Biomedical subjects

Alain Kitzis

Publications and source records attributed to Alain Kitzis.

9 recordsLinked to original sources

PubMed2007

Regulation of capacitative calcium entries by alpha1-syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of alpha1-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 alpha1-syntrophin in muscle cells. TRPC1 was also associated with alpha1-syntrophin in dystrophic muscle cells independently of dystrophin. Furthermore, glutathione S-transferase (GST) pull-down assays showed that TRPC1 binds to the alpha1-syntrophin PDZ domain. Transfected recombinant alpha1-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 alpha1-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.

Animals↗

PubMed2006

Mini-dystrophin expression down-regulates overactivation of G protein-mediated IP3 signaling pathway in dystrophin-deficient muscle cells.

We present here evidence for the enhancement of an inositol 1,4,5-trisphosphate (IP3) mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(-)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, we demonstrated that calcium rise, induced by the perifusion of a solution containing a high potassium concentration, was higher in SolC1(-) than in SolD(+) myotubes. The analysis of amplitude and kinetics of the calcium increase in SolC1(-) and in SolD(+) myotubes during the exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) suggested the presence of two mechanisms of SR calcium release: (1) a fast SR calcium release that depended on ryanodine receptors and (2) a slow SR calcium release mediated by IP3 receptors. Detection analyses of mRNAs (reverse transcriptase [RT]-PCR) and proteins (Western blot and immunolocalization) demonstrated the presence of the three known isoforms of IP3 receptors in both SolC1(-) and SolD(+) myotubes. Furthermore, analysis of the kinetics of the rise in calcium revealed that the slow IP3-dependent release may be increased in the SolC1(-) as compared to the SolD(+), suggesting an inhibitory effect of mini-dystrophin in this signaling pathway. Upon incubation with pertussis toxin (PTX), an inhibitory effect similar to that of the IP3R inhibitor (2-APB) was observed on K+-evoked calcium release. This result suggests the involvement of a Gi protein upstream of the IP3 pathway in these stimulation conditions. A hypothetical model is depicted in which both Gi protein and IP3 production could be involved in K+-evoked calcium release as well as a possible interaction with mini-dystrophin. Our findings demonstrate the existence of a potential relationship between mini-dystrophin and SR calcium release as well as a regulatory role of mini-dystrophin on intracellular signaling.

Animals↗

PubMed2004

The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein.

Cystic fibrosis (CF) is a common lethal genetic disease caused by autosomal recessive mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that belongs to the ATP-Binding Cassette (ABC) family of transporters. The class III CF mutations G551D and G1349D are located within the "signature" sequence LSGGQ and LSHGH of NBD1 and NBD2, respectively. We have constructed by site-directed mutagenesis vectors encoding green fluorescent protein (GFP)-tagged wild-type (wt) CFTR or CFTR containing delF508, G551D, G1349D and G551D/G1349D to study their pharmacology after transient expression in COS-7 cells. We show that IBMX and the benzo[c]quinolizinium derivative MPB-91 stimulates the activity of G1349D-, G551D- and G551D/G1349D-CFTR only in the presence of cAMP-promoting agents like forskolin or cpt-cAMP. Similar half-maximal effective concentrations (EC(50)) of MPB-91 (22-36microM) have been determined for wt-, G551D-, G1349D- and G551D/G1349D-CFTR. The isoflavone genistein stimulates wild-type (wt)- and delF508-CFTR channel activity in a non-Michaelis-Menten manner. By contrast, the response of G1349D- and G551D-CFTR to genistein is dramatically altered. First, genistein is not able to stimulate G1349D- and G551D/G1349D-CFTR. Second, genistein stimulates G551D-CFTR without any inhibition at high concentration. We conclude from these results that whereas G551 in NBD1 is an important molecular site for inhibition of CFTR by genistein, the symmetrical G1349 in NBD2 is also one major site but for the activation of CFTR by genistein. Because both mutations alter specifically the mechanism of CFTR channel activation by genistein, we believe that the signature sequences of CFTR act as molecular switches that upon interaction with genistein turn on and off the channel.

