Increase of the anti-HIV activity of D4T in human T-cell culture by the use of the sate pronucleotide approach

The bis(S-acetyl-2-thioethyl) phosphotriester derivative of 2′,3′-didehydro-2′,3′-dideoxythymidine has been synthesized and evaluated for its inhibitory effects on the replication of HIV-1 in several cell culture systems. This pronucleotide showed potent anti-HIV activity and proved to be significantly superior to the parent nucleoside with regard to the antiviral efficiency.     

Design of new mononucleotide prodrugs: aryl (SATE) phosphotriester derivatives

Synthesis, biological activities and decomposition kinetics of novel phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-tButyl-2-thioethyl (tBuSATE) group and L-tyrosinyl residues are reported. All the derivatives appeared to be potent inhibitors of HIV-1 replication in various cell culture experiments. The proposed decomposition process of these mixed phosphotriesters may involve successively an esterase and then a phosphodiesterase activation.     

DESIGN OF NEW MONONUCLEOTIDE PRODRUGS: ARYL (SATE) PHOSPHOTRIESTER DERIVATIVES

Synthesis, biological activities and decomposition kinetics of novel phosphotriester derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) bearing a S-tButyl-2-thioethyl (tBuSATE) group and L-tyrosinyl residues are reported. All the derivatives appeared to be potent inhibitors of HIV-1 replication in various cell culture experiments. The proposed decomposition process of these mixed phosphotriesters may involve successively an esterase and then a phosphodiesterase activation.     

Crosstalk between Smad2/3 and specific isoforms of ERK in TGF‐β1‐induced TIMP‐3 expression in rat chondrocytes

This study investigated the roles of ERK1 and ERK2 in transforming growth factor‐β1 (TGF‐β1)‐induced tissue inhibitor of metalloproteinases‐3 (TIMP‐3) expression in rat chondrocytes, and the specific roles of ERK1 and ERK2 in crosstalk with Smad2/3 were investigated to demonstrate the molecular mechanism of ERK1/2 regulation of TGF‐β1 signalling. To examine the interaction of specific isoforms of ERK and the Smad2/3 signalling pathway, chondrocytes were infected with LV expressing either ERK1 or ERK2 siRNA and stimulated with or without TGF‐β1. At indicated time‐points, TIMP‐3 expression was determined by real‐time PCR and Western blotting; p‐Smad3, nuclear p‐Smad3, Smad2/3, p‐ERK1/2 and ERK1/2 levels were assessed. And then, aggrecan, type II collagen and the intensity of matrix were examined. TGF‐β1‐induced TIMP‐3 expression was significantly inhibited by ERK1 knock‐down, and the decrease in TIMP‐3 expression was accompanied by a reduction of p‐Smad3 in ERK1 knock‐down cells. Knock‐down of ERK2 had no effect on neither TGF‐β1‐induced TIMP‐3 expression nor the quantity of p‐Smad3. Moreover, aggrecan, type II collagen expression and the intensity of matrix were significantly suppressed by ERK1 knock‐down instead of ERK2 knock‐down. Taken together, ERK1 and ERK2 have different roles in TGF‐β1‐induced TIMP‐3 expression in rat chondrocytes. ERK1 instead of ERK2 can regulate TGF‐β/Smad signalling, which may be the mechanism through which ERK1 regulates TGF‐β1‐induced TIMP‐3 expression.     

Insulin inhibits extracellular regulated kinase 1/2 phosphorylation in a phosphatidylinositol 3-kinase (PI3) kinase-dependent manner in Neuro2a cells

Insulin signalling is well studied in peripheral tissue, but not in neuronal tissue. To gain more insight into neuronal insulin signalling we examined protein kinase B (PKB) and extracellular regulated kinase 1 and 2 (ERK1/2) regulation in serum-deprived Neuro2a cells. Insulin phosphorylated PKB in a dose-dependent manner but reduced phosphorylation of ERK1/2. Both processes were phosphatidylinositol 3-kinase (PI3K) dependent. Interestingly, blockade of PI3K in combination with insulin induced phosphorylation of ERK1/2. The phosphorylation of ERK1/2 could be blocked with a specific inhibitor of mitogen-activated protein/ERK kinase (MEK), suggesting that it was mediated through the highly conserved Ras-Raf-MEK-ERK1/2 pathway. Prolonged exposure to high concentrations of insulin resulted in a desensitized PI3K-PKB route. The insulin-induced inhibition of ERK1/2 phosphorylation was also diminished when the PI3K-PKB route was desensitized. Blockade of PI3K in combination with insulin, however, still resulted in an unaltered MEK-dependent phosphorylation of ERK1/2. We conclude that PI3K is an important integrator of insulin signalling in Neuro2a cells as it regulates activation of PKB and inhibition of ERK1/2, and is sensitive to the duration of the insulin stimulus.     

