A general strategy for producing conditional alleles of Src-like tyrosine kinases.

The Src-like tyrosine kinases require membrane localization for transformation and probably for their normal role in signal transduction. We utilized this characteristic to prepare Src-like tyrosine kinases that can be readily activated with the rationally designed chemical inducer of dimerization FK1012. Dimerization of cytoplasmic Src-like tyrosine kinases was not sufficient for signaling, but their recruitment to the plasma membrane led to the rapid activation of transcription factors identical to those regulated by crosslinking the antigen receptor. Moreover, recruitment of activated Src-like kinases to the membrane replaced signaling by the T-lymphocyte antigen receptor complex, leading to the activation of both the Ras/protein kinase C and Ca2+/calcineurin pathways normally activated by antigen receptor signaling. Since these chemical inducers of dimerization are cell permeable, this approach permits the production of conditional alleles of any of the Src-like tyrosine kinases, thereby allowing a delineation of their developmental roles.

Mutant rat phosphatidylinositol/phosphatidylcholine transfer proteins specifically defective in phosphatidylinositol transfer: implications for the regulation of phospholipid transfer activity.

The mammalian phosphatidylinositol/phosphatidylcholine transfer proteins (PI-TPs) catalyze exchange of phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane bilayers in vitro. We find that Ser-25, Thr-59, Pro-78, and Glu-248 make up a set of rat (r) PI-TP residues, substitution of which effected a dramatic reduction in the relative specific activity for PI transfer activity without significant effect on PC transfer activity. Thr-59 was of particular interest as it is a conserved residue in a highly conserved consensus protein kinase C phosphorylation motif in metazoan PI-TPs. Replacement of Thr-59 with Ser, Gln, Val, Ile, Asn, Asp, or Glu effectively abolished PI transfer capability but was essentially silent with respect to PC transfer activity. These findings identify rPI-TP residues that likely cooperate to form a PI head-group binding/recognition site or that lie adjacent to such a site. Finally, the selective sensitivity of the PI transfer activity of rPI-TP to alteration of Thr-59 suggests a mechanism for in vivo regulation of rPI-TP activity.

Role for ceramide as an endogenous mediator of Fas-induced cytotoxicity.

Triggering of the Fas/APO-1 cell-surface receptor induces apoptosis through an uncharacterized chain of events. Exposure of Fas-sensitive cells to an agonist monoclonal antibody induced cell death and a 200-300% elevation in endogenous levels of the sphingolipid ceramide, a proposed intracellular mediator of apoptosis. In contrast, similar treatment of Fas-resistant cells caused insignificant changes in ceramide levels. Because resistant cell lines expressed the Fas antigen, these results indicate that these cells have a defect in the proximal signaling events leading to ceramide generation. Exposure of the resistant cell lines to a synthetic analog of ceramide induced apoptosis, thus bypassing Fas resistance and indicating that the signaling pathways downstream of ceramide were intact. Furthermore, activation of protein kinase C with the diacylglycerol analog phorbol 12-myristate 13-acetate significantly reduced Fas-induced cytotoxicity, suggesting opposing roles for ceramide and protein kinase C in regulation of apoptosis. These results provide evidence for ceramide as a necessary and sufficient lipid mediator of Fas-mediated apoptosis and suggest this process may be modulated via activation of additional signal-transduction pathways.

Disruption of cellular signaling pathways by daunomycin through destabilization of nonlamellar membrane structures.

Albeit anthracyclines are widely used in the treatment of solid tumors and leukemias, their mechanism of action has not been elucidated. The present study gives relevant information about the role of nonlamellar membrane structures in signaling pathways, which could explain how anthracyclines can exert their cytocidal action without entering the cell [Tritton, T. R. & Yee, G. (1982) Science 217, 248-250]. The anthracycline daunomycin reduced the formation of the nonlamellar hexagonal (HII) phase (i.e., the hexagonal phase propensity), stabilizing the bilayer structure of the plasma membrane by a direct interaction with membrane phospholipids. As a consequence, various cellular events involved in signal transduction, such as membrane fusion and membrane association of peripheral proteins [e.g., guanine nucleotide-binding regulatory proteins (G proteins and protein kinase C-alpha beta)], where nonlamellar structures (negative intrinsic monolayer curvature strain) are required, were altered by the presence of daunomycin. Functionally, daunomycin also impaired the expression of the high-affinity state of a G protein-coupled receptor (ternary complex for the alpha 2-adrenergic receptor) due to G-protein dissociation from the plasma membrane. In vivo, daunomycin also decreased the levels of membrane-associated G proteins and protein kinase C-alpha beta in the heart. The occurrence of such nonlamellar structures favors the association of these peripheral proteins with the plasma membrane and prevents daunomycin-induced dissociation. These results reveal an important role of the lipid component of the cell membrane in signal transduction and its alteration by anthracyclines.

