Tyrosine-specific Protein Kinase Research Articles

Tyrphostins and Other Tyrosine Kinase Inhibitors

The development of tyrosine phosphorylation inhibitors has transformed the approach to cancer therapy and is likely to affect other fields of medicine. In spite of the conservation among protein tyrosine kinases (PTKs), one can develop small molecules that block the activity of a narrow spectrum of PTKs and that exhibit much less toxicity than the currently used chemotherapeutic agents. In this review, we discuss principles for inhibiting specific PTKs. We discuss (a) the birth of the concept of generating targeted, nontoxic signal transduction inhibitors, (b) the potential of substrate-competitive versus the more common ATP-competitive PTK inhibitors, (c) the combination of PTK inhibitors with other signal transduction inhibitors to induce apoptosis-the best way to induce the demise of the cancer cell, and (d) the potential to utilize PTK inhibitors/tyrphostins to attenuate nonmalignant pathological conditions, such as immune disorders, tissue rejection, and restenosis.

Engagement of P‐selectin Glycoprotein Ligand‐1 (PSGL‐1) induces interactions with the actin cytoskeleton

P-selectin glycoprotein ligand-1 (PSGL-1) is a well-studied P-selectin counter receptor. Its role as a rolling receptor has been well established, and interactions between PSGL-1 and P-selectin are known to be critical for a strong inflammatory response. Involvement of PSGL-1 in signal transduction after P-selectin engagement is not well known. The purpose of this study was to investigate potential downstream events following PSGL-1 ligation by either a function blocking monoclonal antibody (KPL-1) or P-selectin. Crosslinking PSGL-1 on HL60 cells with KPL-1, which binds to the P-selectin binding site on PSGL-1, induces PSGL-1 association with the actin cytoskeleton. Conversely, crosslinking of PSGL-1 with KPL-2, an antibody that recognizes a sequence on PSGL-1 near the trans-membrane domain, did not. We have shown that interactions between PSGL-1 and the actin cytoskeleton are required for rolling on P-selectin. To determine the potential pathway that induced associations with the actin cytoskeleton, cells were pre-incubated with various pharmacological inhibitors prior to crosslinking. Reagents tested included inhibitors of tyrosine-specific protein kinases (Genistein), p38 kinase (SB 203580), MAP kinase kinase (PD 98059), protein kinase C (Calphostin C), and Syk (Piceatannol). The results from this investigation will help us define the intracellular signaling pathway required for actin cytoskeletal rearrangement following PSGL-1 ligation. [This work was supported by NIH R01 GM60563 and T32HL007829].

The neurotrophin receptor TrkB is colocalized to mitochondrial membranes

The transmembrane tyrosine-specific protein kinase TrkB has been shown to serve as a receptor for the neurotrophic factors BDNF and NT-4. Neurotrophin binding to TrkB isoformes mediates many intracellular signaling pathways, including calcium signalling. Two truncated isoforms of the receptor, lacking the tyrosine kinase activity, signal through a yet unknown pathway. Specific signals modulate the surface expression of TrkB, which is localized in considerable amounts in intracellular pools. These intracellular pools has not been specified so far. We therefore investigated the intracellular distribution of TrkB by colocalisation studies. In contrast to the unspecific neurotrophin receptor NGFRp75, TrkB immunohistochemistry showed a staining pattern very similar to mitochondrial stainings in adult human skeletal muscle fibers. Immunofluorescence techniques revealed in different types of permeabilized cells that TrkB is bound to mitochondrial membranes. This observation was confirmed on isolated astrocyte mitoplasts. Colocalisation of the TrkB ligand NT-4 and the specific mitochondrial marker cytochrome c oxidase was also observed. Western blot analysis of isolated mitochondria from rat brain and skeletal muscle verified that a truncated isoform of TrkB is present in both, brain and muscle mitochondria, and full-length TrkB is additionally present in brain mitochondria. Our results imply that neurotrophins can be stored in mitochondria and possibly act as signalling molecules on mitochondria.

Tyrphostin‐A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.