Mu opioid receptor activation of ERK1/2 is GRK3 and arrestin dependent in striatal neurons

In this study we investigated the mechanisms responsible for MAP kinase ERK1/2 activation following agonist activation of endogenous mu opioid receptors (MOR) normally expressed in cultured striatal neurons. Treatment with the MOR agonist fentanyl caused significant activation of ERK1/2 in neurons derived from wild type mice. Fentanyl effects were blocked by the opioid antagonist naloxone and were not evident in neurons derived from MOR knock-out (-/-) mice. In contrast, ERK1/2 activation by fentanyl was not evident in neurons from GRK3-/- mice or neurons pretreated with small inhibitory RNA for arrestin3. Consistent with this observation, treatment with the opiate morphine (which is less able to activate arrestin) did not elicit ERK1/2 activation in wild type neurons; however, transfection of arrestin3-(R170E) (a dominant positive form of arrestin that does not require receptor phosphorylation for activation) enabled morphine activation of ERK1/2. In addition, activation of ERK1/2 by fentanyl and morphine was rescued in GRK3-/- neurons following transfection with dominant positive arrestin3-(R170E). The activation of ERK1/2 appeared to be selective as p38 MAP kinase activation was not increased by either fentanyl or morphine treatment in neurons from wild type, MOR-/-, or GRK3-/- mice. In addition, U0126 (a selective inhibitor of MEK kinase responsible for ERK phosphorylation) blocked ERK1/2 activation by fentanyl. These results support the hypothesis that MOR activation of ERK1/2 requires opioid receptor phosphorylation by GRK3 and association of arrestin3 to initiate the cascade resulting in ERK1/2 phosphorylation in striatal neurons.     

Mononucleoside SATE glucosyl phosphorothiolates as a new series of pronucleotides

The synthesis and the study of two phosphorothiolate derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and glucosyl residues associated to the phosphorus atom by a 2-oxyethyl link, are reported. These derivatives could be considered as prototypes of a new series of nucleotide prodrugs (pronucleotides).     

Inhibition of TNF-alpha-induced neutrophil apoptosis by crystals of calcium pyrophosphate dihydrate is mediated by the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways up-stream of caspase 3

The role of protein kinases in the inhibition of TNF-alpha associated apoptosis of human neutrophils by crystals of calcium pyrophosphate dihydrate (CPPD) (25 mg/ml) was investigated. We monitored the activities of the p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3-K)-regulated protein kinase B (Akt) in neutrophils incubated with TNF-alpha and CPPD crystals, separately and in combination, in parallel with the endogenous caspase 3 activity and DNA fragmentation. CPPD crystals were observed to induce a robust and transient activation of ERK1, ERK2, and Akt, whereas TNF-alpha produced only a modest and delayed activation of Akt. In the presence of TNF-alpha, Akt activity was enhanced, and CPPD crystal-induced activation of ERK1 and ERK2 was more sustained than with CPPD crystals alone, but TNF-alpha itself reduced the basal phosphotransferase activities of these MAP kinases. Preincubation with the MAP kinase kinase (MEK1) inhibitors PD98059 (20 ng/ml) and U0126 (250 nM), or the PI3-K inhibitors wortmannin (100 nM) and LY294002 (50 microM) repressed the activation of ERK1, ERK2, and Akt in association with CPPD crystal incubation, in the absence or presence of TNF-alpha. Furthermore, the inhibition of the Mek1/Mek2-->ERK1/ERK2 or PI3-K/Akt pathways reversed CPPD crystal-associated suppression of TNF-alpha-induced caspase 3 activation and neutrophil apoptosis. Together, these results indicate that CPPD crystals function to induce acute inflammatory responses through ERK1/ERK2 and PI3-K/Akt-mediated stimulation of neutrophil activation and repression of apoptosis.     