Overexpression of csk inhibits acid-induced activation of NHE-3.

Opossum kidney OKP cells express an apical membrane Na+/H+ antiporter that is encoded by NHE-3 (for Na+/H+ exchanger 3) and is similar in many respects to the renal proximal tubule apical membrane Na+/H+ antiporter. Chronic incubation of OKP cells in acid medium for 24 hr increases Na+/H(+)-antiporter activity and NHE-3 mRNA abundance. The increase in Na+/H(+)-antiporter activity was not prevented by H7, a protein kinase C/protein kinase A inhibitor, but was prevented by herbimycin A, a tyrosine kinase inhibitor. Incubation of cells in acid medium increased c-src activity, and this was inhibited by herbimycin A. To determine the role of the src family of nonreceptor protein-tyrosine kinases, Csk (for carboxyl-terminal src kinase), a physiologic inhibitor of these kinases, was overexpressed in OKP cells. In three clones overexpressing csk, acid-induced increases in Na+/H(+)-antiporter activity and NHE-3 mRNA abundance were inhibited. In these clones, inhibition of acid activation of Na+/H(+)-antiporter activity paralleled inhibition of acid activation of c-src. Neither herbimycin A nor overexpression of csk inhibited dexamethasone-induced increases in Na+/H(+)-antiporter activity. These studies show that decreases in pH activate c-src and that the src family nonreceptor protein-tyrosine kinases play a key role in acid activation of NHE-3.

Biological response to phorbol ester determined by alternative G1 pathways.

A plethora of extracellular signals is known to induce a common set of immediate early genes. The immediate early response, therefore, must not be sufficient to determine the biological outcome. An example of this is found with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). A potent activator of protein kinase C, TPA can either stimulate or inhibit cell proliferation, depending on the cell type. This cell context-dependent response to TPA is observed with two subclones of NIH 3T3 cells, the P- and the N-3T3 clones. TPA is a mitogen for the P-3T3 but an antimitogen for the N-3T3 cells. The immediate early pathway is activated by TPA in both cell types, indicating that this pathway alone does not activate DNA synthesis. The delayed induction of cyclin D1 expression by TPA is observed only in the P-3T3 cells, correlating with mitogenesis. N-Acetylcysteine does not affect the immediate early pathway but can inhibit the TPA-mediated induction of cyclin D1 and DNA synthesis. In the N-3T3 cells, TPA causes an inhibition of the cyclin E-associated kinase at the G1/S transition, correlating with growth inhibition. The growth-inhibitory activity of TPA is not affected by N-acetylcysteine. Thus, the two TPA-regulated G1 pathways can be distinguished by their sensitivity to N-acetylcysteine. These results demonstrate that TPA can activate alternative G1 pathways. Moreover, the selection of the alternative G1 pathways is determined by the cell context, which, in turn, dictates the biological response to TPA.

Neurosecretory habituation in PC12 cells: modulation during parallel habituation.

PC12 cells habituate during repetitive stimulation with acetylcholine, bradykinin, or high potassium. Interspersing these stimulants did not affect the rate of habituation of the others, but it could modulate the amplitude of the norepinephrine secretion each could achieve. Stimulation with acetylcholine inhibited norepinephrine secretion caused by high potassium and bradykinin stimulation, while high potassium had no effect on acetylcholine or bradykinin, and bradykinin increased secretion caused by acetylcholine. Changes in norepinephrine secretion resulting from any of these stimulants correlated with changes in internal calcium levels. Cyclic AMP-, protein kinase C-, and calmodulin-dependent second messenger pathways all modulated norepinephrine secretion caused by acetylcholine and high potassium and showed a distinct hierarchy in their effectiveness. These data demonstrate that different receptor pathways can change the norepinephrine response of one another while not changing the levels of the molecules responsible for habituation.

