Biomedical subjects

T Arnould

Publications and source records attributed to T Arnould.

At least 19 recordsLinked to original sources

PubMed2003

mtCLIC is up-regulated and maintains a mitochondrial membrane potential in mtDNA-depleted L929 cells.

To explain why mitochondrial DNA (mtDNA)-depleted or rho0 cells still keep a mitochondrial membrane potential (Delta(psi)m) in the absence of respiration, several hypotheses have been proposed. The principal and well accepted one involves a reverse of action for ANT combined to F1-ATPase activity. However, the existence of other putative electrogenic channels has been speculated. Here, using mRNA differential display reverse transcriptase-polymerase chain reaction on L929 mtDNA-depleted cells, we identified mtCLIC as a differentially expressed gene in cells deprived from mitochondrial ATP production. Mitochondrial chloride intracellular channel (mtCLIC), a member of a recently discovered and expanding family of chloride intracellular channels, is up-regulated in mtDNA-depleted and rho0 cells. We showed that its expression is dependent on CREB and p53 and is sensitive to calcium and tumor necrosis factor alpha. Interestingly, up- or down-regulation of mtCLIC protein expression changes Delta(psi)m whereas the chloride channel inhibitor NPPB reduces the Delta(psi)m in mtDNA-depleted L929 cells, measured with the fluorescent probe rhodamine 123. Finally, we demonstrated that purified mitochondria from mtDNA-depleted cells incorporate, in a NPPB-sensitive manner, more 36chloride than parental mitochondria. These findings suggest that mtCLIC could be involved in mitochondrial membrane potential generation in mtDNA-depleted cells, a feature required to prevent apoptosis and to drive continuous protein import into mitochondria.

Animals↗

PubMed2002

CREB activation induced by mitochondrial dysfunction is a new signaling pathway that impairs cell proliferation.

We characterized a new signaling pathway leading to the activation of cAMP-responsive element-binding protein (CREB) in several cell lines affected by mitochondrial dysfunction. In vitro kinase assays, inhibitors of several kinase pathways and overexpression of a dominant-negative mutant for calcium/calmodulin kinase IV (CaMKIV), which blocks the activation of CREB, showed that CaMKIV is activated by a mitochondrial activity impairment. A high calcium concentration leading to the disruption of the protein interaction with protein phosphatase 2A explains CaMKIV activation in these conditions. Transcrip tionally active phosphorylated CREB was also found in a rho0 143B human osteosarcoma cell line and in a MERRF cybrid cell line mutated for tRNA(Lys) (A8344G). We also showed that phosphorylated CREB is involved in the proliferation defect induced by a mitochondrial dysfunction. Indeed, cell proliferation inhibition can be prevented by CaMKIV inhibition and CREB dominant-negative mutants. Finally, our data suggest that phosphorylated CREB recruits p53 tumor suppressor protein, modifies its transcriptional activity and increases the expression of p21(Waf1/Cip1), a p53-regulated cyclin-dependent kinase inhibitor.

Animals↗

PubMed2001

Hypoxia-induced increase in intracellular calcium concentration in endothelial cells: role of the Na(+)-glucose cotransporter.

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet-activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca(2+)](i)) which then activates the phospholipase A(2) (PLA(2)). The link between the decrease in ATP and the increase in [Ca(2+)](i) was not known and is investigated in this work. We first showed that the presence of extracellular Na(+) was necessary to observe the hypoxia-induced increase in [Ca(2+)](i) and the activation of PLA(2). This increase was not due to the release of Ca(2+) from intracellular stores, since thapsigargin did not inhibit this process. The Na(+)/Ca(2+) exchanger was involved since dichlorobenzamil inhibited the [Ca(2+)](i) and the PLA(2) activation. The glycolysis was activated, but the intracellular pH (pH(i)) in hypoxic cells did not differ from control cells. Finally, the hypoxia-induced increase in [Ca(2+)](i) and PLA(2) activation were inhibited by phlorizin, an inhibitor of the Na(+)-glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na(+) through the activated Na(+)-glucose cotransport followed by the activation of the Na(+)/Ca(2+) exchanger, resulting in a net influx of Ca(2+).

Adenosine Triphosphate↗

PubMed2001

PGF(2alpha), a prostanoid released by endothelial cells activated by hypoxia, is a chemoattractant candidate for neutrophil recruitment.