Amino Acid Motifs↗

PubMed2004

Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent chloride channel that mediates electrolyte transport across the luminal surface of epithelial cells. In this paper, we describe the CFTR regulation by syntaxin 8, a t-SNARE protein (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) involved in the SNARE endosomal complex. Syntaxin family members are key molecules implicated in diverse vesicle docking and membrane fusion events. We found that syntaxin 8 physically interacts with CFTR: recombinant syntaxin 8 binds CFTR in vitro and both proteins co-immunoprecipitate in HT29 cells. Syntaxin 8 regulates CFTR-mediated currents in chinese hamster ovary (CHO) cells stably expressing CFTR and syntaxin 8. Iodide efflux and whole-cell patch-clamp experiments on these cells indicate a strong inhibition of CFTR chloride current by syntaxin 8 overexpression. At the cellular level, we observed that syntaxin 8 overexpression disturbs CFTR trafficking. Confocal microscopy shows a dramatic decrease in green fluorescent protein-tagged CFTR plasma membrane staining, when syntaxin 8 is coexpressed in COS-7 cells. Using antibodies against Lamp-1, TfR or Rab11 we determined by immunofluorescence assays that both proteins are mainly accumulated in recycling endosomes. Our results evidence that syntaxin 8 contributes to the regulation of CFTR trafficking and chloride channel activity by the SNARE machinery.

Animals↗

PubMed2004

Regulation of the cystic fibrosis transmembrane conductance regulator channel by beta-adrenergic agonists and vasoactive intestinal peptide in rat smooth muscle cells and its role in vasorelaxation.

The signaling events that regulate vascular tone include voltage-dependent Ca(2+) influx and the activities of various ionic channels; which molecular entities are involved and their role are still a matter of debate. Here we show expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel in rat aortic smooth muscle cells. Immunoprecipitation and in vitro protein kinase A phosphorylation show the appearance of mature band C of CFTR. An immunohistochemistry study shows CFTR proteins in smooth muscles of aortic rings but not in skeletal muscles. Using the iodide efflux method, a combination of agonists and pharmacological agents was used to dissect the function of CFTR. Agonists of the cAMP pathway, the beta-adrenergic agonist isoproterenol, and the neuropeptide vasoactive intestinal peptide activate CFTR-dependent transport from cells maintained in a high but not low extracellular potassium-rich saline, suggesting that depolarization of smooth muscle is critical to CFTR activation. Smooth muscle CFTR possesses all of the pharmacological attributes of its epithelial homologues: stimulation by the CFTR pharmacological activators MPB-07 (EC(50) = 158 microm) and MPB-91 (EC(50) = 20 microm) and inhibition by glibenclamide and diphenylamine-2-carboxylic acid but not by 5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene. Contraction measurements on isolated aortic rings were performed to study the contribution of CFTR to vascular tone. With aortic rings (without endothelium) preconstricted by high K(+) saline or by the alpha-adrenergic agonist norepinephrine, CFTR activators produced a concentration-dependent relaxation. These results identify for the first time the expression and function of CFTR in smooth muscle where it plays an unexpected but fundamental role in the autonomic and hormonal regulation of the vascular tone.

Adrenergic beta-Agonists↗

PubMed2004

Characterization of the different BCR-ABL transcripts with a single multiplex RT-PCR.

The diagnosis of chronic myeloid leukemia is based on detection of the Philadelphia (Ph) chromosome or the BCR-ABL gene. The junction present in the transcript may vary according to the reciprocal translocation t(9;22)(q34;11). Identification of the transcript (p190, p210 or p230) does not reveal the type of junction but this information is very important for classification of patients in clinical trials. Most identification kits do not explore p230 transcripts and are unable to determine exotic breakpoints. We have developed a clinical molecular diagnosis assay, able to identify all of the BCR-ABL transcripts and, by single assay, to characterize all of the possible transcript junctions. This technique is based on RT-PCR and PCR-capillary electrophoresis. For each patient sample, we performed RT-PCR with three different BCR primers each coupled to a specific different fluorochrome and a unique reverse ABL primer. Depending on the transcript, only one BCR primer was used for each RT-PCR. After capillary electrophoresis and fluorescence determination, we were able to identify both the transcript and its junction at the same time.

