Signal transduction pathways activated by RET oncoproteins in PC12 pheochromocytoma cells

Gene alterations in the ret proto-oncogene, which encodes a receptor tyrosine kinase, have been found to associate with several human diseases. In this study, we showed that induction of the vgf promoter activity is a good molecular indicator for RET activation in PC12 cells, a rat pheochromocytoma cell line. We demonstrated that all forms of RET oncoprotein, including RET chimeric oncoproteins found in human papillary thyroid carcinomas (RET/PTC) as well as RET oncoproteins found in patients with multiple endocrine neoplasia type 2A and 2B (2A/RET and 2B/RET) can induce vgf promoter activity in PC12 cells. In contrast, a RET mutant found in a patient with Hirschsprung's disease, as well as a RET/PTC1 mutant with deletion of the dimerization domain, failed to induce vgf promoter activity in PC12 cells. We further determined that the signaling events mediated by phosphorylated Tyr294 and phosphorylated Tyr451 binding sites are essential for RET/PTC1 to induce vgf promoter activity in PC12 cells. We also showed that RET/PTC1, 2A/RET, and 2B/RET induce ELK-, cAMP-responsive element binding protein (CREB), or JUN-mediated gene expression in PC12 cells, and these three signaling events are mediated by phosphorylated Tyr294 and phosphorylated Tyr451 binding sites in RET/PTC1.     

Fading and rebound of currents induced by ATP in PC12 cells

1. Patch clamp recording (whole cell configuration) was used to study the action of ATP on rat phaeochromocytoma (PC12) cells usually held at -70 mV and rapidly superfused with buffered saline. ATP (0.5, 1 or 5 mM), applied from micropipettes by pressure application with brief (< or = 50 ms) pulses, induced inward currents with rapid onset and decay. ADP and alpha, beta-methylene ATP were ineffective. 2. ATP (5 mM) applied with pulses > 200 ms long elicited a complex current response characterized by a rapid peak which faded and was followed by a strong current rebound (lasting several s) as soon as the application was terminated. This type of response was readily replicated as long as ATP applications were spaced at 2-3 min intervals. The amplitude of peak and rebound currents was dependent on the length of pressure pulse and was similarly depressed by bath application of a threshold dose (25 microM) of ATP. Rapid fading and rebound of ATP-induced membrane currents were also observed when the Y-tube method was used for applying this agonist. 3. The reversal potential for peak and rebound currents was the same while the time constant values for peak fading and rebound onset were insensitive to changes in membrane potential between -70 and -40 mV. When ATP was applied to a cell clamped at depolarized potential, no current was observed but rapid return of the membrane potential to -70 mV immediately at the end of ATP application was associated with a large rebound current. 4. Brief (20 ms) application of ATP during the onset of the rebound current strongly and transiently suppressed it. The same application performed during the gradual decay of the rebound wave elicited a transient inward current which was much smaller and shorter than the one observed when the cell was in its resting state. Application of 2 s ATP pulses at 20 s intervals equally reduced the initial peak and rebound currents which recovered at the same rate. 5. The present data are interpreted according to a scheme which suggests two types of ATP receptor desensitization. The first one (D1) would be characterized by fast kinetics and low agonist affinity; rapid recovery from D1 would then be manifested as current rebound presumably due to receptor reactivation. The second desensitized state (D2) has slow kinetics and high affinity for the agonist: it is therefore typically seen with sustained application of a low dose of ATP. It is proposed that desensitization and its recovery can influence the time course of membrane responses mediated by purinoceptors.     

Diverse signal transduction pathways mediated by endogenous P2 receptors in cultured rat cerebral cortical neurons

The present study was conducted to assess the intracellular signaling pathways mediated by receptors for ATP, uridine triphosphate (UTP), and 2-methylthio ATP (2-MeSATP), by monitoring patch-clamp currents and intracellular calcium mobilization in cultured rat cortical cerebral neurons. All three agonists evoked potassium currents and increased the intracellular free Ca2+ concentration ([Ca2+]i), and these effects were inhibited by the broad G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) but not by the Gi/o-protein inhibitor pertussis toxin (PTX). UTP-evoked currents were inhibited by either the phospholipase C inhibitor neomycin or the selective protein kinase C (PKC) inhibitor GF109203X, and the rise in cytosolic Ca2+ was inhibited by either neomycin or the inositol 1,4,5-trisphosphate (IP3) receptor antagonist heparin, indicating that the UTP receptor involved phospholipase C-mediated phosphatidylinositol signaling. In contrast, 2-MeSATP-induced currents and rise in cytosolic Ca2+ were not inhibited by either neomycin, or GF109203X, or heparin. 2-MeSATP elicited single-channel currents in the cell-attached patch-clamp configuration and also in excised patches. The G-protein activator GTP gamma S induced single-channel currents in a fashion that mimicked the effect of 2-MeSATP. These data suggest that 2 MeSATP activated potassium channels by a direct action of G-protein beta gamma subunits and increased [Ca2+]i by a mechanism independent of phospholipase C stimulation and IP3 production. ATP-evoked currents were partially inhibited by either neomycin or GF109203X, although the rise in cytosolic Ca2+ was not affected by these inhibitors. ATP produced single-channel currents with two major classes of the slope conductance (86 and 95 pS) in cell-attached patches, each of which is consistent with that achieved by 2-MeSATP (85 pS) or UTP (96 pS); the currents with the lower conductance were observed in the outside-out patch-clamp configuration. These results indicate that P2 receptors for UTP and 2-MeSATP are linked to a PTX-insensitive G-protein involving different signal transduction pathways and that ATP responses are mediated by both of these P2 receptors.     

