalling pathways, syntrophins play a central role in organizing signalplex anchored to the dystrophin scaffold. The dystrophin associated complex, can bind up to four syntrophin through binding domains of dystrophin and dystrobrevin, allowing the scaffold of multiple signalling proteins in close proximity. Multiple interactions mediated by PH and PDZ domains of syntrophin also contribute to build a complete signalplex which may include ion channels, such as voltage-gated sodium channels or TRPC cation channels, together with, trimeric G protein, G protein-coupled receptor, plasma membrane calcium pump, and NOS, to enable efficient and regulated signal transduction and ion transport. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Regulation of TRPC1 and TRPC4 Cation Channels Requires an α1-Syntrophin-dependent Complex in Skeletal Mouse Myotubes

The dystrophin-associated protein complex (DAPC) is essential for skeletal muscle, and the lack of dystrophin in Duchenne muscular dystrophy results in a reduction of DAPC components such as syntrophins and in fiber necrosis. By anchoring various molecules, the syntrophins may confer a role in cell signaling to the DAPC. Calcium disorders and abnormally elevated cation influx in dystrophic muscle cells have suggested that the DAPC regulates some sarcolemmal cationic channels. We demonstrated previously that mini-dystrophin and α1-syntrophin restore normal cation entry in dystrophin-deficient myotubes and that sarcolemmal TRPC1 channels associate with dystrophin and the bound PDZ domain of α1-syntrophin. This study shows that small interfering RNA (siRNA) silencing of α1-syntrophin dysregulated cation influx in myotubes. Moreover, deletion of the PDZ-containing domain prevented restoration of normal cation entry by α1-syntrophin transfection in dystrophin-deficient myotubes. TRPC1 and TRPC4 channels are expressed at the sarcolemma of muscle cells; forced expression or siRNA silencing showed that cation influx regulated by α1-syntrophin is supported by TRPC1 and TRPC4. A molecular association was found between TRPC1 and TRPC4 channels and the α1-syntrophin-dystrophin complex. TRPC1 and TRPC4 channels may form sarcolemmal channels anchored to the DAPC, and α1-syntrophin is necessary to maintain the normal regulation of TRPC-supported cation entry in skeletal muscle. Cation channels with DAPC form a signaling complex that modulates cation entry and may be crucial for normal calcium homeostasis in skeletal muscles.

Differential interaction and activation of Rho family GTPases by p210bcr-abl and p190bcr-abl.

The p210bcr-abl and p190bcr-abl fusion proteins, respectively responsible for chronic myelogenous leukemia and acute lymphoblastic leukemia, present deregulated tyrosine kinase activity and abnormal localization. The Dbl homology domain of Bcr, activating Rho GTPases, is present in p210bcr-abl, but absent in p190bcr-abl. We investigated the interaction of Bcr-Abl chimeras and Rho proteins by coimmunoprecipitation, pull-down experiments and GEF activity measurement. RhoA, Rac1 and Cdc42 interact in vivo with p210bcr-abl only. Moreover, the three types of GTPases are activated in vitro and in vivo by p210bcr-abl. Nevertheless, Rac1 and Cdc42, but not RhoA, are activated by p190bcr-abl in vitro and in vivo. Part of this GEF activity of p190bcr-abl is probably attributable to p95vav, which is complexed with both p190bcr-abl and p210bcr-abl in an activated form. p160bcr, also in complex with Bcr-Abl, presents no GEF activity in p190bcr-abl-expressing cells. These results suggest that differential activation of Rho proteins should play a major role in Bcr-Abl-induced leukemogenesis.

Plasma membrane calcium channels in cancer: Alterations and consequences for cell proliferation and migration.

