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Biomedical subjects

P Crespo

Publications and source records attributed to P Crespo.

At least 37 records · Page 2Linked to original sources
PubMed1999

Regulation of c-Myc and Max in megakaryocytic and monocytic-macrophagic differentiation of K562 cells induced by protein kinase C modifiers: c-Myc is down-regulated but does not inhibit differentiation.

We have studied the regulation and role of c-Myc and Max in the differentiation pathways induced in K562 cells by 12-O-tetradecanoyl phorbol-13 acetate (TPA) and staurosporine, an activator and inhibitor, respectively, of protein kinase C (PKC). We found that staurosporine induced megakaryocytic differentiation, as revealed by the cellular ultrastructure, platelet formation, and DNA endoreduplication. In contrast, TPA induced a differentiated phenotype that more closely resembled that of the monocyte-macrophage lineage. c-myc expression was down-regulated in K562 differentiated by both TPA and staurosporine, whereas max expression did not change in either case. Although PKC enzymatic activity was low in cells terminally differentiated with TPA and staurosporine, inhibition of PKC activity by itself did not induce c-myc down-regulation. We conclude that the c-myc gene is switched off as a consequence of the differentiation process triggered by these drugs in a manner independent from PKC. Ectopic overexpression of c-Myc in K562 cells did not affect the monocytic-macrophagic and megakaryocytic differentiation, indicating that c-Myc suppression is not required for these processes in K562. Similarly, both differentiation pathways were not affected by Max overexpression or by concomitant overexpression of c-Myc and Max. This result is in contrast with the inhibition of erythroid differentiation of K562 exerted by c-Myc, suggesting divergent roles for c-Myc/Max, depending on the differentiation pathway.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1998

Transforming G protein-coupled receptors block insulin and ras-induced adipocytic differentiation in 3T3-L1 cells: evidence for a PKC and MAP kinase independent pathway.

We have used the expression of muscarinic m1 receptors in the preadipocytic 3T3-L1 cell line for dissecting the nature of the G protein-linked pathways governing adipocytic differentiation, a complex process controlled by many stimuli and their downstream targets. 3T3-L1 cells can be differentiated by insulin or by ras oncogenes, and MAP kinase has been implicated in this process. However, m1 stimulation failed to induce differentiation of 3T3-L1 cells. Furthermore, it prevented insulin or v-ras-induced adipocytic differentiation, utilizing a protein kinase C-independent pathway. m1 stimulation did not alter the phosphorylation state of the insulin receptor substrates IRS-1 and SHC, nor the recruitment of Grb-2. Interestingly, whereas m1 receptors potently activated MAP kinase, another differentiation-inhibitor, TNF alpha, did not affect it. These results suggest that the control of adipocytic differentiation can occur utilizing a biochemical route independent of protein kinase C, and acting downstream, or independently from the Ras-MAP kinase pathway.

3T3 Cells↗
PubMed1998

[Restenosis and remodeling after percutaneous transluminal carotid angioplasty].

INTRODUCTION: The characteristics of restenosis and remodeling after carotid percutaneous transluminal angioplasty (PTA) were badly known. OBJECTIVE: To describe these characteristics in our series of carotid PTA. PATIENTS AND METHODS: A total of 78 cases of PTA for symptomatic > 70% atherosclerotic stenosis of the extracranial internal carotid artery, were selected from our series of PTA if follow-up was > 12 months. All of them were followed with extracranial continuous-wave Doppler. RESULTS: Restenosis of any degree was found in 17 cases (21.79%) and always asymptomatic. A restenosis > or = 70% was found in 5 cases (6.4%). Restenosis was mainly found in cases without residual stenosis nor dissection after PTA (p = 0.002). Restenosis was found in 16 cases (94.11%) in the first 6-months, with no progression thereafter. Remodeling of residual stenosis was frequent (17 cases; 53.11%) and found mainly during the first month after PTA. Its incidence was highest in patients with dissection treated with heparin. In cases with restenosis, remodeling was infrequent, incomplete and occurred after 18-24 months. CONCLUSIONS: 1. Significant restenosis after PTA due to myointimal proliferation, was infrequent. All cases were asymptomatic, under antiplatelet treatment. A new interventional procedure might not be necessary. 2. Complete remodeling was frequently found after 1-month control, mainly in arteries with some residual stenosis and dissection after PTA.

