Biomedical subjects

Stéphane Schurmans

Publications and source records attributed to Stéphane Schurmans.

12 recordsLinked to original sources

PubMed2005

Targeted calretinin expression in granule cells of calretinin-null mice restores normal cerebellar functions.

Ca2 binding proteins such as calretinin, characterized by the presence of EF-hand motifs that bind Ca2+ ions, are involved in the shaping of intraneuronal Ca2+ fluxes. In the cerebellar cortex, information processing tightly relies on variations in intracellular Ca2+ concentration in Purkinje and granule cells. Calretinin-deficient (Cr-/-) mice present motor discoordination, suggesting cellular and network cerebellar dysfunctions. To determine the cell specificity of these alterations, we constructed transgenic Cr-/- mice exhibiting a selective reexpression of calretinin in granule cells through the promoter function of the GABAA receptor alpha6 subunit gene. Normal granule cell excitability and wild-type Purkinje cell firing behavior in awake mice were restored while the emergence of high-frequency oscillations was abolished. Behavioral analysis of these calretinin-rescue mice revealed that normal motor coordination was restored as compared with Cr-/- mice. These results demonstrate that calretinin is required specifically in granule cells for correct computation in the cerebellar cortex and indicate that the finetuning of granule cell excitability through regulation of Ca2+ homeostasis plays a crucial role for information coding and storage in the cerebellum.

Animals↗

PubMed2005

Phosphatidylinositol 3,4,5-trisphosphate modulation in SHIP2-deficient mouse embryonic fibroblasts.

SHIP2, the ubiquitous SH2 domain containing inositol 5-phosphatase, includes a series of protein interacting domains and has the ability to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]in vitro. The present study, which was undertaken to evaluate the impact of SHIP2 on PtdIns(3,4,5)P(3) levels, was performed in a mouse embryonic fibroblast (MEF) model using SHIP2 deficient (-/-) MEF cells derived from knockout mice. PtdIns(3,4,5)P(3) was upregulated in serum stimulated -/- MEF cells as compared to +/+ MEF cells. Although the absence of SHIP2 had no effect on basal PtdIns(3,4,5)P(3) levels, we show here that this lipid was significantly upregulated in SHIP2 -/- cells but only after short-term (i.e. 5-10 min) incubation with serum. The difference in PtdIns(3,4,5)P(3) levels in heterozygous fibroblast cells was intermediate between the +/+ and the -/- cells. In our model, insulin-like growth factor-1 stimulation did not show this upregulation. Serum stimulated phosphoinositide 3-kinase (PI 3-kinase) activity appeared to be comparable between +/+ and -/- cells. Moreover, protein kinase B, but not mitogen activated protein kinase activity, was also potentiated in SHIP2 deficient cells stimulated by serum. The upregulation of protein kinase B activity in serum stimulated cells was totally reversed in the presence of the PI 3-kinase inhibitor LY-294002, in both +/+ and -/- cells. Altogether, these data establish a link between SHIP2 and the acute control of PtdIns(3,4,5)P(3) levels in intact cells.

Animals↗

PubMed2005

Normal thyroid structure and function in rhophilin 2-deficient mice.

Rhophilin 2 is a Rho GTPase binding protein initially isolated by differential screening of a chronically thyrotropin (TSH)-stimulated dog thyroid cDNA library. In thyroid cell culture, expression of rhophilin 2 mRNA and protein is enhanced following TSH stimulation of the cyclic AMP (cAMP) transduction cascade. Yeast two-hybrid screening and coimmunoprecipitation have revealed that the GTP-bound form of RhoB and components of the cytoskeleton are protein partners of rhophilin 2. These results led us to suggest that rhophilin 2 could play an important role downstream of RhoB in the control of endocytosis during the thyroid secretory process which follows stimulation of the TSH/cAMP pathway. To validate this hypothesis, we generated rhophilin 2-deficient mice and analyzed their thyroid structure and function. Mice lacking rhophilin 2 develop normally, have normal life spans, and are fertile. They have no visible goiter and no obvious clinical signs of hyper- or hypothyroidism. The morphology of thyroid cells and follicles in these mice were normal, as were the different biological tests performed to investigate thyroid function. Our results indicate that rhophilin 2 does not play an essential role in thyroid physiology.

Adaptor Proteins, Signal Transducing↗

PubMed2004

SHIP2 is recruited to the cell membrane upon macrophage colony-stimulating factor (M-CSF) stimulation and regulates M-CSF-induced signaling.