The study of calcium channels in molecular mechanisms of cancer transformation is still a novel area of research. Several studies, mostly conducted on cancer cell lines, however support the idea that a diversity of plasma membrane channels participates in the remodeling of Ca2+ homeostasis, which regulates various cancer hallmarks such as uncontrolled multiplication and increase in migration and invasion abilities. However few is still understood concerning the intracellular signaling cascades mobilized by calcium influx participating to cancer cell behavior. This review intends to gather some of these pathways dependent on plasma membrane calcium channels and described in prostate, breast and lung cancer cell lines. In these cancer cell types, the calcium channels involved in calcium signaling pathways promoting cancer behaviors are mostly non-voltage activated calcium channels and belong to the TRP superfamily (TRPC, TPRPV and TRPM families) and the Orai family. TRP and Orai channels are part of many signaling cascades involving the activation of transmembrane receptors by extracellular ligand from the tumor environment. TRPV can sense changes in the physical and chemical environment of cancer cells and TRPM7 are stretch activated and sensitive to cholesterol. Changes in activation and or expression of plasma-membrane calcium channels affect calcium-dependent signaling processes relevant to tumorigenesis. The studies cited in this review suggest that an increase in plasma membrane calcium channel expression and/or activity sustain an elevated calcium entry (constitutive or under the control of extracellular signals) promoting higher cell proliferation and migration in most cases. A variety of non-voltage-operated calcium channels display change expression and/or activity in a same cancer type and cooperate to the same process relevant to cancer cell behavior, or can be involved in a different sequence of events during the tumorigenesis. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

Myoblast fusion requires cytosolic calcium elevation but not activation of voltage-dependent calcium channels

Many studies of in vitro skeletal myogenesis have demonstrated that fusion of myoblasts into multinucleated myotubes is regulated by calcium-dependent processes. Calcium ions appear to be necessary at the outer face of the membrane, and an additional internal calcium increase seems required to promote fusion of aligned myoblasts. It has been proposed that a calcium influx could take place prior to fusion and that this may be mediated by voltage-dependent calcium channels. Previously, we showed that two types of voltage-dependent calcium currents were expressed in multinucleated myotubes but not in rat myoblasts growing in primary culture before the withdrawal of the growth medium. We also showed that the previous formation of multinucleated synticia was not a prerequisite of developmental appearance of calcium currents, suggesting that the two events were time-correlated but not sequentially dependent. These features led us to investigate changes in internal calcium activity and the possible appearance of voltage-dependent calcium influx pathways just after the promotion of fusion by the change of culture medium. The results confirm that a rise in cytosolic calcium activity occurs slightly before fusion in confluent myoblasts and remained in newly formed myotubes. Reducing this elevation by internal calcium buffering lowered myoblast fusion and, reciprocally, blocking cell fusion prevented calcium increase. Treatment with the organic calcium channel blockers nifedipine (5 microM) and PN 200-110 (1 microM) did not alter cytosolic calcium changes nor cell fusion, and voltage-dependent calcium currents were never observed by the perforated patch-clamp technique in aligned fusion-competent myoblasts. Other voltage-operated mechanisms of calcium rise were not detected since depolarization with hyperpotassium solutions failed to elicit increases in intracellular calcium. On the contrary, acetylcholine was able to promote extracellular calcium-dependent calcium transients. Our results confirm the requirement of an increase in resting calcium during fusion, but do not support the hypothesis of an influx through voltage-dependent channels or other voltage-operated pathways. The elevation of internal calcium activity may result from other mechanisms, such as a cholinergic action for example.

New insights in the regulation of calcium transfers by muscle dystrophin-based cytoskeleton: implications in DMD

Calcium mishandling in Duchenne muscular dystrophy (DMD) suggested that dystrophin, a membrane-associated cytoskeleton protein, may regulate calcium-signalling cascades such as calcium entries. Calcium overload in human DMD myotubes is dependent on their contractile activity suggesting the involvement of channels being activated during contraction and/or calcium release. Forced expression of mini-dystrophin in dystrophin-deficient myotubes, reactivates appropriate sarcolemmal expression of dystrophin-associated proteins and restores normal calcium handling in the cytosol. Furthermore, the recombinant mini-dystrophin reduced the store-operated calcium influx across the sarcolemma, and the mitochondrial calcium uptake during this influx. A slow component of calcium release dependent on IP3R, as well as the production of IP3, were also reduced to normal levels by expression of mini-dystrophin. Our studies provide a new model for the convergent regulation of transmembrane calcium influx and IP3-dependent calcium release by the dystrophin-based cytoskeleton (DBC). We also suggest molecular association of such channels with DBC which may provide the scaffold for assembling a multiprotein-signalling complex that modulates the channel activity. This suggests that the loss of this molecular association could participate in the alteration of calcium homeostasis observed in DMD muscle cells.