Glutamate receptors: brain function and signal transduction

Glutamate receptors are important in neural plasticity, neural development and neurodegeneration. N-methyl-d-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors act as glutamate-gated cation channels, whereas metabotropic receptors (mGluRs) modulate the production of second messengers via G proteins. Molecular studies from our and other laboratories indicated that NMDA receptors and mGluRs exist as multiple subunits (NMDAR1 and NMDAR2A-2D) and multiple subtypes (mGluR1-mGluR8). In light of the molecular diversity of glutamate receptors, we explored the function and intracellular signaling mechanisms of different members of glutamate receptors. In the visual system, retinal bipolar cells receive glutamate transmission from photoreceptors and contribute to segregating visual signals into ON and OFF pathways. The molecularly cloned mGluR6 is restrictedly expressed at the postsynaptic site of ON-bipolar cells in both rod and cone systems. Gene targeting of mGluR6 results in a loss of ON responses without changing OFF responses and severely impairs detecting visual contrasts. Since AMPA receptors mediate OFF responses in OFF-bipolar cells, two distinct types of glutamate receptors effectively operate for ON and OFF responses. mGluR1 and mGluR5 are both coupled to inositol triphosphate (IP3)/calcium signal transduction with an identical agonist selectivity. Single-cell intracellular calcium ([Ca2+]i) recordings indicated that glutamate evokes a non-oscillatory and oscillatory [Ca2+]i response in mGluR1-expressing and mGluR5-expressing cells, respectively. This difference results from a single amino acid substitution, aspartate of mGluR1 or threonine of mGluR5, at the G protein-interacting carboxy-terminal domains. Protein kinase C phosphorylation of the threonine of mGluR5 is responsible for inducing [Ca2+]i oscillations in mGluR5-expressing cells and cultured glial cells. Thus, the two closely related mGluR subtypes mediate diverging intracellular signaling in glutamate transmission.     

Protection of PC12 cells glutathione peroxidase in L-DOPA induced cytotoxicity

L-DOPA may cause side-effects during the treatment of Parkinson's disease. We investigated the role of glutathione peroxidase (GSHPx) in cellular defense against L-DOPA cytotoxicity. A line of PC12 cells overexpressing GSHPx with plasmid pRc/CMV-GSHPx was established and stable transfectants overexpressing GSHPx were used for this study. GSHPx activity was found to be 1.5-fold higher in GSHPx-transfectants than in mock-controlled transfectants. Transfectants over expressing GSHPx were also significantly more resistant to exposure to either L-DOPA or t-butyl hydroperoxide than mock-transfected cells. Results suggested that L-DOPA may cause neuronal cell death by an oxidative pathway and GSHPx may play an important role in cellular defense against oxidative stress.     

Recent progress in the molecular biology of receptors and intra-cellular signal transduction

Extracellular signals are recognized by the receptors on the surface of cytoplasmic membrane and are transmitted through the complex array of intracellular signal transduction networks. Through recent advances in molecular and genetic analyses, we are beginning to understand the outline of the signaling machineries leading from receptors to targets at molecular level. This review intends to describe the general characteristics of receptors and components of intra-cellular signal transduction systems. Future prospects of the studies on signal transduction in medicine are also discussed.     

Roles of the JAK-STAT system in signal transduction via cytokine receptors

JAK-STAT signaling pathways are known to play an essential role in the specific activation of interferon-inducible genes. Many cytokines interacting with the cytokine receptor superfamily also appear to activate these pathways. Recent evidence indicates that JAKs play an essential role(s) in cytokine receptor signaling, including both specific pathways linked to STATs and general pathways regulating cell growth and functions.     