Epstein-Barr Virus (EBV) Latent Membrane Protein 2A Regulates B-Cell Receptor-Induced Apoptosis and EBV Reactivation through Tyrosine Phosphorylation

Epstein-Barr virus (EBV) is a human herpesvirus that establishes a lifelong latent infection of B cells. Within the immune system, apoptosis is a central mechanism in normal lymphocyte homeostasis both during early lymphocyte development and in response to antigenic stimuli. In this study, we found that latent membrane protein 2A (LMP2A) inhibited B-cell receptor (BCR)-induced apoptosis in Burkitt's lymphoma cell lines. Genistein, a specific inhibitor of tyrosine-specific protein kinases, blocked BCR-induced apoptosis and EBV reactivation in the cells. These findings indicate that LMP2A blocks BCR-induced cell apoptosis and EBV reactivation through the inhibition of activation of tyrosine kinases by BCR cross-linking.

Protein kinase C translocation and Src protein tyrosine kinase activation mediate isoflurane-induced preconditioning in vivo: potential downstream targets of mitochondrial adenosine triphosphate-sensitive potassium channels and reactive oxygen species.

The authors tested the hypotheses that protein kinase C (PKC)-specific isoform translocation and Src protein tyrosine kinase (PTK) activation play important roles in isoflurane-induced preconditioning in vivo. Rats (n = 125) instrumented for measurement of hemodynamics underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion and received 0.9% saline (control); PKC inhibitors chelerythrine (5 mg/kg), rottlerin (0.3 mg/kg), or PKC-epsilonV1-2 peptide (1 mg/kg); PTK inhibitors lavendustin A (1 mg/kg) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; 1 mg/kg); mitochondrial adenosine triphosphate-sensitive potassium channel antagonist 5-hydroxydecanote (10 mg/kg); or reactive oxygen species scavenger N-acetylcysteine (150 mg/kg) in the absence and presence of a 30-min exposure to isoflurane (1.0 minimum alveolar concentration) in separate groups. Isoflurane was discontinued 15 min before coronary occlusion (memory period). Infarct size was determined using triphenyltetrazolium staining. Immunohistochemistry and confocal microscopic imaging were performed to examine PKC translocation in separate groups of rats. Isoflurane significantly (P < 0.05) reduced infarct size (40 +/- 3% [n = 13]) as compared with control experiments (58 +/- 2% [n = 12]). Chelerythrine, rottlerin, PKC-epsilonV1-2 peptide, lavendustin A, PP1, 5-hydroxydecanote, and N-acetylcysteine abolished the anti-ischemic actions of isoflurane (58 +/- 2% [n = 8], 50 +/- 3% [n = 9], 53 +/- 2% [n = 9], 59 +/- 3% [n = 6], 57 +/- 3% [n = 7], 60 +/- 3% [n = 7], and 53 +/- 3% [n = 6], respectively). Isoflurane stimulated translocation of the delta and epsilon isoforms of PKC to sarcolemmal and mitochondrial membranes, respectively. Protein kinase C-delta, PKC-epsilon, and Src PTK mediate isoflurane-induced preconditioning in the intact rat heart. Opening of mitochondrial adenosine triphosphate-sensitive potassium channels and generation of reactive oxygen species are upstream events of PKC activation in this signal transduction process.

United States Food and Drug Administration Drug Approval summary: Gefitinib (ZD1839; Iressa) tablets.