The paired activation of the two components of the muscarinic M3 receptor dimer is required for induction of ERK1/2 phosphorylation

Muscarinic M(3) receptors stimulate ERK1/2, the mitogen-activated protein kinase pathway. A mutant of the muscarinic M(3) receptor in which most of the third intracellular (i3) loop had been deleted (M(3)-short) completely lost the ability to stimulate the ERK1/2 phosphorylation in COS-7 cells. This loss was evident despite the fact that the receptor was able to couple efficiently to the phospholipase C second messenger pathway. In co-transfected cells, M(3)-short greatly reduced the ability of M(3) to activate ERK1/2. In another set of experiments we tested the ability of a mutant M(3)/M(2)(16aa) receptor, in which the first 16 amino acids of the i3 loop of the M(3) receptor were replaced with the corresponding segment of the muscarinic M(2) receptor to stimulate ERK1/2 phosphorylation. This mutant is not coupled to Galpha(q), but it is weakly coupled to Galpha(i). Despite its coupling modification this receptor was able to stimulate ERK1/2 phosphorylation. Again, M(3)-short greatly reduced the ability of M(3)/M(2)(16aa) to activate ERK1/2 in co-transfected cells. Similar results were obtained in stable-transfected Chinese hamster ovary (CHO) cells lines. In CHO M(3) cells carbachol induced a biphasic increase of ERK1/2 phosphorylation; a first increase at doses as low as 0.1 microm and a second increase starting from 10 microm. In CHO M(3)-short and in double-transfected CHO M(3)/M(3)-short cells we observed only the lower doses increase of ERK1/2 phosphorylation; no further increase was observed up to 1 mm carbachol. This suggests that in double-transfected CHO cells M(3)-short prevents the effect of the higher doses of carbachol on the M(3) receptor. In a final experiment we tested the ability of co-transfected chimeric alpha(2)/M(3) and M(3)/alpha(2) receptors to activate the ERK1/2 pathway. When given alone, carbachol and, to a lesser extent, clonidine, stimulated the coupling of the co-transfected chimeric receptors to the phospholipase C second messenger pathway, but they were unable to stimulate ERK1/2 phosphorylation. On the contrary, a strong stimulation of ERK1/2 phosphorylation was observed when the two agonists were given together despite the fact that the overall increase in phosphatidylinositol hydrolysis was not dissimilar from that observed in cells treated with carbachol alone. Our data suggest that the activation of the ERK1/2 pathway requires the coincident activation of the two components of a receptor dimer.     

Akt down-regulates ERK1/2 nuclear localization and angiotensin II-induced cell proliferation through PEA-15

Angiotensin II (AngII) type 1 receptors (AT1) regulate cell growth through the extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol 3-kinase (PI3K) pathways. ERK1/2 and Akt/protein kinase B, downstream of PI3K, are independently activated but both required for mediating AngII-induced proliferation when expressed at endogenous levels. We investigate the effect of an increase in the expression of wild-type Akt1 by using Chinese hamster ovary (CHO)-AT1 cells. Unexpectedly, Akt overexpression inhibits the AT1-mediated proliferation. This effect could be generated by a cross-talk between the PI3K and ERK1/2 pathways. A functional partner is the phosphoprotein enriched in astrocytes of 15 kDa (PEA-15), an Akt substrate known to bind ERK1/2 and to regulate their nuclear translocation. We report that Akt binds to PEA-15 and that Akt activation leads to PEA-15 stabilization, independently of PEA-15 interaction with ERK1/2. Akt cross-talk with PEA-15 does not affect ERK1/2 activation but decreases their nuclear activity as a result of the blockade of ERK1/2 nuclear accumulation. In response to AngII, PEA-15 overexpression displays the same functional consequences on ERK1/2 signaling as Akt overactivation. Thus, Akt overactivation prevents the nuclear translocation of ERK1/2 and the AngII-induced proliferation through interaction with and stabilization of endogenous PEA-15.     

Increase of the adenallene anti-HIV activity in cell culture using its bis(tBuSATE) phosphotriester derivative.

The bis(S-pivaloyl-2-thioethyl) phosphotriester derivative of 9-(4'-hydroxy-1',2'-butadienyl)adenine (adenallene) was synthesized. This mononucleotide prodrug proved to be more effective than the parent nucleoside in inhibiting HIV-1 replication in several human T4 lymphoblastoid cell lines.     

SATE (aryl) phosphotriester series. II. Stability studies and physicochemical parameters

The stability of phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and various aryl residues derived from L-tyrosine was evaluated in biological media. The results demonstrate that such compounds give rise to intracellular delivery of the parent mononucleotide through esterase and phosphodiesterase hydrolytic steps, successively.     