Cholesterol protects PC12 cells from beta-amyloid induced calcium disordering and cytotoxicity

THE neurotoxic action of beta-amyloid seems to play an important role in the pathogenesis of Alzheimer's disease. The disruption of calcium homeostasis by beta-amyloid has been suspected to be the mechanism of its neurotoxicity. We found that beta-amyloid 25-35 induces a rapid increase in cytosolic calcium of PC12 cells, and subsequently, a dramatic decrease in cell viability. The increase in cytosolic calcium induced by beta-amyloid is effectively blocked by cholesterol in a dose-dependent manner. Furthermore, pretreatment of PC12 cells with cholesterol also significantly attenuates the neurotoxicity induced by beta-amyloid. These findings suggest that extracellular free cholesterol can protect neurones from beta-amyloid neurotoxicity mediated by the disruption of calcium homeostasis.     

Heterodimeric neurotrophins induce phosphorylation of Trk receptors and promote neuronal differentiation in PC12 cells

Neurotrophins are a family of highly conserved proteins that affect the development and maintenance of distinct neuronal populations. Neurotrophins exist in vivo as homodimers, but we show that neurotrophins can exist as heterodimers in vitro and are pluripotent, being able to bind and to activate different Trk tyrosine kinase receptors as well as promote neuronal differentiation in PC12 cells as effectively as wild type homodimers. These asymmetric neurotrophin dimers allow unique characterization of neurotrophin structure-function relationships with Trk receptors. The chimeric Trk activities of these heterodimers suggest an alternative model of neurotrophin-Trk receptor activation in which the critical Trk-interacting elements may be attributed to a single protomer.     

Redox regulation of signal transduction in vascular smooth muscle cells: thiol oxidizing agents induced phospholipase D

Decrease in intracellular thiols leads to oxidative stress and thus may cause alterations in the activity of redox-sensitive enzymes required for signal transduction. Here, we report that, N-ethylmaleimide and phenylarsine oxide, which are known to oxidize free thiols as well as protein thiols, induced phosphatidyl ethanol generation in the micromolar range suggesting activation of phospholipase D in vascular smooth muscle cells. These agents also induced significant phosphatidic acid and diacylglycerol generation without causing protein kinase C activation. Phenylarsine oxide and N-ethyl maleimide induced phospholipase D activation is protein kinase C independent as it was not inhibited by compound-3 and bisindolylmaleimide, potent protein kinase C inhibitors. Tyrosine kinase inhibitor herbimycin A by itself activated PLD, but inhibited the phospholipase D activation by phenylarsine oxide and N-ethylmaleimide. These results suggest that oxidation of the cellular thiols activates phospholipase D independent of protein kinase C.     

NO is not involved in the simvastatin induced cell division and differentiation in PC12 cells

Simvastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor has been reported to inhibit cell division and induce neurite-like outgrowth in PC12 cells [Sato-Suzuki, I. and Murota, S., Neurosci. Lett., 220 (1996) 21-24]. In the present paper, we examined whether the induced nitric oxide (NO) in the simvastatin-treated PC12 cells is involved in the growth arrest and differentiation as reported in nerve growth factor (NGF) treated PC12 cells. Treatment of PC12 cells with simvastatin caused peripherin formation and enhanced NO production just like NGF-treated PC12 cells. Different from NGF, however, NO synthase inhibitors could not affect the growth arrest and differentiation in simvastatin-treated PC12 cells. In conclusion, NO had nothing to do with cell division and differentiation in simvastatin-treated PC12 cells.     

5-Azacytidine induces toxicity in PC12 cells by apoptosis

5-Azacytidine (5 Az)is a potent inhibitor of DNA methylation, and it may allow inactive genes to become expressed. In a previous study, we demonstrated that 5 Az administered to the dam induced apoptosis in the brains of fetal mice. In this study, the 5 Az-induced apoptosis was further characterized in differentiated PC 12 cells as a model for neuronal apoptosis. Cell death, determined by the activity of released lactate dehydrogenase (LDH) into the medium, occurred from 24 to 48 hrs after 5 Az treatment. Toxicity for differentiated PC 12 cells was observed on treatment with more than 10(-1) micrograms/ml of 5 Az, and it reached the maximal level at 10 micrograms/ml. Cycloheximide, an inhibitor of protein synthesis, prevented 5 Az toxicity, suggesting that this cell death required protein synthesis which could be related to the activation of a dormant gene(s). Electrophoresis of DNA from 5 Az-treated cells evoked ladder formation, indicating the cleavage of DNA into nucleosomes. Scanning electron microscopy demonstrated bleb formation, the so-called apoptotic bodies on the cell surface. The biochemical and morphological findings indicated that 5 Az-induced cell death occurred in the form of apoptosis. 5 Az-induced cell death was prevented by treatment with cAMP but not by treatment with high K+ or deoxycytidine. These results suggest that a cAMP-sensitive mechanism is involved in 5 Az-induced cell death. PC 12 cells should be of value in elucidating the molecular mechanism of 5 Az-induced neuronal apoptosis.     