Adult↗
PubMed1997

Cbl-b, a member of the Sli-1/c-Cbl protein family, inhibits Vav-mediated c-Jun N-terminal kinase activation.

We have used the yeast two-hybrid system to identify proteins that interact with Vav, a GDP/GTP exchange factor for the Rac-1 GTPase that plays an important role in cell signaling and oncogenic transformation. This experimental approach resulted in the isolation of Cbl-b, a signal transduction molecule highly related to the mammalian c-cbl proto-oncogene product and to the C. elegans Sli-1 protein, a negative regulator of the EGF-receptor-like Let23 protein. The interaction between Vav and Cbl-b requires the entire SH3-SH2-SH3 carboxy-terminal domain of Vav and a long stretch of proline-rich sequences present in the central region of Cbl-b. Stimulation of quiescent rodent fibroblasts with either epidermal or platelet-derived growth factors induces an increased affinity of Vav for Cbl-b and results in the subsequent formation of a Vav-dependent trimeric complex with the ligand-stimulated tyrosine kinase receptors. During this process, Vav, but not Cbl-b, becomes highly phosphorylated on tyrosine residues. Overexpression of Cbl-b inhibits the signal transduction pathway of Vav that leads to the stimulation of c-Jun N-terminal kinase. By contrast, expression of truncated Cbl-b proteins and of missense mutants analogous to those found in inactive Sli-1 proteins have no detectable effect on Vav activity. These results indicate that Vav and Cbl-b act coordinately in the first steps of tyrosine protein kinase receptor-mediated signaling and suggest that members of the Sli-1/Cbl family are also negative regulators of signal transduction in mammalian cells.

Animals↗
PubMed1997

Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI 3-kinase gamma.

The tyrosine kinase class of receptors induces mitogen-activated protein kinase (MAPK) activation through the sequential interaction of the signaling proteins Grb2, Sos, Ras, Raf, and MEK. Receptors coupled to heterotrimeric guanine triphosphate-binding protein (G protein) stimulate MAPK through Gbetagamma subunits, but the subsequent intervening molecules are still poorly defined. Overexpression of phosphoinositide 3-kinase gamma (PI3Kgamma) in COS-7 cells activated MAPK in a Gbetagamma-dependent fashion, and expression of a catalytically inactive mutant of PI3Kgamma abolished the stimulation of MAPK by Gbetagamma or in response to stimulation of muscarinic (m2) G protein-coupled receptors. Signaling from PI3Kgamma to MAPK appears to require a tyrosine kinase, Shc, Grb2, Sos, Ras, and Raf. These findings indicate that PI3Kgamma mediates Gbetagamma-dependent regulation of the MAPK signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product.

The oncogenic protein Vav harbours a complex array of structural motifs, including leucine-rich, Dbl-homology, pleckstrin-homology, zinc-finger, SH2 and SH3 domains. Upon stimulation by antigens or mitogens, Vav becomes phosphorylated on key tyrosine residues and associates with other signalling proteins, including the mitogen receptors Zap-70 (ref. 6), Vap-1 (ref. 5) and Slp-76 (ref. 7). Disruption of the vav locus by homologous recombination causes severe defects in signalling by primary antigen receptors, leading to abnormal lymphocyte proliferation and lymphopenia. Despite the importance of Vav cell signalling, the function of this protein remains unknown. Here we show that tyrosine-phosphorylated Vav, but not the non-phosphorylated protein, catalyses GDP/GTP exchange on Rac-1, a protein implicated in cell proliferation and cytoskeletal organization, causing this GTPase to switch from its inactive to its active state. Transfection experiments also show that phosphorylation of Vav on tyrosine residues leads to nucleotide exchange on Rac-1 in vivo and stimulates c-Jun kinase, a downstream element in the signalling pathway involving this GTPase. Our results have identified a function for Vav and define a mechanism in which engaged membrane receptors activate its signalling pathway.

