Morphology of Abdominal Aortic Aneurysms and Correlation with Biomechanical Tests of Aneurysmal Wall Fragments.

BACKGROUND: Evaluate how specific morphologic aspects of abdominal aortic aneurysms (AAAs), including asymmetries, curvatures, tortuosities, and angulations, among others can influence the intrinsic biomechanical properties of the AAA's wall. This study analyzed the correlation of geometric measurements (1-dimensional, 2-dimensional, and 3-dimensional) of preoperative tomographic images of AAA with uniaxial biomechanical tests of the arterial wall fragments of these AAA obtained in open surgical repair of aneurysms. METHODS: It was a multicenter, experimental, and observational study, and initially 54 individuals were selected who underwent open surgical of AAA, with valid biomechanical tests of the anterior wall of the AAA. Seven individuals were excluded because they had poor preoperative quality computed tomography scans and/or artifacts that impeded image segmentation and extraction of AAA geometric indices. The aortic fragments were subjected to uniaxial biomechanical destructive tests to obtain the following data: maximum load, failure stress, failure tension, failure strain energy, strain, and fragment thickness. In the same patients, preoperative computed tomography scans were performed with the extraction of 26 geometric indices, subdivided into 9 1-dimensional indices, 6 2-dimensional indices, and 11 3-dimensional indices. Data were subjected to statistical analysis using SPSS version 28. RESULTS: Comparing ruptured and unruptured AAA, no statistical difference was observed between the biomechanical and geometric parameters. The fragment thickness of the ruptured AAA was lower than that of the unruptured AAA (P < 0.05). By comparing tomographic geometric indices and biomechanical parameters of the aortic fragments using Pearson's coefficient, positive and linear correlations (P < 0.05) were observed between the geometric variable maximum diameter (Dmax) of the AAA with maximum load (r = 0.408), failure tension (r = 0.372), and failure stress (r = 0.360). Positive and linear correlations were also observed between the variable diameter/height ratio (DHr) and the maximum load (r = 0.360), failure tension (r = 0.354), and failure stress (r = 0.289). The geometric variable DHr was dependent and correlated with Dmax. Simple regression analysis showed that R2 varied between 8.3% and 16.7%, and all models were significant (P < 0.05). CONCLUSIONS: Dmax and DHr were linearly and positively correlated with the resistance parameters (maximum load, failure tension, and failure stress) of the AAA fragments. The DHr variable is dependent and correlated with Dmax. There was no correlation between the other geometric indices and the biomechanical parameters of the AAA wall. The asymmetries did not globally influence the biomechanics of AAA wall; however, they may influence regionally. Larger AAAs were stronger than smaller ones. Therefore, such findings may point toward Dmax is still the main geometric parameter, which influences the anterior wall, and possibly globally in the AAA.

Neural stem cell self-renewal stimulation by store-operated calcium entries in adult mouse area postrema: influence of leptin.

Neural stem cells (NSCs) persist in specific brain germinative niches and sustain neurogenesis throughout life in adult mammals. In addition to the two major stem cell niches in the subventricular zone and the hippocampal dentate gyrus, the area postrema located in the brainstem has been identified as a neurogenic zone as well. NSCs are regulated by signals from the microenvironment that adjust stem cell response to the needs of the organism. Evidence accumulated over the past decade indicates that Ca2+ channels play pivotal functions in NSC maintenance. In this study, we explored in area postrema NSCs the presence and roles of a subset of Ca2+ channels, the store-operated Ca2+ channels (SOCs) that have the capacity to transduce extracellular signals into Ca2+ signals. Our data show that NSCs derived from the area postrema express TRPC1 and Orai1, known to form SOCs, as well as their activator STIM1. Ca2+ imaging indicated that NSCs exhibit store-operated Ca2+ entries (SOCEs). Pharmacological blockade of SOCEs with SKF-96365, YM-58483 (also known as BTP2) or GSK-7975A resulted in decreased NSC proliferation and self-renewal, indicating a major role for SOCs in maintaining NSC activity within the area postrema. Furthermore, our results show that leptin, an adipose tissue-derived hormone whose ability to control energy homeostasis is dependent on the area postrema, decreased SOCEs and reduced self-renewal of NSCs in the area postrema. As aberrant SOC function has been linked to an increasing number of diseases, including brain disorders, our study opens new perspectives for NSCs in brain pathophysiology.