Despite increasing evidence supporting the involvement of neutrophils in ischemic and postischemic damages, the mechanisms underlying the early recruitment of these cells are not completely understood. In this report, the effects of conditioned media from hypoxic endothelial cells on neutrophil chemotaxis were investigated by biochemical and morphological studies. We showed that conditioned media collected from several endothelial cell origins submitted to hypoxia as well as ischemic rat liver perfusion liquids have a chemotactic activity for neutrophils. The role of various chemoattractant molecules like HETEs, platelet-activating factor, and cytokines such as interleukin-8 and interleukin-1 was examined in the same model. Chemotactic peptide contribution was ruled out as boiled conditioned media still trigger chemotaxis. However, cell treatment with cyclooxygenase inhibitors, neutralization of PGF(2alpha) biological activity with polyclonal antibodies, and the neutrophil preincubation with a specific PGF(2alpha) antagonist, all dramatically inhibited neutrophil chemotaxis. A strong chemoattractant effect of pure exogenous PGF(2alpha) or of a synthetic analog was also observed. The major effect of PGF(2alpha) on neutrophil chemotaxis was confirmed ex vivo in a rat liver perfusion ischemic model. These results suggest that PGF(2alpha), a prostanoid abundantly released by the endothelium of hypoxic or ischemic tissues, is a chemoattractant molecule that might be involved in the early recruitment of neutrophils in ischemic organs.

Animals↗

PubMed2000

14-3-3 interacts with regulator of G protein signaling proteins and modulates their activity.

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) that stimulate the inactivation of heterotrimeric G proteins. We have recently shown that RGS proteins may be regulated on a post-translational level (Benzing, T., Brandes, R., Sellin, L., Schermer, B., Lecker, S., Walz, G., and Kim, E. (1999) Nat. Med. 5, 913-918). However, mechanisms controlling the GAP activity of RGS proteins are poorly understood. Here we show that 14-3-3 proteins associate with RGS7 and RGS3. Binding of 14-3-3 is mediated by a conserved phosphoserine located in the Galpha-interacting portion of the RGS domain; interaction with 14-3-3 inhibits the GAP activity of RGS7, depends upon phosphorylation of a conserved residue within the RGS domain, and results in inhibition of GAP function. Collectively, these data indicate that phosphorylation-dependent binding of 14-3-3 may act as molecular switch that controls the GAP activity keeping a substantial fraction of RGS proteins in a dormant state.

14-3-3 Proteins↗

PubMed2000

Endothelial cell responses to hypoxia: initiation of a cascade of cellular interactions.

The origin of several vascular pathologies involves sudden or recurrent oxygen deficiency. In this review, we examine what the biochemical and molecular responses of the endothelial cells to the lack of oxygen are and how these responses may account for the features observed in pathological situations, mainly by modifications of cell-cell interactions. Two major responses of the endothelial cells have been observed depending on the degree and duration of the oxygen deficiency. Firstly, acute hypoxia rapidly activates the endothelial cells to release inflammatory mediators and growth factors. These inflammatory mediators are able to recruit and promote the adherence of neutrophils to the endothelium where they become activated. The synthesis of platelet-activating factor plays a key role in this adherence process. Secondly, longer periods of hypoxia increase the expression of specific genes such as those encoding some cytokines as well as for the growth factors platelet-derived growth factor and vascular endothelial growth factor. The transcriptional induction of these genes is mediated through the activation of several transcription factors, the most important one being hypoxia inducible factor-1. The link between our knowledge of the signalling cascade of the cellular and molecular events initiated by hypoxia and their involvement in several vascular pathological situations, varicose veins, tumor angiogenesis and pulmonary hypertension is discussed briefly.

Cell Communication↗

PubMed2000

ERK activation upon hypoxia: involvement in HIF-1 activation.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor activated by hypoxia. The HIF-1 activation transduction pathway is poorly understood. In this report, we investigated the activation of extracellular regulated kinases (ERK) in hypoxia and their involvement in HIF-1 activation. We demonstrated that in human microvascular endothelial cells-1 (HMEC-1), ERK kinases are activated during hypoxia. Using dominant negative mutants, we showed that ERK1 is needed for hypoxia-induced HIF-1 transactivation activity. Moreover, using a kinase assay and Western blot experiments, we showed that HIF-1alpha is phosphorylated in hypoxia by an ERK-dependent pathway. These results evidence the role of mitogen-activated protein kinase in the transcriptional response to hypoxia.

Blotting, Western↗

PubMed1999

Interaction between RGS7 and polycystin.