Animals↗
PubMed1997

The pathway connecting m2 receptors to the nucleus involves small GTP-binding proteins acting on divergent MAP kinase cascades.

m1 and m2 receptors are traditionally linked to tissue specific functions performed by fully differentiated cells. However, these receptors have been also implicated in growth stimulation. The mechanisms whereby these receptors regulate proliferative signaling pathways are still poorly understood. Furthermore, pharmacological evidence suggest that many growth promoting agents act on Gi coupled receptors, but there is no formal proof that induction of DNA-synthesis results from decreased intracellular levels of cAMP. In our laboratory, we have used the expression of ml and m2 receptors as a model for studying proliferative signaling through G protein-coupled receptors. Currently available evidence suggest that these receptors signal to distinct members of the MAP kinase superfamily, MAP kinase and JNK, through betagamma subunits of heterotrimeric G proteins acting, respectively, on a Ras and Rac1 dependent pathway.

Animals↗
PubMed1996

The small GTP-binding protein rho activates c-Jun N-terminal kinases/stress-activated protein kinases in human kidney 293T cells. Evidence for a Pak-independent signaling pathway.

Work from a number of laboratories has established a role for certain small GTP-binding proteins in controlling the enzymatic activity of a family of serine-threonine kinases known as mitogen-activated protein kinases (MAPKs). MAPKs have been classified into three subfamilies: extracellular signal-regulated kinases (ERKs), also known as MAPKs; c-Jun N-terminal kinases (JNKs); and p38 kinase. Whereas Ras controls the activation of MAPKs, we and others have recently observed that in certain cells, the small GTP-binding proteins Rac1 and Cdc42 but not Rho regulate the activity of JNKs. Furthermore, because Rac1 and Cdc42 but not Rho bind and activate a kinase known as Pak1, it has been suggested that Pak1 is the most upstream component of the pathway linking these GTPases to JNK. However, in both yeast and mammalian cells, Rho1p, a Rho homologue, and RhoA, respectively, directly interact with a number of proteins, including kinases related to protein kinase C. In addition, in yeast, Rho1p controls the activity of a MAPK cascade involved in bud formation. Considering this diversity of target molecules for small GTP-binding proteins, their likely tissue specific distribution, and the potential role for Rho in signaling to a kinase cascade, we decided to extend our initial analysis, exploring the ability of Ras and Rho-related GTP-binding proteins to activate MAPK or JNK in a variety of cell lines. We found that in the human kidney epithelial cell line, 293T, Cdc42 and all Rho proteins, RhoA, RhoB, and RhoC, but not Rac or Ras can induce activation of JNK. Furthermore, we provide evidence that signaling from Rho proteins to JNK in 293T cells does not involve Pak1. Taken together these findings demonstrate that Rho signals to JNK in a cell type-specific manner and suggest the existence of a novel, Pak1-independent signaling route communicating the Rho family of small GTP-binding proteins to the JNK pathway.

Animals↗
PubMed1996

Rac-1 dependent stimulation of the JNK/SAPK signaling pathway by Vav.