The mechanosensitive TRPV2 calcium channel promotes human melanoma invasiveness and metastatic potential.

Melanoma is a highly aggressive cancer endowed with a unique capacity of rapidly metastasizing, which is fundamentally driven by aberrant cell motility behaviors. Discovering "migrastatics" targets, specifically controlling invasion and dissemination of melanoma cells during metastasis, is therefore of primary importance. Here, we uncover the prominent expression of the plasma membrane TRPV2 calcium channel as a distinctive feature of melanoma tumors, directly related to melanoma metastatic dissemination. In vitro as well as in vivo, TRPV2 activity is sufficient to confer both migratory and invasive potentials, while conversely TRPV2 silencing in highly metastatic melanoma cells prevents aggressive behavior. In invasive melanoma cells, TRPV2 channel localizes at the leading edge, in dynamic nascent adhesions, and regulates calcium-mediated activation of calpain and the ensuing cleavage of the adhesive protein talin, along with F-actin organization. In human melanoma tissues, TRPV2 overexpression correlates with advanced malignancy and poor prognosis, evoking a biomarker potential. Hence, by regulating adhesion and motility, the mechanosensitive TRPV2 channel controls melanoma cell invasiveness, highlighting a new therapeutic option for migrastatics in the treatment of metastatic melanoma.

Challenges in glioblastoma research: focus on the tumor microenvironment.

Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.

Store-Operated Calcium Channels Control Proliferation and Self-Renewal of Cancer Stem Cells from Glioblastoma.

Glioblastoma is the most frequent and deadly form of primary brain tumors. Despite multimodal treatment, more than 90% of patients experience tumor recurrence. Glioblastoma contains a small population of cells, called glioblastoma stem cells (GSC) that are highly resistant to treatment and endowed with the ability to regenerate the tumor, which accounts for tumor recurrence. Transcriptomic studies disclosed an enrichment of calcium (Ca2+) signaling transcripts in GSC. In non-excitable cells, store-operated channels (SOC) represent a major route of Ca2+ influx. As SOC regulate the self-renewal of adult neural stem cells that are possible cells of origin of GSC, we analyzed the roles of SOC in cultures of GSC previously derived from five different glioblastoma surgical specimens. Immunoblotting and immunocytochemistry experiments showed that GSC express Orai1 and TRPC1, two core SOC proteins, along with their activator STIM1. Ca2+ imaging demonstrated that SOC support Ca2+ entries in GSC. Pharmacological inhibition of SOC-dependent Ca2+ entries decreased proliferation, impaired self-renewal, and reduced expression of the stem cell marker SOX2 in GSC. Our data showing the ability of SOC inhibitors to impede GSC self-renewal paves the way for a strategy to target the cells considered responsible for conveying resistance to treatment and tumor relapse.

Natural History of Splanchnic Artery Aneurysms.

INTRODUCTION: Splanchnic artery aneurysms (SAAs) represent a rare and potential life-threatening disease with a documented incidence of 0.1-2.0%. The risk of rupture and the diameter to recommend surgery are still controversial. The purpose of this study was to review surveillance computed tomography scans (CTs) at a high-volume institution in order to better define the natural history of the SAA. METHODS: Between January 2000 and February 2019, all SAAs patients in follow-up at a single center institution were selected for analysis. CTs from patients managed nonoperatively and CTs before surgery from patients submitted to surgery were studied. The first CTs were used to determine aneurysm size, morphology, and anatomic characteristics, and the last CTs performed during nonoperative follow-up were used to compare the diameter with the previous CTs. Primary endpoint included growth rate for all SAAs location, and secondary endpoint included the clinical or anatomical characteristic associated with a faster growth rate. RESULTS: In total, 116 consecutive patients were identified with SAAs and 74 patients with 87 SAAs who had at least 2 CTs during follow-up were analyzed. From those 74 patients, 12 were submitted to surgery and only their preoperative CTs were analyzed. The SAAs' locations were: splenic (55.4%), hepatic (12.2%), superior mesenteric artery (17.6%), celiac trunk (27.0%), gastric and gastroepiploic arteries (1.4%), pancreaticoduodenal and gastroduodenal arteries (4.1%). The median follow-up for all patients was 46.7 months (±35.3), and the median of growth for all aneurysms was 0.63 mm/year (±2.19). Only the splenic aneurysms presented growth with statistic significance of 1.08 mm per/year (±1.99) (P < 0.001). Only portal hypertension showed statistically significance to splenic aneurysm growth (P = 0.002). Multivariate analysis for variables associated with splenic aneurysm growth ≥1 mm/year showed that portal hypertension was the only variable with statistical significance (P < 0.01, IC 95% 2.0-186.9, ß = 19.5). CONCLUSIONS: Although longer-term follow-up and larger sample size are needed to better understand the natural history of SAAs, the majority of SAAs tends to remain stable in size through follow-up. Portal hypertension was the only risk factor found for true splenic aneurysm growth, and so those patients must have a closer follow-up.