Regulators of G protein signaling (RGS) proteins accelerate the intrinsic GTPase activity of certain Galpha subunits and thereby modulate a number of G protein-dependent signaling cascades. Currently, little is known about the regulation of RGS proteins themselves. We identified a short-lived RGS protein, RGS7, that is rapidly degraded through the proteasome pathway. The degradation of RGS7 is inhibited by interaction with a C-terminal domain of polycystin, the protein encoded by PKD1, a gene involved in autosomal-dominant polycystic kidney disease. Furthermore, membranous expression of C-terminal polycystin relocalized RGS7. Our results indicate that rapid degradation and interaction with integral membrane proteins are potential means of regulating RGS proteins.

Amino Acid Sequence↗

PubMed1999

Specific association of the gene product of PKD2 with the TRPC1 channel.

The function(s) of the genes (PKD1 and PKD2) responsible for the majority of cases of autosomal dominant polycystic kidney disease is unknown. While PKD1 encodes a large integral membrane protein containing several structural motifs found in known proteins involved in cell-cell or cell-matrix interactions, PKD2 has homology to PKD1 and the major subunit of the voltage-activated Ca2+ channels. We now describe sequence homology between PKD2 and various members of the mammalian transient receptor potential channel (TRPC) proteins, thought to be activated by G protein-coupled receptor activation and/or depletion of internal Ca2+ stores. We show that PKD2 can directly associate with TRPC1 but not TRPC3 in transfected cells and in vitro. This association is mediated by two distinct domains in PKD2. One domain involves a minimal region of 73 amino acids in the C-terminal cytoplasmic tail of PKD2 shown previously to constitute an interacting domain with PKD1. However, distinct residues within this region mediate specific interactions with TRPC1 or PKD1. The C-terminal domain is sufficient but not necessary for the PKD2-TRPC1 association. A more N-terminal domain located within transmembrane segments S2 and S5, including a putative pore helical region between S5 and S6, is also responsible for the association. Given the ability of the TRPC to form functional homo- and heteromultimeric complexes, these data provide evidence that PKD2 may be functionally related to TRPC proteins and suggest a possible role of PKD2 in modulating Ca2+ entry in response to G protein-coupled receptor activation and/or store depletion.

Amino Acid Sequence↗

PubMed1999

The polycystic kidney disease 1 gene product modulates Wnt signaling.

Two distinct signaling pathways, involving Wnt signaling and polycystin, have been found to be critical for normal kidney development. Renal tubulogenesis requires the presence of certain Wnt proteins, whereas mutations in polycystin impede the terminal differentiation of renal tubular epithelial cells, causing the development of large cystic kidneys that characterize autosomal dominant polycystic kidney disease. Polycystin is an integral membrane protein, consisting of several extracellular motifs indicative of cell-cell and cell-matrix interactions, coupled through multiple transmembrane domains to a functionally active cytoplasmic domain. We report here that expression of the C-terminal cytoplasmic domain of polycystin stabilizes soluble endogenous beta-catenin and stimulates TCF-dependent gene transcription in human embryonic kidney cells. Microinjection of the polycystin C-terminal cytoplasmic domain induces dorsalization in zebrafish. Our findings suggest that polycystin has the capacity to modulate Wnt signaling during renal development.

Animals↗

PubMed1999

Cellular activation triggered by the autosomal dominant polycystic kidney disease gene product PKD2.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by germ line mutations in at least three ADPKD genes. Two recently isolated ADPKD genes, PKD1 and PKD2, encode integral membrane proteins of unknown function. We found that PKD2 upregulated AP-1-dependent transcription in human embryonic kidney 293T cells. The PKD2-mediated AP-1 activity was dependent upon activation of the mitogen-activated protein kinases p38 and JNK1 and protein kinase C (PKC) epsilon, a calcium-independent PKC isozyme. Staurosporine, but not the calcium chelator BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N', N'-tetraacetate], inhibited PKD2-mediated signaling, consistent with the involvement of a calcium-independent PKC isozyme. Coexpression of PKD2 with the interacting C terminus of PKD1 dramatically augmented PKD2-mediated AP-1 activation. The synergistic signaling between PKD1 and PKD2 involved the activation of two distinct PKC isozymes, PKC alpha and PKC epsilon, respectively. Our findings are consistent with others that support a functional connection between PKD1 and PKD2 involving multiple signaling pathways that converge to induce AP-1 activity, a transcription factor that regulates different cellular programs such as proliferation, differentiation, and apoptosis. Activation of these signaling cascades may promote the full maturation of developing tubular epithelial cells, while inactivation of these signaling cascades may impair terminal differentiation and facilitate the development of renal tubular cysts.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1999

Modulation of renal tubular cell function by RGS3.