The protein product of the human vav oncogene, Vav exhibits a number of structural motifs suggestive of a role in signal transduction pathways, including a leucine-rich region, a plekstrin homology (PH) domain, a cysteine-rich domain, two SH3 regions, an SH2 domain, and a central Dbl homology (DH) domain. However, the transforming pathway(s) activated by Vav has not yet been elucidated. Interestingly, DH domains are frequently found in guanine nucleotide-exchange factors for small GTP-binding proteins of the Ras and Rho families, and it has been recently shown that, whereas Ras controls the activation of mitogen activated kinases (MAPKs), two members of the Rho family of small GTPases, Rac 1 and Cdc42, regulate activity of stress activated protein kinases (SAPKs), also termed c-jun N-terminal kinases (JNKs). The structural similarity between Vav and other guanine nucleotide exchange factors for small GTP-binding proteins, together with the recent identification of biochemical routes specific for members of the Ras and Rho family of GTPases, prompted us to explore whether MAPK or JNK are downstream components of the Vav signaling pathways. Using the COS-7 cell transient expression system, we have found that neither Vav nor the product of the vav proto-oncogene, proto-Vav, can enhance the enzymatic activity of a coexpressed, epitope tagged MAPK. On the other hand, we have observed that, whereas proto-Vav can slightly elevate JNK/SAPK activity, oncogenic Vav potently activates JNK/SAPK to an extent comparable to that elicited by two guanine-nucleotide exchange factors for Rho family members, Dbl and Ost. We also show that point mutations in conserved residues within the cysteine rich and DH domains of Vav both prevent its ability to activate JNK/SAPK and render Vav oncogenically inactive. In addition, we found that coexpression of the Rac-1 N17 dominant inhibitory mutant dramatically diminishes JNK/SAPK stimulation by Vav, as well as reduces the focus-forming ability of Vav in NIH3T3 murine fibroblasts. Taken together, these findings provide the first evidence that Rac-1 and JNK are integral components of the Vav signaling pathway.

3T3 Cells↗
PubMed1996

Protein kinase C-zeta reverts v-raf transformation of NIH-3T3 cells.

We have identified protein kinase C-zeta (PKC-zeta) as a novel suppressor of neoplastic transformation caused by the v-raf oncogene. PKC-zeta overexpression drastically retards proliferation, abolishes anchorage-independent growth, and reverts the morphological transformation of v-raf-transformed NIH-3T3 cells. The molecular basis for this effect appears to be a specific induction of junB and egr-1 expression, triggered synergistically by PKC-zeta via a Raf/Mek/MAPK-independent mechanism and v-raf. junB-promoter/CAT assays revealed that PKC-zeta directly targets the junB promoter. The induction of junB and egr-1 is linked to the v-raf transformation-suppressing effect of PKC-zeta as constitutive expression of junB and egr-1 but not of c-jun also abolishes anchorage-independent growth of v-raf-transformed NIH-3T3 cells. Moreover, junB overexpression leads to a retardation of proliferation in these cells. PKC-zeta interferes with the serum inducibility of an AP-1 reporter plasmid in v-raf-transformed NIH-3T3 cells, indicating that PKC-zeta antagonizes transformation and proliferation by down-modulating AP-1 function via induction of junB. In summary, our data suggest that PKC-zeta counteracts v-raf transformation by modulating the expression of the transcription factors junB and egr-1.

3T3 Cells↗
PubMed1996

[The effect of angioplasty on cerebral vasomotor reactivity in patients with important stenosis of the internal carotid artery].

INTRODUCTION: We present the preliminary results of the effects on percutaneous translumenal angioplasty (PTA) cerebral haemodynamics in thirty patients with important stenosis of the internal carotidal artery. MATERIALS AND METHODS: Using transcranial doppler (TCD) with acetazolamide (ACZ) as a vasodilatory stimulus, we assessed vasomotor reactivity (VR and pulsatility index (Pl) both before and three months after angioplasty. We also carried out single photon emission computerized tomography (SPECT) and cartography and frequency measurement by electroencephalogram (EEG) again both before and after PTA. RESULTS: Of the first twelve patients who completed the study, VR was maintained in nine and PI increased after PTA in seven. In three patients VR was exhausted and in two recovered after PTA. CONCLUSIONS: PTA improved cerebral haemodynamics. Similar more exhaustive studies are needed if revascularization techniques are to be useful in treating patients with this level of stenosis but who are asymptomatic as to the risk of haemodynamic cerebral vasomotor angioplasty.

Acetazolamide↗
PubMed1995

Dual effect of beta-adrenergic receptors on mitogen-activated protein kinase. Evidence for a beta gamma-dependent activation and a G alpha s-cAMP-mediated inhibition.