On May 5, 2003, gefitinib (Iressa; ZD1839) 250-mg tablets (AstraZeneca Inc.) received accelerated approval by the United States Food and Drug Administration as monotherapy for patients with locally advanced or metastatic non-small cell lung cancer after failure of both platinum-based and docetaxel chemotherapies. Information provided in this summary includes chemistry manufacturing and controls, clinical pharmacology, and clinical trial efficacy and safety results. Gefitinib is an anilinoquinazoline compound with the chemical name 4-quinazolinamine,N-(3-chloro-4-flurophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]. It has the molecular formula C(22)H(24)ClFN(4)O(3). Gefitinib is often referred to as a "specific" or "selective" inhibitor of epidermal growth factor receptor. Studies demonstrate, however, that gefitinib inhibits the activity of other intracellular transmembrane tyrosine-specific protein kinases at concentrations similar to those at which it inhibits the epidermal growth factor signal. Maximum plasma concentrations resulting from clinically relevant doses are 0.5-1 microM or more, well within the IC(50) values of several tyrosine kinases. No clinical studies have been performed that demonstrate a correlation between epidermal growth factor receptor expression and response to gefitinib. Gefitinib is 60% available after oral administration and is widely distributed throughout the body. Gefitinib is extensively metabolized in the liver by cytochrome P450 3A4 enzyme. Over a 10-day period, approximately 86% of an orally administered radioactive dose is recovered in the feces, with <4% of the dose in the urine. After daily oral administration, steady-state plasma levels are reached in 10 days and are 2-fold higher than those achieved after single doses. Gefitinib effectiveness was demonstrated in a randomized, double-blind, Phase II, multicenter trial comparing two oral doses of gefitinib (250 versus 500 mg/day). A total of 216 patients were enrolled. The 142 patients who were refractory to or intolerant of a platinum and docetaxel comprised the evaluable population for the efficacy analysis. A partial tumor response occurred in 14% (9 of 66) of patients receiving 250 mg/day gefitinib and in 8% (6 of 76) of patients receiving 500 mg/day gefitinib. The overall objective response rate (RR) for both doses combined was 10.6% (15 of 142 patients; 95% confidence interval, 6.0-16.8%). Responses were more frequent in females and in nonsmokers. The median duration of response was 7.0 months (range, 4.6-18.6+ months). Other submitted data included the results of two large trials conducted in chemotherapy-naive, stage III and IV non-small cell lung cancer patients. Patients were randomized to receive gefitinib (250 or 500 mg daily) or placebo, in combination with either gemcitabine plus cisplatin (n = 1093) or carboplatin plus paclitaxel (n = 1037). Results from this study showed no benefit (RR, time to progression, or survival) from adding gefitinib to chemotherapy. Consequently, gefinitib is only recommended for use as monotherapy. Common adverse events associated with gefitinib treatment included diarrhea, rash, acne, dry skin, nausea, and vomiting. Interstitial lung disease has been observed in patients receiving gefitinib. Worldwide, the incidence of interstitial lung disease was about 1% (2% in the Japanese post-marketing experience and about 0.3% in a United States expanded access program). Approximately one-third of the cases have been fatal. Gefitinib was approved under accelerated approval regulations on the basis of a surrogate end point, RR. No controlled gefitinib trials, to date, demonstrate a clinical benefit, such as improvement in disease-related symptoms or increased survival. Accelerated approval regulations require the sponsor to conduct additional studies to verify that gefitinib therapy produces such benefit.

The role of protein tyrosine kinases in CYP1A1 induction by omeprazole and thiabendazole in rat hepatocytes

Benzimidazoles compounds like omeprazole (OME) and thiabendazole (TBZ) mediate CYP1A1 induction differently from classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). To clarify the involvement of an intracellular signal pathway in CYP1A1 induction by OME and TBZ, the TBZ, OME and 3-MC signal-transducing pathways were compared by using specific protein tyrosine kinase inhibitors in primary culture of rat hepatocytes. The effect of OME and TBZ (75–250 μM) on cytochrome P450 1A1 (CYP1A1) expression was therefore studied in primary cultures of rat hepatocytes after 24 h, 48 h and 72 h of exposure. Both compounds provoked a dose- and time-dependent increase in CYP1A1 (EROD activity, protein and mRNA levels), but OME was less effective at all the concentrations and times tested. The mechanism of benzimidazole-mediated induction of CYP1A1 was investigated by comparison with 3-MC, a prototypical AhR ligand. As expected, OME and TBZ were unable to displace [ 3H]-TCDD from its binding sites to the AhR in competitive binding studies. Moreover, classic tyrosine kinase inhibitor herbimycin A (HA) inhibited the two benzimidazoles-mediated CYP1A1 inductions, but only partially inhibited the 3-MC-mediated one. Another two tyrosine kinase inhibitors, Lavendustin A (LA) and genistein (GEN), had no effect on CYP1A1 induction by benzimidazoles and 3-MC. These results are consistent with the implication of a tyrosine kinase, most probably the Src tyrosine kinase, in the mechanism of CYP1A1 induction in rat hepatocytes.