Calcium Channels in Adult Brain Neural Stem Cells and in Glioblastoma Stem Cells.

The brain of adult mammals, including humans, contains neural stem cells (NSCs) located within specific niches of which the ventricular-subventricular zone (V-SVZ) is the largest one. Under physiological conditions, NSCs proliferate, self-renew and produce new neurons and glial cells. Several recent studies established that oncogenic mutations in adult NSCs of the V-SVZ are responsible for the emergence of malignant primary brain tumors called glioblastoma. These aggressive tumors contain a small subpopulation of cells, the glioblastoma stem cells (GSCs), that are endowed with proliferative and self-renewal abilities like NSCs from which they may arise. GSCs are thus considered as the cells that initiate and sustain tumor growth and, because of their resistance to current treatments, provoke tumor relapse. A growing body of studies supports that Ca2+ signaling controls a variety of processes in NSCs and GSCs. Ca2+ is a ubiquitous second messenger whose fluctuations of its intracellular concentrations are handled by channels, pumps, exchangers, and Ca2+ binding proteins. The concerted action of the Ca2+ toolkit components encodes specific Ca2+ signals with defined spatio-temporal characteristics that determine the cellular responses. In this review, after a general overview of the adult brain NSCs and GSCs, we focus on the multiple roles of the Ca2+ toolkit in NSCs and discuss how GSCs hijack these mechanisms to promote tumor growth. Extensive knowledge of the role of the Ca2+ toolkit in the management of essential functions in healthy and pathological stem cells of the adult brain should help to identify promising targets for clinical applications.

Role of the calcium toolkit in cancer stem cells.

Cancer stem cells are a subpopulation of tumor cells that proliferate, self-renew and produce more differentiated tumoral cells building-up the tumor. Responsible for the sustained growth of malignant tumors, cancer stem cells are proposed to play significant roles in cancer resistance to standard treatment and in tumor recurrence. Among the mechanisms dysregulated in neoplasms, those related to Ca2+ play significant roles in various aspects of cancers. Ca2+ is a ubiquitous second messenger whose fluctuations of its intracellular concentrations are tightly controlled by channels, pumps, exchangers and Ca2+ binding proteins. These components support the genesis of Ca2+ signals with specific spatio-temporal characteristics that define the cell response. Being involved in the coupling of extracellular events with intracellular responses, the Ca2+ toolkit is often hijacked by cancer cells to promote notably their proliferation and invasion. Growing evidence obtained during the last decade pointed to a role of Ca2+ handling and mishandling in cancer stem cells. In this review, after a general overview of the concept of cancer stem cells we analyse and discuss the studies and current knowledge regarding the complex roles of Ca2+ toolkit and signaling in these cells. We highlight that numbers of Ca2+ signaling actors promote cancer stem cell state and are associated with cell resistance to current cancer treatments and thus may represent promising targets for potential clinical applications.

Deregulation of calcium homeostasis in Bcr-Abl-dependent chronic myeloid leukemia.

BACKGROUND: Chronic myeloid leukemia (CML) results from hematopoietic stem cell transformation by the bcr-abl chimeric oncogene, encoding a 210 kDa protein with constitutive tyrosine kinase activity. In spite of the efficiency of tyrosine kinase inhibitors (TKI; Imatinib), other strategies are explored to eliminate CML leukemia stem cells, such as calcium pathways. RESULTS: In this work, we showed that Store-Operated Calcium Entry (SOCE) and thrombin induced calcium influx were decreased in Bcr-Abl expressing 32d cells (32d-p210). The 32d-p210 cells showed modified Orai1/STIM1 ratio and reduced TRPC1 expression that could explain SOCE reduction. Decrease in SOCE and thrombin induced calcium entry was associated to reduced Nuclear Factor of Activated T cells (NFAT) nucleus translocation in 32d-p210 cells. We demonstrated that SOCE blockers enhanced cell mobility of 32d-p210 cells and reduced the proliferation rate in both 32d cell lines. TKI treatment slightly reduced the thrombin-induced response, but imatinib restored SOCE to the wild type level. Bcr-Abl is also known to deregulate Protein Kinase C (PKC), which was described to modulate calcium entries. We showed that PKC enhances SOCE and thrombin induced calcium entries in control cells while this effect is lost in Bcr-Abl-expressing cells. CONCLUSION: The tyrosine kinase activity seems to regulate calcium entries probably not directly but through a global cellular reorganization involving a PKC pathway. Altogether, calcium entries are deregulated in Bcr-Abl-expressing cells and could represent an interesting therapeutic target in combination with TKI.