Biopacemaker acceleration without increased synchronization by chronic exposure to phorbol myristate acetate

L'activité électrique du coeur est initiée par la génération spontanée de potentiels d'action venant des cellules pacemaker du noeud sinusal (SN). Toute dysfonction au niveau de cette région entraîne une instabilité électrique du coeur. La majorité des patients souffrant d'un noeud sinusal déficient nécessitent l'implantation chirurgicale d'un pacemaker électronique; cependant, les limitations de cette approche incitent à la recherche d'une alternative thérapeutique. La base moléculaire des courants ioniques jouant un rôle crucial dans l'activité du noeud sinusal sont de plus en plus connues. Une composante importante de l'activité des cellules pacemakers semble être le canal HCN, responsable du courant pacemaker If. Le facteur T-box 3 (Tbx3), un facteur de transcription conservé durant le processus de l'évolution, est nécessaire au développement du système de conduction cardiaque. De précédentes études ont démontré que dans différentes lignées cellulaires le Phorbol 12-myristate 13-acetate (PMA) active l'expression du gène codant Tbx3 via des réactions en cascade partant de la protéine kinase C (PKC). L'objectif principal de cette étude est de tester si le PMA peut augmenter la fréquence et la synchronisation de l'activité spontanée du pacemaker biologique en culture. Plus précisément, nous avons étudié les effets de l'exposition chronique au PMA sur l'expression du facteur de transcription Tbx3, sur HCN4 et l'activité spontanée chez des monocouches de culture de myocytes ventriculaires de rats néonataux (MVRN). Nos résultats démontrent que le PMA augmente significativement le facteur transcription de Tbx3 et l'expression ARNm de HCN4, favorisant ainsi l'augmentation du rythme et de la stabilité de l'activité autonome. De plus, une diminution significative de la vitesse de conduction a été relevée et est attribuée à la diminution du couplage intercellulaire. La diminution de la vitesse de conduction pourrait expliquer l'effet négatif du PMA sur la synchronisation de l'activité autonome du pacemaker biologique. Ces résultats ont été confirmés par un modèle mathématique multicellulaire suggérant que des fréquences et résistances intercellulaires plus élevée pourraient induire une activité plus stable et moins synchrone. Cette étude amène de nouvelles connaissances très importantes destinées à la production d'un pacemaker biologique efficient et robuste.

Thiamine Deficiency Results in Downregulation of the GLAST Glutamate Transporter in Cultured Astrocytes

Pyrithiamine-induced thiamine deficiency (TD) is a well-established model of Wernicke's encephalopathy in which a glutamate-mediated excitotoxic mechanism may play an important role in determining selective vulnerability. In order to examine this possibility, cultured astrocytes were exposed to TD and effects on glutamate transport and metabolic function were studied. TD led to decreases in cellular levels of thiamine and thiamine diphosphate (TDP) after 24 h of treatment and decreased activities of the TDP-dependent enzymes alpha-ketoglutarate dehydrogenase and transketolase after 4 and 7 days, respectively. TD treatment for 10 days led to a reversible decrease in the uptake of [H-3]-D-aspartate, a nonmetabolizable analogue of glutamate. Kinetic analysis revealed that the uptake inhibition was caused by a 47% decrease in the V-max for uptake of [H-3]-D-aspartate, with no change in the K-m value. Immunoblotting showed that this decrease in uptake was due to an 81% downregulation of the astrocyte-specific GLAST glutamate transporter. Loss of uptake activity and GLAST protein were blocked by treatment with the protein kinase C inhibitor H7, while exposure to DCG IV, a group II metabotropic glutamate receptor (mGluR) agonist, resulted in improvement of [H-3]-D-aspartate uptake and a partial reversal of transporter downregulation. These results are consistent with our recent in vivo findings of a loss of astrocytic glutamate transporters in TD and provide evidence that TD conditions may increase phosphorylation. of GLAST, contributing to its downregulation. In addition, manipulation of group II mGluR activity may provide an important strategy in the treatment of this disorder. (C) 2003 Wiley-Liss, Inc.