Na/H exchange regulatory factor 1, a novel AKT-associating protein, regulates extracellular signal-regulated kinase signaling through a B-Raf-mediated pathway

Na/H exchange regulatory factor 1 (NHERF1) is a scaffolding protein that regulates signaling and trafficking of several G protein-coupled receptors (GPCRs), including the parathyroid hormone receptor (PTH1R). GPCRs activate extracellular signal-regulated kinase (ERK)1/2 through different mechanisms. Here, we characterized NHERF1 regulation of PTH1R-stimulated ERK1/2. Parathyroid hormone (PTH) stimulated ERK1/2 phosphorylation by a protein kinase A (PKA)-dependent, but protein kinase C-, cyclic adenosine 5'-monophosphate-, and Rap1-independent pathway in Chinese hamster ovary cells stably transfected with the PTH1R and engineered to express NHERF1 under the control of tetracycline. NHERF1 blocked PTH-induced ERK1/2 phosphorylation downstream of PKA. This suggested that NHERF1 inhibitory effects on ERK1/2 occur at a postreceptor locus. Forskolin activated ERK1/2, and this effect was blocked by NHERF1. NHERF1 interacted with AKT and inhibited ERK1/2 activation by decreasing the stimulatory effect of 14-3-3 binding to B-Raf, while increasing the inhibitory influence of AKT negative regulation on ERK1/2 activation. This novel regulatory mechanism provides a new model by which cytoplasmic adapter proteins modulate ERK1/2 activation through a receptor-independent mechanism involving B-Raf.     

ERK1/2 antagonizes glycogen synthase kinase-3beta-induced apoptosis in cortical neurons

Inhibition of glycogen synthase kinase-3beta (GSK3beta) is one of the mechanisms by which phosphatidylinositol 3-kinase (PI3K) activation protects neurons from apoptosis. Here, we report that inhibition of ERK1/2 increased the basal activity of GSK3beta in cortical neurons and that both ERK1/2 and PI3K were required for brain-derived neurotrophic factor (BDNF) suppression of GSK3beta activity. Moreover, cortical neuron apoptosis induced by expression of recombinant GSK3beta was inhibited by coexpression of constitutively active MKK1 or PI3K. Activation of both endogenous ERK1/2 and PI3K signaling pathways was required for BDNF to block apoptosis induced by expression of recombinant GSK3beta. Furthermore, cortical neuron apoptosis induced by LY294002-mediated activation of endogenous GSK3beta was blocked by expression of constitutively active MKK1 or by BDNF via stimulation of the endogenous ERK1/2 pathway. Although both PI3K and ERK1/2 inhibited GSK3beta activity, neither had an effect on GSK3beta phosphorylation at Tyr-216. Interestingly, PI3K (but not ERK1/2) induced the inhibitory phosphorylation of GSK3beta at Ser-9. Significantly, coexpression of constitutively active MKK1 (but not PI3K) still suppressed neuronal apoptosis induced by expression of the GSK3beta(S9A) mutant. These data suggest that activation of the ERK1/2 signaling pathway protects neurons from GSK3beta-induced apoptosis and that inhibition of GSK3beta may be a common target by which ERK1/2 and PI3K protect neurons from apoptosis. Furthermore, ERK1/2 inhibits GSK3beta activity via a novel mechanism that is independent of Ser-9 phosphorylation and likely does not involve Tyr-216 phosphorylation.     

SATE (aryl) phosphotriester series. I. Synthesis and biological evaluation

Synthesis and biological activities of several phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and aryl residues derived from L-tyrosine are reported. All compounds showed marked anti-HIV activity in thymidine kinase-deficient CEM cells demonstrating their ability to deliver intracellularly the parent 5'-mononucleotide.     

cAMP potentiates H(2)O(2)-induced ERK1/2 phosphorylation without the requirement for MEK1/2 phosphorylation

In Jurkat T lymphocytes, hydrogen peroxide (H(2)O(2)) potentiates the phosphorylation level of extracellular signal-regulated kinase 1 and 2 (ERK1/2) caused by T cell receptor (TCR) stimulation with anti-CD3 and anti-CD28 or anti-CD3 alone. Submillimolar concentrations of H(2)O(2)-induced phosphorylation of ERK1/2 and MAP/ERK kinase 1 and 2 (MEK1/2) without antigenic stimulation. H(2)O(2) also induced the electrophoretic mobility shift of Lck from 56 to 60 kDa. The MEK inhibitor, PD98059 attenuated ERK1/2 and MEK1/2 phosphorylation, as well as the migration shift of Lck induced by H(2)O(2). The phospholipase C (PLC) inhibitor, U73122, and EGTA reduced the phosphorylation of both ERK1/2 and MEK1/2 induced by H(2)O(2). Interestingly, an increase of intracellular cAMP level with forskolin or 8-(4-chlorophenylthio)-cAMP augmented ERK1/2 phosphorylation by H(2)O(2), while inhibiting MEK1/2 phosphorylation by H(2)O(2). These results demonstrate an alternative pathway that results in augmentation of ERK1/2 phosphorylation without concomitant MEK1/2 phosphorylation in T cells.     