Store-Operated Calcium Entries Control Neural Stem Cell Self-Renewal in the Adult Brain Subventricular Zone.

The subventricular zone (SVZ) is the major stem cell niche in the brain of adult mammals. Within this region, neural stem cells (NSC) proliferate, self-renew and give birth to neurons and glial cells. Previous studies underlined enrichment in calcium signaling-related transcripts in adult NSC. Because of their ability to mobilize sustained calcium influxes in response to a wide range of extracellular factors, store-operated channels (SOC) appear to be, among calcium channels, relevant candidates to induce calcium signaling in NSC whose cellular activities are continuously adapted to physiological signals from the microenvironment. By Reverse Transcription Polymerase Chain Reaction (RT-PCR), Western blotting and immunocytochemistry experiments, we demonstrate that SVZ cells express molecular actors known to build up SOC, namely transient receptor potential canonical 1 (TRPC1) and Orai1, as well as their activator stromal interaction molecule 1 (STIM1). Calcium imaging reveals that SVZ cells display store-operated calcium entries. Pharmacological blockade of SOC with SKF-96365 or YM-58483 (also called BTP2) decreases proliferation, impairs self-renewal by shifting the type of SVZ stem cell division from symmetric proliferative to asymmetric, thereby reducing the stem cell population. Brain section immunostainings show that TRPC1, Orai1, and STIM1 are expressed in vivo, in SOX2-positive SVZ NSC. Injection of SKF-96365 in brain lateral ventricle diminishes SVZ cell proliferation and reduces the ability of SVZ cells to form neurospheres in vitro. The present study combining in vitro and in vivo approaches uncovers a major role for SOC in the control of SVZ NSC population and opens new fields of investigation for stem cell biology in health and disease. Stem Cells 2018;36:761-774.

Ca2+ protein alpha 1D of CaV1.3 regulates intracellular calcium concentration and migration of colon cancer cells through a non-canonical activity.

It is generally accepted that voltage-gated Ca2+ channels, CaV, regulate Ca2+ homeostasis in excitable cells following plasma membrane depolarization. Here, we show that the Ca2+ protein α1D of CaV1.3 channel is overexpressed in colorectal cancer biopsies compared to normal tissues. Gene silencing experiments targeting α1D reduced the migration and the basal cytosolic Ca2+ concentration of HCT116 colon cancer cell line and modified the cytosolic Ca2+ oscillations induced by the sodium/calcium exchanger NCX1/3 working in its reverse mode. Interestingly, NCX1/3 regulated membrane potential of HCT116 cells only when α1D was silenced, and blocking NCX1/3 increased cytosolic Ca2+ concentration and cell migration. However, membrane depolarization did not induce an increase in intracellular Ca2+. Patch-clamp experiments clearly showed that the inward Ca2+ current was absent. Finally, flow cytometry and immunofluorescence studies showed that α1D protein was localized at the plasma membrane, in cytosol and cell nuclei. Altogether, we uncover a novel signaling pathway showing that α1D is involved in the regulation of Ca2+ homeostasis and cell migration by a mechanism independent of its plasma membrane canonical function but that involved plasma membrane Na+/Ca2+ exchanger.

Constitutive calcium entry and cancer: updated views and insights.