Role of imatinib mesylate (Gleevec®/Glivec®) in gastrointestinal stromal tumors

Gastrointestinal stromal tumors are soft tissue sarcomas of the gastrointestinal tract that originate from mesenchymal cells. Advances in the systemic therapy of gastrointestinal stromal tumors are highlighted by the rapid development and approval of the molecularly targeted therapy imatinib mesylate (Gleevec®/Glivec®). Mutations of the KIT gene are known to be present in most gastrointestinal stromal tumors and result in gain of function, with permanent activation of the expressed KIT receptor in the absence of binding of the stem cell factor ligand. Imatinib is the first rationally designed selective inhibitor of specific protein tyrosine kinases, including KIT. Inhibiting the downstream signaling of KIT switches the cell balance into apoptosis. Although complete responses have seldom been seen up until now, imatinib has proven to be extremely effective in the treatment of patients with unresectable and/or metastatic gastrointestinal stromal tumors.

Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins

To study the signaling pathway involved in the regulation of galectin-3 expression we used phorbol ester to stimulate macrophage differentiation of THP-1 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) increased significantly the level of expression of galectin-3 in THP-1 cells. PMA-induced galectin-3 overexpression was blocked by: protein kinase C inhibitors staurosporine, calphostin C, and apigenin; tyrosine-specific protein kinase inhibitors genistein and tyrphostin A25; PD 98059, a selective inhibitor of mitogen-activated protein kinase (MAPK) kinase 1 (MEK1 or MKK1); and SB 203580, a specific inhibitor of p38 MAPK. Galectin-3 up-regulation was not affected by exposure to two inhibitors of cAMP-dependent protein kinase (PKA), H-89 and KT5720. Co-transfection of pPG3.5, a plasmid vector containing the rabbit galectin-3 promoter and the constructs pMCL-MKK1 N3 or pRC-RSV-MKK3Glu that constitutively express MKK1 and MKK3, raised the activity of galectin-3 promoter by 185% and 110%, respectively. Co-transfection with a Ha- Ras expression vector stimulated galectin-3 promoter activity approximately 10-fold. Expression of c- Jun or v- Jun raised the level of galectin-3 promoter activity more the three- and fourfold, respectively. Co-transfection of c- Jun and pPG3.5 5′-upstream deletion mutants resulted in a reduction of the galectin-3 promoter activity by 50% to 80%. Transfection of c- Jun, v- Jun or Ha- Ras increased significantly galectin-3 protein in THP-1 cells. These findings indicated that Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway plays an important role in the expression of galectin-3 in PMA-stimulated macrophages. We further investigated the effect of modified lipoproteins on galectin-3 expression in macrophages. Murine resident peritoneal macrophages loaded with acetylated low-density lipoprotein (AcLDL) or oxidized LDL (OxLDL) showed increased galectin-3 protein and mRNA. These results showed that treatment of macrophages with PMA or modified lipoproteins results in galectin-3 overexpression. These findings may explain the enhanced expression of galectin-3 in atherosclerotic foam cells and suggest that Ras/MAPK signal transduction pathway is involved in controlling this gene.

Phytoestrogens and prostate cancer.

Androgcns are required to maintain the integrity of the prostate and the survival of androgen dependent epithelial cells within the gland. Anti-androgens arc the primary treatment strategy for non-localized prostate cancer, but ultimately fail over time with the development of androgen independent tumors. Estrogens affect the growth and development of the prostate and may affect the development of prostate cancer. Because of the side effects of estrogen treatment alternative therapies include the use of phytoestrogens as chemopreventative and chemotherapeutic treatment modalities. Phytoestrogens, can cause growth arrest and in some cases apoptosis in prostate cancer cells in vivo and in vitro. This may be due to the estrogenic properties of the compounds or alternative mechanisms of action. A number of phytoestrogens have been shown to have anti-androgenic effects and anti-oxidant activities. Other mechanisms include inhibition of 5alpha-reductase, 17beta-hydroxysteroid dehydrogenase, aromatase, tyrosine specific protein kinases and DNA topoisomerase II. This review examines the possible relation between phytoestrogens and prostate cancer and their possible use in prostate cancer prevention or management.