Nicotine regulates nicotinic cholinergic receptors and subunit mRNAs in PC 12 cells through protein kinase A

To understand the up-regulation of neuronal nicotinic cholinergic receptors (nAcChRs) that results from chronic in vivo treatment with nicotine, we studied the effect of nicotine on [3H]nicotine binding sites on PC 12 cells. PC 12 cells were grown in nicotine hemisulfate (10(-6) to 10(-3) M) or vehicle for 7 days, and specific [3H]nicotine binding was measured. Nicotine (10(-6) to 10(-4) M) dose-dependently increased specific binding by up to 2.6-fold over basal levels in 5-7 days, whereas a 10(-3) M concentration failed to do so. In contrast, [3H]nicotine binding to PC 12 cell mutants (A126.1B2 and A123.7), deficient in cAMP-responsive protein kinase A Types I and/or II, was unaffected by nicotine. Northern gel analysis of nAcChR subunit mRNAs from wild type PC 12 cells showed that the mRNA encoding the dominant agonist-binding subunit, alpha 3, was significantly reduced by nicotine, as early as 4 h after treatment, whereas mRNA for the structural beta 2 subunit was slightly increased. In contrast, the alpha 3 subunit mRNA from the PC 12 cell mutant A123.7 was not significantly decreased after 4 h and 7 days of nicotine treatment. These studies indicate that nicotine up-regulates expression of nAcChRs on wild type PC 12 cells and reduces the content of alpha 3 subunit mRNA; these effects require an intact protein kinase A system. The divergent effects of nicotine on the nAcChR compared to its alpha 3 subunit mRNA suggests that enhanced expression of nicotinic receptors may not involve synthesis of new receptor subunit proteins.     

Overexpression of cation-dependent mannose 6-phosphate receptor prevents cell death induced by serum deprivation in PC12 cells

Beta1,4-galactosyltransferase (GalTase, EC 2.4.1.38) transfers galactose to the terminal N-acetylglucosamine of complex-type N-glycans, which have great importance for cell-cell interactions during fertilization and early embryogenesis. In this study, the activity of beta1,4-galactosyltransferase in mouse brain during development was measured with the method of reverse HPLC using a fluorescence-labeled biantenary sugar chain, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-2Manalpha1- 3) Manbeta1-4GlcNAcbeta1-4GlcNAc-PA. The level of messenger RNA of this enzyme during the development of mouse brain was also investigated with Northern blot analysis. The results showed that: (1)beta1,4-galactosyltransferase showed similar branch specificity and kinetics for the biantenary substrate during development; (2) GalTase activity in fetal mouse brain was four times higher than that in adult mouse brain and decreased gradually in the course of development; (3) messenger RNA level was highest in fetal mouse and decreased dramatically after birth. However, the contents of mRNA were not parallel to the enzyme activity.     

Activation of FGF receptors by mutations in the transmembrane domain

Oxidative DNA damage by a model Cr(V) complex, [CrO(ehba)2]-, with and without added H2O2, was investigated for the formation of base and sugar products derived from C1', C4', and C5' hydrogen atom abstraction mechanisms. EPR studies with 5,5-dimethylpyrroline N-oxide (DMPO) have shown that Cr(V)-ehba alone can oxidize the spin trap via a direct chromium pathway, whereas reactions of Cr(V)-ehba in the presence of H2O2 generated the hydroxyl radical. Direct (or metal-centered) Cr(V)-ehba oxidation of single-stranded (ss) and double-stranded (ds) calf thymus DNA demonstrated the formation of thiobarbituric acid-reactive species (TBARS) and glycolic acid in an O2-dependent manner, consistent with abstraction of the C4' H atom. A minor C1' H atom abstraction mechanism was also observed for direct Cr(V) oxidation of DNA, but no C5' H atom abstraction product was observed. Direct Cr(V) oxidation of ss- and ds-DNA also caused the release of all four nucleic acid bases with a preference for the pyrimidines cytosine and thymine in ds-DNA, but no base release preference was observed in ss-DNA. This base release was O2-independent and could not be accounted for by the H atom abstraction mechanisms in this study. Reaction of Cr(V)-ehba with H2O2 and DNA yielded products consistent with all three DNA oxidation pathways measured, namely, C1', C4', and C5' H atom abstractions. Cr(V)-ehba and H2O2 also mediated a nonpreferential release of DNA bases with the exception of the oxidatively sensitive purine, guanine. Direct and H2O2-induced Cr(V) DNA oxidation had opposing substrate preferences, with direct Cr(V) oxidation favoring ss-DNA while H2O2-induced Cr(V) oxidative damage favored ds-DNA. These results may help explain the carcinogenic mechanism of chromium(VI) and serve to highlight the differences and similarities in DNA oxidation between high-valent chromium and oxygen-based radicals.