The enzymatic activity of mitogen-activated protein kinases (MAP kinases) increases in response to agents acting on a variety of cell surface receptors, including receptors linked to heterotrimeric G proteins of the Gi and Gq family. Recently, it has been shown that stimulation of beta-adrenergic receptors, which are typical of those that act through Gs to activate adenylyl cyclases, potently activates MAP kinases in the heart, resulting in the hypertrophy of the cardiac muscle (Lazou, A., Bogoyevitch, M.A., Clerk, A., Fuller, S.J., Marshall, C.J., and Sudgen, P.H. (1994) Circ. Res. 75, 938-941). We have observed that exposure of COS-7 cells to a beta-adrenergic agonist, isoproterenol, raises intracellular levels of cAMP and effectively activates protein kinase A (PKA) and an epitope-tagged MAP kinase. However, MAP kinase stimulation by isoproterenol was neither mimicked by expression of an activated mutant of G alpha s, nor by treatment with PKA-stimulating agents. Moreover, pretreatment of COS-7 with a permeable cAMP analog, 8-Br-cAMP, markedly decreased MAP kinase activation by either isoproterenol or epidermal growth factor. Thus, in COS-7 cells cAMP and PKA do not appear to mediate MAP kinase activation by beta-adrenergic receptors. Signaling from beta-adrenergic receptors to MAP kinase was inhibited by transfection of a chimeric molecule consisting of the CD8 receptor and the carboxyl terminus of the beta-adrenergic receptor kinase, which includes the beta gamma-binding domain. MAP kinase activation by isoproterenol was not affected by depletion of protein kinase C, but it was completely abolished by expression of Ras-inhibiting molecules. We conclude that signaling from beta-adrenergic receptors to MAP kinase involves an activating signal mediated by beta gamma subunits acting on a Ras-dependent pathway and a G alpha s-induced inhibitory signal mediated by cAMP and PKA. The balance between these two opposing mechanisms of regulation would be expected to control the MAP kinase response to beta-adrenergic agonists as well as to other biologically active agents known to act on Gs coupled receptors, including a number of hormones, neurotransmitters, and lipid mediators.

Base Sequence↗
PubMed1995

Overexpression of mammalian protein kinase C-zeta does not affect the growth characteristics of NIH 3T3 cells.

The structure of protein kinase C zeta (PKC zeta) is unusual with respect to other PKCs, as it lacks the C2 domain and possesses only one zinc finger region. Consequently, PKC zeta can not be activated by diacylglycerol or phorbol esters and is not downregulated by prolonged treatment by phorbol esters nor blocked by commonly utilized PKC inhibitors. In this study, we have explored the idea that PKC zeta might participate in proliferative pathways. Our findings show that marked overexpression of mammalian PKC zeta does not alter the growth characteristics of NIH 3T3 cells, nor induces cellular transformation. Furthermore, mammalian PKC zeta does not potentiate the transforming ability of oncogenes such as ras, sis and the muscarinic receptor m1. In this context, PKC zeta or its dominant negative mutant do not affect MAP kinase activation by oncogenes or growth factors. Taken together, our findings demonstrate that mammalian PKC zeta does not directly participate in signaling pathways involved in oncogenic transformation.

3T3 Cells↗
PubMed1995

The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway.

c-Jun amino-terminal kinases (JNKs) and mitogen-activated protein kinases (MAPKs) are closely related; however, they are independently regulated by a variety of environmental stimuli. Although molecules linking growth factor receptors to MAPKs have been recently identified, little is known about pathways controlling JNK activation. Here, we show that in COS-7 cells, activated Ras effectively stimulates MAPK but poorly induces JNK activity. In contrast, mutationally activated Rac1 and Cdc42 GTPases potently activate JNK without affecting MAPK, and oncogenic guanine nucleotide exchange factors for these Rho-like proteins selectively stimulate JNK activity. Furthermore, expression of inhibitory molecules for Rho-related GTPases and dominant negative mutants of Rac1 and Cdc42 block JNK activation by oncogenic exchange factors or after induction by inflammatory cytokines and growth factors. Taken together, these findings strongly support a critical role for Rac1 and Cdc42 in controlling the JNK signaling pathway.

Animals↗
PubMed1995

Neuronal activity of primate putamen during categorical perception of somaesthetic stimuli.