Phosphorylation of the Na+,K+-ATPase in skeletal muscle: potential mechanism for changes in pump cell-surface abundance and activity.

In skeletal muscle, insulin stimulation leads to phosphorylation of Na(+),K(+)-ATPase alpha-subunits on both serine/threonine and tyrosine residues, translocation of Na(+),K(+)-ATPase molecules to the plasma membrane, and increased Na(+),K(+)-ATPase activity. The molecular nature of the tyrosine kinase that phosphorylates Na(+),K(+)-ATPase is not yet identified. In vitro phosphorylation experiments show that the alpha-subunit of Na(+),K(+)-ATPase from skeletal muscle is a substrate for the tyrosine-specific protein kinase c-src. Tyrosine phosphorylation of the alpha-subunits of Na(+),K(+)-ATPase may be an important mechanism for insulin-mediated regulation of Na(+),K(+)-ATPase translocation and activity.

Evidence for a role of protein tyrosine kinases in cell death induced by gp120 in CHP100 neuroblastoma cells

HIV-1 coat protein gp120 is able to kill neuronal cells in culture. Here we address the possible role of protein tyrosine kinases (PTKs) in gp120-induced neurotoxicity using the CHP100 human neuroblastoma cell line as experimental model. For this purpose, the effect of specific PTK inhibitors like genistein, herbimycin A and lavendustin A was evaluated on CHP100 cell death elicited by the viral protein. Here we report that genistein (1–10 μM) significantly reduced the cytotoxic effects induced by gp120 (10 pM). The same protective action was offered by a pre-treatment with herbimycin A (0.1–1 μM) or lavendustin A (1–10 μM). Conversely, daidzein (1–100 μM), a genistein analogue devoid of PTK inhibitory properties, failed to reduce CHP100 cell death caused by gp120. These findings suggest that PTKs can be involved in the signal transduction cascade by which the glycoprotein induces neurotoxicity.

Soy Consumption and Phytoestrogens: Effect on Serum Prostate Specific Antigen When Blood Lipids and Oxidized Low-Density Lipoprotein are Reduced in Hyperlipidemic Men

Soy Consumption and Phytoestrogens: Effect on Serum Prostate Specific Antigen When Blood Lipids and Oxidized Low-Density Lipoprotein are Reduced in Hyperlipidemic Men

Signal transduction mechanism leading to enhanced proliferation of primary cultured adult rat hepatocytes treated with royal jelly 57-kDa protein.

A 57-kDa protein in royal jelly (RJ) was previously shown to stimulate hepatocyte DNA synthesis and prolongs the proliferation of hepatocytes as well as increasing albumin production [Kamakura, M., Suenobu, N., and Fukushima, M. (2001) Biochem. Biophys. Res. Commun. 282, 865-874]. In this study, I investigated the signal transduction mechanisms involved in the induction of hepatocyte DNA synthesis and the promotion of cell survival by this 57-kDa protein in primary cultures of adult rat hepatocytes. Hepatocyte DNA synthesis induced by the 57-kDa protein was not influenced by several alpha- and beta-adrenoceptor antagonists, but was dose-dependently abolished by an inhibitor of a tyrosine-specific protein kinase, genistein. A phospholipase C inhibitor (U-73122) and a protein kinase C (PKC) inhibitor (sphingosine) inhibited 57-kDa protein-stimulated he-patocyte DNA synthesis, whereas a protein kinase A inhibitor (H-89) did not. The 57-kDa protein also activated PKC in rat hepatocytes. Various inhibitors of intracellular signal transduction elements (PD98059, p21 ras farnesyltransferase inhibitor, wortmannin and rapamycin) also blocked hepatocyte DNA synthesis induced by the 57-kDa protein. Furthermore, the 57-kDa protein activated mitogen-activated protein (MAP) kinase in rat hepatocytes. The activation of MAP kinase by the 57-kDa protein was inhibited by PD98059 and sphingosine. The 57-kDa protein also activated protein kinase B, which is a key regulator of cell survival. These results suggest that, like growth factors, the 57-kDa protein activates several important intracellular signaling factors involved in the stimulation of hepatocyte DNA synthesis and the protection of cells from apoptosis.