The recently discovered family of regulators of G protein signaling (RGS) accelerates the intrinsic GTPase activity of certain Galpha subunits, thereby terminating G protein signaling. Particularly high mRNA levels of one family member, RGS3, are found in the adult kidney. To establish the temporal and spatial renal expression pattern of RGS3, a polyclonal antiserum was raised against the COOH terminus of RGS3. Staining of mouse renal tissue at different gestational stages revealed high levels of RGS3 within the developing and mature tubular epithelial cells. We tested whether RGS3 can modulate tubular migration, an important aspect of tubular development, in response to G protein-mediated signaling. Several mouse intermedullary collecting duct (mIMCD-3) cell lines were generated that expressed RGS3 under the control of an inducible promoter. Lysophosphatidic acid (LPA) is a potent chemoattractant that mediates its effects through heterotrimeric G proteins. We found that induction of RGS3 significantly reduced LPA-mediated cell migration in RGS3-expressing mIMCD-3 clones, whereas chemotaxis induced by hepatocyte growth factor remained unaffected by RGS3. Our findings suggest that RGS3 modulates tubular functions during renal development and in the adult kidney.

Aging↗

PubMed1998

The polycystic kidney disease 1 gene product mediates protein kinase C alpha-dependent and c-Jun N-terminal kinase-dependent activation of the transcription factor AP-1.

Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary disorder that accounts for 8-10% of end stage renal disease. PKD1, one of two recently isolated ADPKD gene products, has been implicated in cell-cell and cell-matrix interactions. However, the signaling pathway of PKD1 remains undefined. We found that the C-terminal 226 amino acids of PKD1 transactivate an AP-1 promoter construct in human embryonic kidney cells (293T). PKD1-induced transcription is specific for AP-1; promoter constructs containing cAMP response element-binding protein, c-Fos, c-Myc, or NFkappaB-binding sites are unaffected by PKD1. In vitro kinase assays revealed that PKD1 triggers the activation of c-Jun N-terminal kinase (JNK), but not of mitogen-activated protein kinases p38 or p44. Dominant-negative Rac-1 and Cdc42 mutations abrogated PKD1-mediated JNK and AP-1 activation, suggesting a critical role for small GTP-binding proteins in PKD1-mediated signaling. Several protein kinase C (PKC) inhibitors decreased PKD1-mediated AP-1 activation. Conversely, expression of the C-terminal domain of PKD1 increased PKC activity in 293T cells. A dominant-negative PKC alpha, but not a dominant-negative PKC beta or delta, abrogated PKD1-mediated AP-1 activation. These findings indicate that small GTP-binding proteins and PKC alpha mediate PKD1-induced JNK/AP-1 activation, together comprising a signaling cascade that may regulate renal tubulogenesis.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1998

Effect of aescine on hypoxia-induced neutrophil adherence to umbilical vein endothelium.

Although venous stasis due to blood stagnation in lower limbs has been recognised as an important etiological factor for the development of varicose veins, the mechanism linking this ischemic situation to the modifications of the venous wall in varicose veins is still unclear. There is evidence that the activation of the endothelium during blood stasis and its subsequent cascade of interactions with other cell types could alter the structure of the vein wall and could possibly be at the origin of the disease. While phlebotonic drugs are often used to improve symptoms in chronic venous insufficiency, their precise mechanism of action is not well understood. We now tested aescine (Reparil i.v. form) in an ex vivo model which mimics this situation, i.e., perfused human umbilical vein exposed to hypoxic conditions. To study the effect of aescine on neutrophil activation and adhesion to the endothelium, human umbilical veins were incubated under hypoxic conditions with or without aescine and the interactions between the endothelium and neutrophil-like cells, HL60, were investigated. We observed that a large number of HL60 became adherent to the endothelium of veins after 2 h hypoxia and that these adherent HL60 were activated: they released high amounts of superoxide anion and of leukotriene B4. Aescine (250 ng/ml or 0.22 microM) was shown to markedly inhibit HL60 adherence to hypoxic endothelium. By decreasing the number of adherent HL60, aescine also decreased the subsequent production of superoxide anion and of leukotriene B4. Scanning electron microscopy confirmed the increased HL60 adherence to the endothelium, as well as the inhibitory effect of aescine. These results support results of in vitro studies on isolated endothelial cells in which aescine was shown to inhibit the hypoxia-induced activation of endothelial cells and the subsequent increased adherence of neutrophils. In vivo, the activated and infiltrated leukocytes release free radicals, chemotactic molecules such as leukotriene B4 and proteases which then can degrade the extracellular matrix. These processes could contribute to alterations of the venous wall similar to those observed in varicose veins. By maintaining an intact endothelium during in vivo blood stasis in the lower limbs and preventing neutrophil recruitment, adherence and activation, aescine could prevent the resulting alterations of the venous wall. These results could explain at least in part the potential benefit of the drug in the prevention of venous insufficiency.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1998

Effect of hydroxyethylrutosides on hypoxial-induced neutrophil adherence to umbilical vein endothelium.