Effects of pathophysiological concentrations of albumin on NHE3 activity and cell proliferation in primary cultures of human proximal tubule cells

The progression of renal disease correlates strongly with hypertension and the degree of proteinuria, suggesting a link between excessive Na+ reabsorption and exposure of the proximal tubule to protein. The present study investigated the effects of albumin on cell growth and Na+ uptake in primary cultures of human proximal tubule cells (PTC). Albumin (1.0 mg/ml) increased cell proliferation to 134.1 +/- 11.8% (P < 0.001) of control levels with no change in levels of apoptosis. Exposure to 0.1 and 1.0 mg/ml albumin increased total Na-22(+) uptake to 119.1 &PLUSMN; 6.3% (P = 0.005) and 115.6 &PLUSMN; 5.3% (P < 0.006) of control levels, respectively, because of an increase in Na+/H+ exchanger isoform 3 (NHE3) activity. This was associated with an increase in NHE3 mRNA to 161.1 +/- 15.1% (P < 0.005) of control levels in response to 0.1 mg/ml albumin. Using confocal microscopy with a novel antibody raised against the predicted extracellular NH2 terminus of human NHE3, we observed in nonpermeabilized cells that exposure of PTC to albumin (0.1 and 1.0 mg/ml) increased NHE3 at the cell surface to 115.4 &PLUSMN; 2.7% (P < 0.0005) and 122.4 +/- 3.7% (P < 0.0001) of control levels, respectively. This effect was paralleled by significant increases in NHE3 in the subplasmalemmal region as measured in permeabilized cells. These albumin-induced increases in expression and activity of NHE3 in PTC suggest a possible mechanism for Na+ retention in response to proteinuria.

IRS-1 regulation in health and disease

The global incidence of diabetes is increasing at epidemic rates. Estimates suggest there are currently 150 million people with diabetes and this number is expected to double in the next 20 years. Type 2 diabetes accounts for 95% of all cases and is characterized in part by impaired sensitivity to insulin or 'insulin resistance'. Defects in the insulin signalling pathways underpin this resistance. In the current article we discuss the regulation of Insulin Receptor Substrate-1 (IRS-1), a protein that plays a pivotal role in insulin signalling and whose function is impaired in subjects with insulin resistance. Coordination of IRS-1 function is multi-faceted, involving phosphorylation of IRS-1 at multiple serine/threonine residues. This controls many aspects of IRS-1, including its interaction with the insulin receptor and subsequent tyrosine phosphorylation, as well as its subcellular distribution and targeting for degradation by the proteasome. Such tight control ensures appropriate transduction and attenuation of the insulin signal, thereby regulating insulin action in healthy individuals. Emerging evidence indicates that `diabetogenic factors' associated with insulin resistance, such as TNFalpha and elevated circulating fatty acids, impact on insulin signalling at the level of IRS-1 serine/threonine phosphorylation. The expression and/or activity of several kinases, such as IkappaB kinase beta (IKKbeta) and salt-induced kinase 2 (SIK2), and the phosphorylation of IRS-1 at key sites, such as Ser307 and Ser789, are increased in states of insulin resistance. Identifying the pathways by which such factors activate these and other kinases, and de. ning the precise roles of specific serine/threonine phosphorylation events in IRS-1 regulation, represent important goals which may eventually provide a rationale for therapeutic intervention.

Antitumor activity of 3-ingenyl angelate: Plasma membrane and mitochondrial disruption and necrotic cell death

Options for skin cancer treatment currently include surgery, radiotherapy, topical chemotherapy, cryosurgery, curettage, and electrodes-sication. Although effective, surgery is costly and unsuitable for certain patients. Radiotherapy can leave a poor cosmetic effect, and current chemotherapy is limited by low cure rates and extended treatment schedules. Here, we describe the preclinical activity of a novel topical chemotherapeutic agent for the treatment of skin cancer, 3-ingenyl angelate (PEP005), a hydrophobic diterpene ester isolated from the plant Euphorbia peplus. Three daily topical applications of 42 nmol (18 mug) of PEP005 cured a series of s.c. mouse tumors (B16 melanoma, LK2 UV-induced squamous cell carcinoma, and Lewis lung carcinoma; it = >14 tumors/group) and human tumors (DO4 melanoma, HeLa cervical carcinoma, and PC3 and DU145 prostate carcinoma; it = >4 tumors/group) previously established (5-10 mm(3)) on C57BL/6 or Fox1(nu) mice. The treatment produced a mild, short-term erythema and eschar formation but, ultimately, resulted in excellent skin cosmesis. The LD90 for PEP005 for a panel of tumor cell lines was 180-220 muM. Electron microscopy showed that treatment with PEP005 both ill vitro (230 tot) and ill vivo (42 nmol) rapidly caused swelling of mitochondria and cell death by primary necrosis. Cr-51 release, uptake of propidium iodide, and staining with the mitochondria dye JC1, revealed that PEP005 (230 muM) treatment of tumor cells ill vitro resulted in a rapid plasma membrane perturbation and loss of mitochondrial membrane potential. PEP005 thus emerges as a new topical anti-skin cancer agent that has a novel mode of action involving plasma membrane and mitochondrial disruption and primary necrosis, ultimately resulting in an excellent cosmetic outcome.