Activation of distinct signal transduction pathways in Trypanosoma cruzi isolates with differential capacity to invade host cells

Mammalian cell invasion by Trypanosoma cruzi requires the activation of signal transduction pathways that result in a Ca 2+ response both in the parasite and the host cell. By using drugs that interfere with the signalling processes, we investigated if the difference in the ability of T. cruzi isolates to invade host cells was associated with the activation of distinct signalling routes in the parasites. Experiments were performed with metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, using the highly invasive isolate CL and the poorly infective isolate G, which belong to distinct phylogenetic lineages. Treatment of parasites with adenylyl cyclase activator forskolin increased the infectivity of the G but not of the CL isolate towards HeLa cells. On the other hand, a specific protein tyrosine kinase inhibitor genistein reduced by ∼75% the penetration of CL but not of G isolate into HeLa cells. In the CL but not in the G isolate, protein tyrosine kinase mediated the phosphorylation of a 175 kDa protein in a manner inducible by a HeLa cell extract. Upon treatment with the phospholipase C inhibitor U73122, or with drugs such as caffeine, which affects Ca 2+ release from inositol-1,4,5-triphosphate-sensitive stores, or thapsigargin, an inhibitor of intracellular Ca 2+ transport ATPases, the infectivity of the CL but not of the G isolate diminished significantly ( P<0.005). In both isolates, a combination of ionomycin plus NH 4Cl or nigericin released Ca 2+ from acidic vacuoles containing a Ca 2+/H + exchange system. This treatment reduced the infectivity of metacyclic forms of the G but not of the CL isolate. Taken together, these data suggest that, for host cell invasion, distinct signalling pathways are activated in metacyclic trypomastigotes of the two isolates, leading to Ca 2+ release from different intracellular compartments.

Second messengers in platelet aggregation evoked by serotonin and A23187, a calcium ionophore

We investigated the combined effect of 5-hydroxytryptamine (5-HT, serotonin) and calcium ionophore (A23187) on human platelet aggregation. Aggregation, monitored at 37°C using a Dual-channel Lumi-aggregometer, was recorded for 5 min after challenge by a change in light transmission as a function of time. 5-HT (2–200 μM) alone did not cause platelet aggregation, but markedly potentiated A23187 (low dose) induced aggregation. Inhibitory concentration (IC 50) values for a number of compounds were calculated as means ± SEM from dose-response determinations. Synergism between 5--HT (2–5 μM) and A23187 (0.5–2 μM) was inhibited by 5-HT receptor blockers, methysergide (IC 50=18 μM) and cyproheptadine (IC 50=20 μM), and calcium channel blockers (verapamil and diltiazem, IC 50=20 μM and 40 μM respectively). Interpretation of the effects of these blockers is complicated by their lack of specificity. Similarly, U73122, an inhibitor of phospholipase C (PLC), blocked the synergistic effect at an IC 50 value of 9.2 μM. Wortmannin, a phosphatidylinositide 3-kinase (PI 3-K) inhibitor, also blocked the response (IC 50 = 2.6 μM). However, neither genistein, a tyrosine-specific protein kinase inhibitor, nor chelerythrine, a protein kinase C inhibitor, affected aggregation at concentrations up to 10 μM. We conclude that the synergistic interaction between 5-HT and ionophore may be mediated by activation of PLC/Ca 2+ and PI 3-kinase signalling pathways, but definitive proof will require other enzyme inhibitors with greater specificity.