The Src homology 2-containing inositol phosphatase SHIP1 functions in hemopoietic cells to limit activation events mediated by PI3K products, including Akt activation and cell survival. In contrast to the limited cellular expression of SHIP1, the related isoform SHIP2, is widely expressed in both parenchymal and hemopoietic cells. The goal of this study was to determine how SHIP2 functions to regulate M-CSF signaling. We report that 1) SHIP2 was tyrosine-phosphorylated in M-CSF-stimulated human alveolar macrophages, human THP-1 cells, murine macrophages, and the murine macrophage cell line RAW264; 2) SHIP2 associated with the M-CSF receptor after M-CSF stimulation; and 3) SHIP2 associated with the actin-binding protein filamin and localization to the cell membrane, requiring the proline-rich domain, but not on the Src homology 2 domain of SHIP2. Analyzing the function of SHIP2 in M-CSF-stimulated cells by expressing either wild-type SHIP2 or an Src homology 2 domain mutant of SHIP2 reduced Akt activation in response to M-CSF stimulation. In contrast, the expression of a catalytically deficient mutant of SHIP2 or the proline-rich domain of SHIP2 enhanced Akt activation. Similarly, the expression of wild-type SHIP2 inhibited NF-kappaB-mediated gene transcription. Finally, fetal liver-derived macrophages from SHIP2 gene knockout mice enhanced activation of Akt in response to M-CSF treatment. These data suggest a novel regulatory role for SHIP2 in M-CSF-stimulated myeloid cells.

Animals↗

PubMed2004

Identification and subcellular distribution of endogenous Ins(1,4,5)P(3) 3-kinase B in mouse tissues.

Inositol 1,4,5-trisphosphate 3-kinase (IP(3)-3K) catalyses the phosphorylation of inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. cDNAs encoding three mammalian isoforms have been reported and referred to as IP(3)-3KA, IP(3)-3KB, and IP(3)-3KC. IP(3)-3KB is particularly sensitive to proteolysis at the N-terminus, a mechanism known to generate active fragments of lower molecular mass. Endogenous IP(3)-3KB has therefore not been formally identified in tissues. We have probed a series of murine tissues with an antibody directed against the C-terminus of IP(3)-3KB and used IP(3)-3KB deficient mouse tissues as negative controls. IP(3)-3KB was shown to be particularly well expressed in brain, lung, and thymus with molecular masses of 110-120kDa. The identification of the native IP(3)-3KB by Western blotting for the first time will facilitate further studies of regulation of its activity by specific proteases and/or phosphorylation.

Amino Acid Sequence↗

PubMed2004

Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3.

We produced and analyzed mice deficient for Na/Ca exchanger 3 (NCX3), a protein that mediates cellular Ca(2+) efflux (forward mode) or Ca(2+) influx (reverse mode) and thus controls intracellular Ca(2+) concentration. NCX3-deficient mice (Ncx3(-/-)) present a skeletal muscle fiber necrosis and a defective neuromuscular transmission, reflecting the absence of NCX3 in the sarcolemma of the muscle fibers and at the neuromuscular junction. The defective neuromuscular transmission is characterized by the presence of electromyographic abnormalities, including low compound muscle action potential amplitude, a decremental response at low-frequency nerve stimulation, an incremental response, and a prominent postexercise facilitation at high-frequency nerve stimulation, as well as neuromuscular blocks. The analysis of quantal transmitter release in Ncx3(-/-) neuromuscular junctions revealed an important facilitation superimposed on the depression of synaptic responses and an elevated delayed release during high-frequency nerve stimulation. It is suggested that Ca(2+) entering nerve terminals is cleared relatively slowly in the absence of NCX3, thereby enhancing residual Ca(2+) and evoked and delayed quantal transmitter release during repetitive nerve stimulation. Our findings indicate that NCX3 plays an important role in vivo in the control of Ca(2+) concentrations in the skeletal muscle fibers and at the neuromuscular junction.

Alleles↗

PubMed2004

Polymorphisms in type II SH2 domain-containing inositol 5-phosphatase (INPPL1, SHIP2) are associated with physiological abnormalities of the metabolic syndrome.

Type II SH2 domain-containing inositol 5-phosphatase (INPPL1, or SHIP2) plays an important role in the control of insulin sensitivity. INPPL1 mutations affecting gene function have been found in rat models of type 2 diabetes and hypertension and in type 2 diabetic patients. We investigated the influence of nucleotide variation in INPPL1 on components of the metabolic syndrome. Following comprehensive resequencing of the gene, we genotyped 12 informative polymorphisms in 1,304 individuals from 424 British type 2 diabetes families that were characterized for several metabolic phenotypes. We have found highly significant associations of single nucleotide polymorphisms (SNPs) and haplotypes of INPPL1 with hypertension as well as with other components of the metabolic syndrome. In a cohort of 905 French type 2 diabetic patients, we found evidence of association of INPPL1 SNPs with the presence of hypertension. We conclude that INPPL1 variants may impact susceptibility to disease and/or to subphenotypes involved in the metabolic syndrome in some diabetic patients.

Aged↗

PubMed2003

SH2-containing inositol 5-phosphatases 1 and 2 in blood platelets: their interactions and roles in the control of phosphatidylinositol 3,4,5-trisphosphate levels.