Emerging roles of urotensin-II in cardiovascular disease

Urotensin-II (UII) is a highly potent endogenous peptide within the cardiovascular system. Through stimulation of Galphaq-coupled UT receptors, UII mediates contraction of vascular smooth muscle and endothelial-dependent vasorelaxation, and positive inotropy in human right atrium and ventricle. A pathogenic role of the UT receptor system is emerging in cardiovascular disease states, with evidence for upregulation of the UT receptor system in patients with congestive heart failure (CHF), pulmonary hypertension, cirrhosis and portal hypertension, and chronic renal failure. In vitro and in vivo studies show that under pathophysiological conditions, UII might contribute to cardiomyocyte hypertrophy, extracellular matrix production, enhanced vasoconstriction, vascular smooth muscle cell hyperplasia, and endothelial cell hyper-permeability. Single nucleotide polymorphisms of the UII gene may also impart a genetic predisposition of patients to diabetes. Therefore, the UT receptor system is a potential therapeutic target in the treatment of cardiac, pulmonary, and renal diseases. UT receptor antagonists are currently being developed to prevent and/or reverse the effects of over-activated UT receptors by the endogenous ligand. This review describes UII peptide and converting enzymes, and UT receptors in the cardiovascular system, focusing on pathophysiological roles of UII in the heart and blood vessels. (C) 2004 Elsevier Inc. All rights reserved,

Retinal glutamate transporter activity persists under simulated ischemic conditions

Elevated extracellular concentrations of the neurotransmitter glutamate are neurotoxic and directly contribute to CNS damage as a result of ischemic pathologies. However, the main contributors to this uncontrolled rise in glutamate are still unconfirmed. It has been reported that the reversal of high-affinity glutamate transporters is a significant contributing factor. Conversely, it has also Peen observed that these transporters continue to take up glutamate, albeit at a reduced saturation concentration, under ischemic conditions. We sought to determine whether glutamate transporters continue to remove glutamate from the extracellular space under ischemic conditions by pharmacologically modulating the activity of high-affinity retinal glutamate transporters during simulated ischemia in vitro. Retinal glutamate transporter activity was significantly reduced under these ischemic conditions. The suppression of retinal glutamate transporter activity, with the protein kinase C inhibitor chelerythrine, significantly reduced ischemic glutamate uptake and enhanced retinal neurodegeneration. These findings imply a limited but protective role for retinal glutamate transporters under certain ischemic conditions, suggesting that pharmacological enhancement of high-affinity glutamate transporter activity may reduce tissue damage and loss of function resulting from toxic extracellular glutamate concentrations. (C) 2004 Wiley-Liss, Inc.

The ins and outs of E-cadherin trafficking

One way of controlling the activity of E-cadherin - a protein that is, simultaneously, a major cell-adhesion molecule, a powerful tumour suppressor, a determinant of cell polarity and a partner to the potent catenin signalling molecules - is to keep it on the move. During the past two decades, many insights into the fundamental role of E-cadherin in these processes have been garnered. Studies during the past five years have begun to reveal the importance of intracellular trafficking as a means of regulating the functions of E-cadherin. E-cadherin is trafficked to and from the cell surface by exocytic and multiple endocytic pathways. In this article, we survey the vesicle-trafficking machinery that is responsible for the sorting, transport, actin association and vesicle targeting of E-cadherin to regulate its movement and function during growth and development and, possibly, in cancer.