We have studied neuronal activity in the putamen of two monkeys trained to discriminate the speed of moving tactile stimuli. Animals pressed one of two target switches to indicate whether the speed of the probe across the skin was low or high. The activity of single neurones was recorded in the putamen ipsilateral to the glabrous skin of the stimulated hand and contralateral to the responding arm. During the task, we recorded neurones in the putamen that showed responses confined exclusively to the stimulus period of all speeds. A second class of putamen neurones responded during the stimulus period but continued discharging during the reaction and movement time period. None of these two classes of putamen neurones discharged when the same set of stimuli were delivered passively. A third class of putamen neurones responded differentially in the discrimination task and predicted whether the speed of the stimulus was low or high. A number of these neurones, which responded differentially during the categorization task, were tested in a light instruction task. This tested the possibility that these differential responses were associated with the intention to move the arm to one of the two target switches. Few neurones responded in this situation. These results indicate that the putamen, in addition to its role in motor regulation, is also involved in higher order aspects of sensory-motor behaviour and in the sensory decision process in this learned somaesthetic task.

Animals↗
PubMed1995

Expression of apolipoprotein E in cholesterol-loaded macrophages of extrahepatic tissues during experimental hypercholesterolemia.

To study the expression of extrahepatic apolipoprotein E (apoE) under hypercholesterolemic conditions, apoE mRNA levels were evaluated in 14 tissues of rabbits fed on a cholesterol rich diet and compared to age-matched control animals. In hypercholesterolemic rabbits apoE expression was significantly induced in adipose tissue, adrenals, aorta, lung and spleen. The increase in apoE mRNA levels in lung and spleen was associated with the presence of cholesterol-loaded macrophages. These cells were found to express high levels of apoE mRNA as demonstrated by in situ mRNA hybridization. Our results suggest that extrahepatic tissues would be partially responsible for the rise in serum apoE levels detected under hypercholesterolemic conditions.

Adipose Tissue↗
PubMed1995

Representation of moving tactile stimuli in the somatic sensory cortex of awake monkeys.

1. We have studied how neurons of primary somatic sensory (SI) cortex encode the direction and speed of moving tactile stimuli delivered to the glabrous skin of the contralateral hand. 2. From a total of 178 neurons recorded in SI cortex of 3 awake Macaca mulatta monkeys, 103 were selected for quantitative analysis. Forty-six neurons had slowly adapting (SA) responses, 43 quickly adapting (QA), and 14 mixed SA-QA properties. All possessed cutaneous receptive fields on the distal segments of digits 2, 3, or 4. Receptive fields were scanned with a metal probe (2 mm diam, hemispheric tip) in four different directions (0, 90, 180, and 270 degrees), over a fixed traverse distance of 6 mm, at a variety of speeds (4-100 mm/s), and with a static normal force of 20 g by means of a computer-controlled tactile stimulator. 3. Most neurons gave statistically significant differences in mean impulse rate during the moving stimuli (Wilcoxon, P < 0.01), in at least one of the four directions, compared with the control (nonstimulus) period. The Kruskal-Wallis test (P < 0.01) and the direction index (DI > 35%) determined that about one-half of SI cortical neurons showed significant differences in mean impulse rates associated with the direction of the stimuli at the speeds of 23, 50, and 100 mm/s, and about one-third at 4 mm/s. 4. We determined how the temporal covariance of the neural activity was associated with the parameters of the moving stimuli by calculating the coefficients of the Karhunen-Loève (KL) transform for each set of stimulus responses. Decomposition of the neural activity into principal components indicated that approximately 85% of the impulse train variance during the stimulus responses was contained in the 1st 10 coefficients of the KL transform for the speeds of 23, 50, and 100 mm/s, and approximately 75% at the speed of 4 mm/s. The line spectra calculated from the coefficients of the KL transform showed that the variance contained in the impulse trains in about one-half of the neurons is related to the stimuli. 5. We investigated how the temporal covariance of the neuronal activity was correlated with the direction of the stimulus, by fitting the first coefficient of the KL transform to a weighting function model. This analysis showed that the first coefficient of the KL transform varied as an orderly function of the direction of the moving stimuli.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