Src homology domain 2-containing inositol 5-phosphatases 1 and 2 (SHIP1 and SHIP2) are capable of dephosphorylating the second messenger PtdIns(3,4,5) P3 (phosphatidylinositol 3,4,5-trisphosphate) and interacting with several signalling proteins. SHIP1 is essentially expressed in haematopoietic cells, whereas SHIP2, a closely related enzyme, is ubiquitous. In the present study, we show that SHIP1 and SHIP2 are expressed as functional PtdIns(3,4,5) P3 5-phosphatases in human blood platelets and are capable of interacting when these two lipid phosphatases are co-expressed, either naturally (platelets and A20 B lymphoma cells) or artificially (COS-7 cells). Using COS-7 cells transfected with deletion mutants of SHIP2, we demonstrate that the Src homology domain 2 of SHIP2 is the minimal and sufficient protein motif responsible for the interaction between the two phosphatases. These results prompted us to investigate the relative importance of SHIP1 and SHIP2 in the control of PtdIns(3,4,5) P3 levels in platelets using homozygous or heterozygous SHIP1- or SHIP2-deficient mice. Our results strongly suggest that SHIP1, rather than SHIP2, plays a major role in controlling PtdIns(3,4,5) P3 levels in response to thrombin or collagen activation of mouse blood platelets.

Animals↗

PubMed2003

Altered neuronal excitability in cerebellar granule cells of mice lacking calretinin.

Calcium-binding proteins such as calretinin are abundantly expressed in distinctive patterns in the CNS, but their physiological function remains poorly understood. Calretinin is expressed in cerebellar granule cells, which provide the major excitatory input to Purkinje cells through parallel fibers. Calretinin-deficient mice exhibit dramatic alterations in motor coordination and Purkinje cell firing recorded in vivo through unknown mechanisms. In the present study, we used patch-clamp recording techniques in acute slice preparation to investigate the effect of a null mutation of the calretinin gene on the intrinsic electroresponsiveness of cerebellar granule cells at a mature developmental stage. Calretinin-deficient granule cells exhibit faster action potentials and generate repetitive spike discharge showing an enhanced frequency increase with injected currents. These alterations disappear when 0.15 mm of the exogenous fast-calcium buffer BAPTA is infused in the cytosol to restore the calcium-buffering capacity. A proposed mathematical model demonstrates that the observed alterations of granule cell excitability can be explained by a decreased cytosolic calcium-buffering capacity resulting from the absence of calretinin. This result suggests that calcium-binding proteins modulate intrinsic neuronal excitability and may therefore play a role in information processing in the CNS.

Animals↗

PubMed2003

Inositol 1,3,4,5-tetrakisphosphate is essential for T lymphocyte development.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) is phosphorylated by Ins(1,4,5)P(3) 3-kinase, generating inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). The physiological function of Ins(1,3,4,5)P(4) is still unclear, but it has been reported to be a potential modulator of calcium mobilization. Disruption of the gene encoding the ubiquitously expressed Ins(1,4,5)P(3) 3-kinase isoform B (Itpkb) in mice caused a severe T cell deficiency due to major alterations in thymocyte responsiveness and selection. However, we were unable to detect substantial defects in Ins(1,4,5)P(3) amounts or calcium mobilization in Itpkb(-/-) thymocytes. These data indicate that Itpkb and Ins(1,3,4,5)P(4) define an essential signaling pathway for T cell precursor responsiveness and development.

Animals↗

PubMed2003

Loss of nucleotide regulation of epithelial chloride transport in the jejunum of P2Y4-null mice.

The P2Y(4) receptor is responsive to UTP in human and to ATP and UTP in rodents. With the aim of identifying its pharmacotherapeutic interest, we generated P2Y(4)-null mice by a classic gene targeting method. The proportion of genotypes was consistent with X-linked Mendelian transmission. Gene inactivation was checked by the complete disappearance of P2Y(4) receptor mRNA from liver, stomach, and intestine. The P2Y(4)-null mice had a grossly normal behavior, growth, and reproduction. Chloride secretion by the jejunal epithelium was assessed in Ussing chambers by the measurement of the short circuit current in the presence of phlorizin. We show here that the UTP- and ATP-induced chloride secretory responses observed in wild-type mice are abolished in P2Y(4)-null mice. This is the first clearcut demonstration of a biological role of the P2Y(4) receptor.

Adenosine Triphosphate↗

PubMed2002

The gene INPPL1, encoding the lipid phosphatase SHIP2, is a candidate for type 2 diabetes in rat and man.

Genetic susceptibility to type 2 diabetes involves many genes, most of which are still unknown. The lipid phosphatase SHIP2 is a potent negative regulator of insulin signaling and sensitivity in vivo and is thus a good candidate gene. Here we report the presence of SHIP2 gene mutations associated with type 2 diabetes in rats and humans. The R1142C mutation specifically identified in Goto-Kakizaki (GK) and spontaneously hypertensive rat strains disrupts a potential class II ligand for Src homology (SH)-3 domain and slightly impairs insulin signaling in cell culture. In humans, a deletion identified in the SHIP2 3' untranslated region (UTR) of type 2 diabetic subjects includes a motif implicated in the control of protein synthesis. In cell culture, the deletion results in reporter messenger RNA and protein overexpression. Finally, genotyping of a cohort of type 2 diabetic and control subjects showed a significant association between the deletion and type 2 diabetes. Altogether, our results show that mutations in the SHIP2 gene contribute to the genetic susceptibility to type 2 diabetes in rats and humans.

3' Untranslated Regions↗