DNA Primers↗

PubMed2004

The ink4a/arf locus evolution in primates: characterization of three ARF sequences.

The human ink4a/arf locus encodes two cell cycle regulatory proteins - the cyclin-dependent kinase inhibitor (p16(ink4a)), and the p53 activator (ARF) - through the use of alternative first exons. This genomic organization is unique in eukaryotes, with two different proteins obtained using different reading frames. The divergence between mouse or opossum and human ARF is very high, whereas proteins have the same nucleolar localization and function. To gain further insights into the relative importance of ARF in different settings, we characterized here the exon 1beta of ARF in 12 different species of primates. We did not find any polymorphism in studied species (monkeys, apes, and humans). These sequences are very similar, with few amino acids substitutions compared to the human sequence. It is strange to find such a high degree of conservation among primates when there is such a low degree of conservation between the human pig, rat, or mouse, chicken exon 1beta sequences. More surprisingly, we observe a threonine at position 31 in all human sequences, whereas an alanine is always present in other sequences. We suggest that when the radiation human/simian appeared or after, a selection of threonine occurred. Moreover, the modifications detected could play a role in different interactions between ARF and other proteins to stabilize or not these complexes.

Amino Acid Sequence↗

PubMed2003

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

The p210(bcr-abl) and p190(bcr-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 p210(bcr-abl), but absent in p190(bcr-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 p210(bcr-abl) only. Moreover, the three types of GTPases are activated in vitro and in vivo by p210(bcr-abl). Nevertheless, Rac1 and Cdc42, but not RhoA, are activated by p190(bcr-abl) in vitro and in vivo. Part of this GEF activity of p190(bcr-abl) is probably attributable to p95(vav), which is complexed with both p190(bcr-abl) and p210(bcr-abl) in an activated form. p160(bcr), also in complex with Bcr-Abl, presents no GEF activity in p190(bcr-abl)-expressing cells. These results suggest that differential activation of Rho proteins should play a major role in Bcr-Abl-induced leukemogenesis.

Animals↗

PubMed2003

A Novel voltage-dependent chloride current activated by extracellular acidic pH in cultured rat Sertoli cells.

Sertoli cells from mammalian testis are key cells involved in development and maintenance of stem cell spermatogonia as well as secretion of a chloride- and potassium-rich fluid into the lumen of seminiferous tubules. Using whole-cell patch clamp experiments, a novel chloride current was identified. It is activated only in the presence of an extracellular acidic pH, with an estimated half-maximal activation at pH 5.5. The current is strongly outwardly rectifying, activated with a fast time-dependent onset of activation but a slow time-dependent kinetic at depolarization pulses. The pH-activated chloride current was not detected at physiological or basic pH and is not sensitive to intracellular or extracellular Ca2+ variation. Diphenylamine-2-carboxylic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid blocked the induced currents, and its anionic selectivity sequence was Cl- > Br- > I-> gluconate. We have performed a reverse transcription-PCR analysis to search for voltage-dependent chloride rClC channels in cultured rat Sertoli cells. Among the nine members of the family only rClC-2, rClC-3, rClC-6, and rClC-7 have been identified. The inwardly rectifying rClC-2 chloride current was activated by hyperpolarization but not by pH variation. A different depolarization-activated outwardly rectifying chloride current was activated only by hypotonic challenge and may correspond either to rClC-3 or rClC-6. Immunolocalization experiments demonstrate that rClC-7 resides in the intracellular compartment of Sertoli cells. This study provides the first functional identification of a native acid-activated chloride current. Based on our molecular analysis of rClC proteins, this new chloride current does not correspond to rClC-2, rClC-3, rClC-6, or rClC-7 channels. The potential physiological role of this native current in an epithelial cell from the reproductive system is discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