Tight control of basal cytosolic Ca2+ concentration is essential for cell survival and to fine-tune Ca2+-dependent cell functions. A way to control this basal cytosolic Ca2+ concentration is to regulate membrane Ca2+ channels including store-operated Ca2+ channels and secondary messenger-operated channels linked to G-protein-coupled or tyrosine kinase receptor activation. Orai, with or without its reticular STIM partner and Transient Receptor Potential (TRP) proteins, were considered to be the main Ca2+ channels involved. It is well accepted that, in response to cell stimulation, opening of these Ca2+ channels contributes to Ca2+ entry and the transient increase in cytosolic Ca2+ concentration involved in intracellular signaling. However, in various experimental conditions, Ca2+ entry and/or Ca2+ currents can be recorded at rest, without application of any experimental stimulation. This led to the proposition that some plasma membrane Ca2+ channels are already open/activated in basal condition, contributing therefore to constitutive Ca2+ entry. This article focuses on direct and indirect observations supporting constitutive activity of channels belonging to the Orai and TRP families and on the mechanisms underlying their basal/constitutive activities.

Role of Scaffolding Proteins in the Regulation of TRPC-Dependent Calcium Entry.

Plasma membrane ion channels, and in particular TRPC channels need a specific membrane environment and association with scaffolding, signaling, and cytoskeleton proteins in order to play their important functional role. The molecular composition of TRPC channels is an important factor in determining channel activation mechanisms. TRPC proteins are incorporated in macromolecular complexes including several key Ca(2 +) signaling proteins as well as proteins involved in vesicle trafficking, cytoskeletal interactions, and scaffolding. Evidence has been provided for association of TRPC with calmodulin (CaM), IP3R, PMCA, Gq/11, RhoA, and a variety of scaffolding proteins. The interaction between TRPC channels with adaptor proteins, determines their mode of regulation as well as their cellular localization and function. Adaptor proteins do not display any enzymatic activity but act as scaffold for the building of signaling complexes. The scaffolding proteins are involved in the assembling of these Ca(2+) signaling complexes, the correct sub-cellular localization of protein partners, and the regulation of the TRPC channelosome. In particular, these proteins, via their multiple protein-protein interaction motifs, can interact with various ion channels involved in the transmembrane potential, and membrane excitability. Scaffolding proteins are key components for the functional organization of TRPC channelosomes that serves as a platform regulating slow Ca(2+) entry, spatially and temporally controlled [Ca(2+)]i signals and Ca(2+) -dependent cellular functions.

SK3/TRPC1/Orai1 complex regulates SOCE-dependent colon cancer cell migration: a novel opportunity to modulate anti-EGFR mAb action by the alkyl-lipid Ohmline.

BACKGROUND: Barely 10-20% of patients with metastatic colorectal cancer (mCRC) receive a clinical benefit from the use of anti-EGFR monoclonal antibodies (mAbs). We hypothesized that this could depends on their efficiency to reduce Store Operated Calcium Entry (SOCE) that are known to enhance cancer cells. RESULTS: In the present study, we demonstrate that SOCE promotes migration of colon cancer cell following the formation of a lipid raft ion channel complex composed of TRPC1/Orai1 and SK3 channels. Formation of this complex is stimulated by the phosphorylation of the reticular protein STIM1 by EGF and activation of the Akt pathway. Our data show that, in a positive feedback loop SOCE activates both Akt pathway and SK3 channel activity which lead to SOCE amplification. This amplification occurs through the activation of Rac1/Calpain mediated by Akt. We also show that Anti-EGFR mAbs can modulate SOCE and cancer cell migration through the Akt pathway. Interestingly, the alkyl-lipid Ohmline, which we previously showed to be an inhibitor of SK3 channel, can dissociated the lipid raft ion channel complex through decreased phosphorylation of Akt and modulation of mAbs action. CONCLUSIONS: This study demonstrates that the inhibition of the SOCE-dependent colon cancer cell migration trough SK3/TRPC1/Orai1 channel complex by the alkyl-lipid Ohmline may be a novel strategy to modulate Anti-EGFR mAb action in mCRC.

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.

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 ß-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.

Involvement of TRPV2 and SOCE in calcium influx disorder in DMD primary human myotubes with a specific contribution of α1-syntrophin and PLC/PKC in SOCE regulation.