Phosphorylation is involved in the activation of metal-regulatory transcription factor 1 in response to metal ions.

We have studied the role of phosphorylation in the activation of metal-regulatory transcription factor-1 (MTF-1) and metallothionein (MT) gene expression. We showed that MTF-1 is phosphorylated in vivo and that zinc stimulates MTF-1 phosphorylation 2-4-fold. Several kinase inhibitors were used to examine the possible involvement of kinase cascades in the activation of MTF-1. Metal-induced MT gene expression was abrogated by protein kinase C (PKC), c-Jun N-terminal kinase (JNK), phosphoinositide 3-kinase, and tyrosine-specific protein kinases inhibitors, as assayed by Northern analysis and by cotransfection experiments using a metal regulatory element-luciferase reporter plasmid. The extracellular signal-activated protein kinase and the p38 kinase cascades did not appear to be essential for the activation of MT gene transcription by metals. By using dominant-negative mutants of PKC, JNK, mitogen-activated kinase kinase 4 (MKK4), and MKK7, we provide further evidence supporting a role for PKC and JNK in the activation of MTF-1 in response to metals. Notably, increased MTF-1 DNA binding in response to zinc and MTF-1 nuclear localization was not inhibited in cells preincubated with the different kinase inhibitors despite strong inhibition of MTF-1-mediated gene expression. This suggests that phosphorylation is essential for MTF-1 transactivation function. We hypothesize that metal-induced phosphorylation of MTF-1 is one of the primary events leading to increased MTF-1 activity. Thus, metal ions such as cadmium could activate MTF-1 and induce MT gene expression by stimulating one or several kinases in the MTF-1 signal transduction pathway.

PTP-epsilon, a tyrosine phosphatase expressed in endothelium, negatively regulates endothelial cell proliferation.

The vascular endothelium is a dynamic interface between the blood vessel and circulating factors and, as such, plays a critical role in vascular events like inflammation, angiogenesis, and hemostasis. Whereas specific protein tyrosine kinases have been identified in these processes, less is known about their protein tyrosine phosphatase (PTP) counterparts. We utilized a RT-PCR/differential hybridization assay to identify PTP-epsilon as a highly abundant endothelial cell PTP. PTP-epsilon mRNA expression is growth factor responsive, suggesting a role for this enzyme in endothelial cell proliferation. Overexpression of PTP-epsilon decreases proliferation by 60% in human umbilical vein endothelial cells (HUVEC) but not in smooth muscle cells or fibroblasts. In contrast, overexpression of PTP-epsilon (D284A), a catalytically inactive mutant, has no significant effect on HUVEC proliferation. These data provide the first functional characterization of PTP-epsilon in endothelial cells and identify a novel pathway that negatively regulates endothelial cell growth. Such a pathway may have important implications in vascular development and angiogenesis.

Natural Protection from Apoptosis by Surfactant Protein A in Type II Pneumocytes

Surfactant-associated protein A (SP-A) is a component of pulmonary surfactant that binds to a specific receptor (SPAR) on the surface of type II alveolar cells of the lung and regulates gene expression and surfactant secretion. Previously we have shown that activation of SPAR by SP-A binding initiates a signal through pathways that involve tyrosine phosphorylation, include IRS-1, and entail activation of phosphatidylinositol 3-kinase (PI3K). In other cell types, cytokines that activate the PI3K signaling pathway promote cell survival. Therefore we investigated whether there was an effect of SP-A on apoptosis as measured by DNA laddering, FACS analysis, TUNEL assay, and annexin V binding. SP-A protected primary cultures of rat type II alveolar cells against the apoptotic effects of etoposide and UV light and also protected the H441 human Clara lung tumor cell line against staurosporine-induced apoptosis. The protective effects of SP-A were abrogated by inhibition of either tyrosine-specific protein kinase activity or PI3K. SP-A/SPAR interaction thus initiates a signaling pathway that regulates apoptosis in type II cells. These findings may be important in understanding the pathogenesis of acute lung injury and pulmonary tumorigenesis and may suggest new therapeutic options.