A clinically available mixture of hydroxyethylrutosides (HR) was examined as a protector against endothelial cell activation by hypoxia in perfused human umbilical vein. The results showed that 500 micrograms/mL HR totally inhibited the adherence of human unstimulated neutrophils to the endothelium of umbilical vein incubated in hypoxic conditions. This inhibition was confirmed by a morphological study performed by scanning electron microscopy. In addition, neutrophils adherent to the hypoxic umbilical vein endothelium became activated, as evidence by the increased release of superoxide anions and synthesis of leukotriene B4. These processes could also be inhibited by HR. In conclusion, the results of this study suggest that the improvement in venous insufficiency observed clinically with HR could, in part, be the result of their ability to inhibit the recruitment and activation of neutrophils by endothelium activated during blood stasis.

Cell Adhesion↗

PubMed1998

Effect of Ginkor Fort on hypoxia-induced neutrophil adherence to human saphenous vein endothelium.

This study was performed to evaluate the effects of Ginkor Fort, a venotropic drug composed of Ginkgo biloba extract, troxerutine, and heptaminol, on neutrophil adherence to the endothelium of saphenous veins. When saphenous veins were incubated 2 h in hypoxic conditions, they showed a five- to sixfold increase in neutrophil adherence to the endothelium. Ginkor Fort at 0.3 mg/ml was able to inhibit this increase by 69%. These results were confirmed by observations in scanning electron microscopy. Ginkor Fort also inhibited the subsequent activation of these neutrophils, as evidenced by the inhibition of superoxide anion release. The biochemical mechanism of this inhibition of neutrophil adherence was studied on endothelial cells in culture. We observed that Ginkor Fort was able to inhibit the different steps of the activation of endothelial cells by hypoxia: the activation of phospholipase A2 and the decrease in adenosine triphosphate (ATP) content. By preventing the first step of the activation cascade, the decrease in ATP content, Ginkor Fort blocks the subsequent increase in neutrophil adherence as well as neutrophil activation. The biochemical mechanism evidenced in this work might explain the beneficial effect of this drug in the treatment of patients with chronic venous insufficiency.

Cell Adhesion↗

PubMed1998

Role of PECAM-1 in the adherence of PMN to hypoxic endothelial cells.

Hypoxia induces an increase in PMN adherence to endothelial cells for which an interaction between ICAM-1 and CD18/CD11b has been demonstrated. Since PECAM-1 has been shown to be involved in PMN transmigration through the endothelium and to increase the binding capacity of leukocyte CD18/CD11b, the role of this molecule in the hypoxia-induced PMN adherence was investigated. Hypoxia did not change the total surface expression of PECAM-1 on HUVEC and did not change the cell-cell border localization of this molecule as TNF-alpha did. In addition, blocking anti-PECAM-1 antibodies could not inhibit the increased adherence of unstimulated human PMN to hypoxia-incubated HUVEC while anti-ICAM-1 partially inhibited this process. These results indicate that PECAM-1 is probably not involved in the hypoxia-induced PMN adherence to endothelial cells.

Antibodies, Monoclonal↗

PubMed1997

Homo- and heterodimeric interactions between the gene products of PKD1 and PKD2.

PKD1 and PKD2 are two recently identified genes that are responsible for the vast majority of autosomal polycystic kidney disease, a common inherited disease that causes progressive renal failure. PKD1 encodes polycystin, a large glycoprotein that contains several extracellular motifs indicative of a role in cell-cell or cell-matrix interactions, and the PKD2 encodes a protein with homology to a voltage-activated calcium channel and to PKD1. It is currently unknown how mutations of either protein functionally cause autosomal polycystic kidney disease. We show that PKD1 and PKD2 interact through their C-terminal cytoplasmic tails. This interaction resulted in an up-regulation of PKD1 but not PKD2. Furthermore, the cytoplasmic tail of PKD2 but not PKD1 formed homodimers through a coiled-coil domain distinct from the region required for interaction with PKD1. These interactions suggest that PKD1 and PKD2 may function through a common signaling pathway that is necessary for normal tubulogenesis and that PKD1 may require the presence of PKD2 for stable expression.

Dimerization↗