Calcium homeostasis is critical for several vital functions in excitable and nonexcitable cells and has been shown to be impaired in many pathologies including Duchenne muscular dystrophy (DMD). Various studies using murine models showed the implication of calcium entry in the dystrophic phenotype. However, alteration of store-operated calcium entry (SOCE) and transient receptor potential vanilloid 2 (TRPV2)-dependant cation entry has not been investigated yet in human skeletal muscle cells. We pharmacologically characterized basal and store-operated cation entries in primary cultures of myotubes prepared from muscle of normal and DMD patients and found, for the first time, an increased SOCE in DMD myotubes. Moreover, this increase cannot be explained by an over expression of the well-known SOCE actors: TRPC1/4, Orai1, and stromal interaction molecule 1 (STIM1) mRNA and proteins. Thus we investigated the modes of regulation of this cation entry. We firstly demonstrated the important role of the scaffolding protein α1-syntrophin, which regulates SOCE in primary human myotubes through its PDZ domain. We also studied the implication of phospholipase C (PLC) and protein kinase C (PKC) in SOCE and showed that their inhibition restores normal levels of SOCE in DMD human myotubes. In addition, the involvement of TRPV2 in calcium deregulation in DMD human myotubes was explored. We showed an abnormal elevation of TRPV2-dependant cation entry in dystrophic primary human myotubes compared with normal ones. These findings show that calcium homeostasis mishandling in DMD myotubes depends on SOCE under the influence of Ca(2+)/PLC/PKC pathway and α1-syntrophin regulation as well as on TRPV2-dependant cation influx.

p210bcr-abl induces amoeboid motility by recruiting ADF/destrin through RhoA/ROCK1.

We previously demonstrated that the Bcr-Abl oncogene, p210(bcr-abl), through its unique GEF domain, specifically activates RhoA and induces spontaneous amoeboid motility. We intend to study the pathways downstream RhoA controlling amoeboid motility. Mouse prolymphoblastic cells (Ba/F3 cell line) expressing different forms of Bcr-Abl were embedded in 3-dimensional (3D) Matrigel to study motility and explore the effects of inhibiting Rho pathway (inhibitors and siRNAs). The phosphorylation levels of cofilin-1 and destrin were analyzed by 2-dimensional electrophoresis. Composition of Bcr-Abl signalplex in different conditions was determined by coimmunoprecipitation. Ba/F3p190 and Ba/F3 expressing a mutant form of p210(bcr-abl) (unable to activate RhoA) cells presented a spontaneous motility, but not an amoeboid type. p210(bcr-abl)-induced amoeboid motility in a 3D matrix requires isoform-specific RhoA/ROCK-1/destrin signaling. Next to the conventional Rho/ROCK/MLC/myosin pathway, this pathway is a crucial determinant for amoeboid motility, specific for the destrin isoform (and not its coexpressed homologue cofilin-1). Also, the presence of destrin (and not cofilin-1) in the p210(bcr-abl) complex is dependent on ROCK1, and this signalplex is required for amoeboid motility. This underscores isoform-specific function within the ADF/cofilin family and provides new insight into Bcr-Abl signaling to amoeboid motility and possible impact on understanding chronic myeloid leukemia progression.

Dystrophin/α1-syntrophin scaffold regulated PLC/PKC-dependent store-operated calcium entry in myotubes.

In skeletal muscles from patient suffering of Duchenne Muscular Dystrophy and from mdx mice, the absence of the cytoskeleton protein dystrophin has been shown to be essential for maintaining a normal calcium influx. We showed that a TRPC store-dependent cation influx is increased by loss of dystrophin or a scaffolding protein α1-syntrophin, however the mechanisms of this calcium mishandling are incompletely understood. First of all, we confirmed that TRPC1 but also STIM1 and Orai1 are supporting the store-operated cation entry which is enhanced in dystrophin-deficient myotubes. Next, we demonstrated that inhibition of PLC or PKC in dystrophin-deficient myotubes restores elevated cation entry to normal levels similarly to enforced minidystrophin expression. In addition, silencing α1-syntrophin also increased cation influx in a PLC/PKC dependent pathway. We also showed that α1-syntrophin and PLCß are part of a same protein complex reinforcing the idea of their inter-relation in calcium influx regulation. This elevated cation entry was decreased to normal levels by chelating intracellular free calcium with BAPTA-AM. Double treatments with BAPTA-AM and PLC or PKC inhibitors suggested that the elevation of cation influx by PLC/PKC pathway is dependent on cytosolic calcium. All these results demonstrate an involvement in dystrophin-deficient myotubes of a specific calcium/PKC/PLC pathway in elevation of store-operated cation influx supported by the STIM1/Orai1/TRPC1 proteins, which is normally regulated by the α1-syntrophin/dystrophin scaffold.