ERK1/2-driven and MKP-mediated inhibition of EGF-induced ERK5 signaling in human proximal tubular cells

The MEK1-ERK1/2 signaling pathway has been implicated in the regulation of renal epithelial cell proliferation, epithelial-to-mesenchymal transition and the induction of an invasive cell phenotype. Much less information is available about the MEK5-ERK5 module and its role in renal epithelial cell proliferation and differentiation. In the present study we have investigated the regulation of these two families of extracellular signal-regulated kinases in epidermal growth factor (EGF)-stimulated human kidney-2 (HK-2) cells and a possible interaction between ERK1/2 and ERK5. Here we report that 5 ng/ml EGF led to a strong stimulation of HK-2 cell proliferation, which was largely U0126-sensitive. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at 10 and 1 microM, respectively, inhibited basal and EGF-induced ERK1/2 phosphorylation but not ERK5 phosphorylation. Long-term inhibition of MEK1/2-ERK1/2 signaling and/or vanadate-sensitive protein phosphatases enhanced and prolonged EGF-induced ERK5 phosphorylation, while transient expression of an adenoviral constitutively active MEK1 (Ad-caMEK1) construct completely blocked EGF-induced ERK5 phosphorylation. Expression of Ad-caMEK1 in HK-2 cells resulted in the upregulation of the dual-specificity phosphatases MKP-3/DUSP6, MKP-1/DUSP1, and DUSP5. The EGF-mediated time-dependent induction of MKP-3, MKP-1 and DUSP5 mRNA levels was U0126-sensitive at a concentration, which blocked EGF-mediated ERK1/2 phosphorylation but not ERK5 phosphorylation. Furthermore, U0126 inhibited EGF-induced MKP-3 and MKP-1 protein expression. Both MKP-3 and MKP-1 co-immunoprecipitated with ERK5 in unstimulated as well as in EGF-stimulated HK-2 cells. These results suggest the existence of an ERK1/2-driven negative feed-back regulation of ERK5 signaling in EGF-stimulated HK-2 cells, which is mediated by MKP-3, DUSP5 and/or MKP-1.     

Mechanisms for vascular cell adhesion molecule-1 activation of ERK1/2 during leukocyte transendothelial migration

Background

During inflammation, adhesion molecules regulate recruitment of leukocytes to inflamed tissues. It is reported that vascular cell adhesion molecule-1 (VCAM-1) activates extracellular regulated kinases 1 and 2 (ERK1/2), but the mechanism for this activation is not known. Pharmacological inhibitors of ERK1/2 partially inhibit leukocyte transendothelial migration in a multi-receptor system but it is not known whether VCAM-1 activation of ERK1/2 is required for leukocyte transendothelial migration (TEM) on VCAM-1.

Methodology/Principal Findings

In this study, we identified a mechanism for VCAM-1 activation of ERK1/2 in human and mouse endothelial cells. VCAM-1 signaling, which occurs through endothelial cell NADPH oxidase, protein kinase Cα (PKCα), and protein tyrosine phosphatase 1B (PTP1B), activates endothelial cell ERK1/2. Inhibition of these signals blocked VCAM-1 activation of ERK1/2, indicating that ERK1/2 is activated downstream of PTP1B during VCAM-1 signaling. Furthermore, VCAM-1-specific leukocyte migration under physiological laminar flow of 2 dynes/cm² was blocked by pretreatment of endothelial cells with dominant-negative ERK2 K52R or the MEK/ERK inhibitors, PD98059 and U0126, indicating for the first time that ERK regulates VCAM-1-dependent leukocyte transendothelial migration.

Conclusions/Significance

VCAM-1 activation of endothelial cell NADPH oxidase/PKCα/PTP1B induces transient ERK1/2 activation that is necessary for VCAM-1-dependent leukocyte TEM.