neuropeptide-y and Neuroectodermal-Tumors--Primitive--Peripheral

Mammalian cells often receive multiple extracellular stimuli under physiological conditions, and the various signaling inputs have to be integrated for the processing of complex biological responses. G protein-coupled receptors (GPCRs) are critical players in converting extracellular stimuli into intracellular signals. In this report, we examined the integration of different GPCR signals by mitogen-activated protein kinases (MAPKs) using the SK-N-MC human brain neuroepithelioma cells as a neuronal model. Stimulation of the Gi-coupled neuropeptide Y1 and Gq-coupled muscarinic M1 acetylcholine receptors, but not the Gs-coupled dopamine D1 receptor, led to the activation of extracellular signal-regulated kinase (ERK). All three receptors were also capable of stimulating c-Jun NH2-terminal kinases (JNK) and p38 MAPK. The Gi-mediated ERK activation was completely suppressed upon inhibition of Src tyrosine kinases by PP1, while the Gq-induced response was suppressed by both PP1 and the Ca2+ chelator, BAPTA-AM. In contrast, activations of JNK and p38 by Gs-, Gi-, and Gq-coupled receptors were sensitive to PP1 and BAPTA-AM pretreatments. Simultaneous stimulation of Gi- and Gq-coupled receptors resulted in the synergistic activation of ERK, but not JNK or p38 MAPK. The Gi/Gq-induced synergistic ERK activation was PTX-sensitive, and appeared to be a co-operative effect between Ca2+ and Src family tyrosine kinases. Enhanced ERK activation was associated with an increase in CREB phosphorylation, while the JNK and p38-responsive transcription factor ATF-2 was weakly enhanced upon Gi/Gq-induction. This report provides evidence that G protein signals can be integrated at the level of MAPK, resulting in differential effects on ERK, JNK and p38 MAPK in SK-N-MC cells.

Topics: Adenylyl Cyclases; Brain Neoplasms; Calcium Signaling; Carbachol; Cyclic AMP Response Element-Binding Protein; Drug Combinations; Drug Synergism; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; GTP-Binding Proteins; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Neuroectodermal Tumors, Primitive, Peripheral; Neurons; Neuropeptide Y; p38 Mitogen-Activated Protein Kinases; Receptor, Muscarinic M1; Receptors, G-Protein-Coupled; Receptors, Neuropeptide Y; Signal Transduction; src-Family Kinases; Transcription Factors; Tumor Cells, Cultured; Type C Phospholipases

We selected three human cancer cell lines [human melanoma (SEKI), human melanoma (G361), and human neuroepithelioma (NAGAI)] that have an ability to develop cancer cachexia syndrome with and without accompanying anorexia and examined the hypothalamic levels of mRNAs for neuropeptide Y (NPY), melanin-concentrating hormone, and orexin. The body weight of sham-operated mice continued to increase, while mice of all tumor-bearing groups lost weight. Competitive reverse transcription-polymerase chain reaction analysis showed that, regardless of feeding status, NPY mRNA levels were elevated in all tumor-bearing mice compared with sham-operated mice, although to a lesser degree than weight-matched pair-weight mice. Melanin-concentrating hormone and orexin mRNA in the hypothalamus followed the same pattern as NPY, although most of the differences did not reach statistical significance. These results support the notion that the response of NPY mRNA to a negative energy balance is less sensitive in these rodent models of cancer cachexia.

Topics: Animals; Cachexia; Carrier Proteins; Eating; Energy Metabolism; Female; Gene Expression; Growth Inhibitors; Humans; Hypothalamic Hormones; Hypothalamus; Interleukin-6; Intracellular Signaling Peptides and Proteins; Leukemia Inhibitory Factor; Lymphokines; Melanins; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Neuroectodermal Tumors, Primitive, Peripheral; Neuropeptide Y; Neuropeptides; Orexins; Organ Size; Pituitary Hormones; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Weight Loss

Neuropeptide Y (NPY) acts through specific receptors to inhibit adenyl cyclase and may have a role in neuroblastomas and neuroepitheliomas (NE) as a regulator of cell growth and differentiation. The authors have examined the status of NPY receptors in the NE cell line SK-N-MC and the effect of retinoic acid (RA), a known differentiating agent, on their expression and function.. Competitive NPY binding studies were performed on normal and RA-treated cells, followed by Scatchard analysis. NPY receptor function in the absence of or following RA treatment was determined by the ability of various concentrations of NPY to attenuate the forskolin-stimulated accumulation of intracellular cAMP. The mitogenic effect of NPY was evaluated by growing normal or RA-treated cell in the presence of various concentrations of NPY.. Scatchard analysis showed a Kd of 2.3 nmol/L and a Bmax of 91,000 receptors per cell for untreated cells. RA treatment decreased receptor expression to 11,700 per cell without a significant change in receptor affinity (3.6 nmol/L). The effect of forskolin was inhibited by NPY in a dose-dependent fashion in both untreated and treated cells indicating functional receptors in both NPY stimulates the growth of normal SK-N-MC cells. NPY stimulated growth was significantly attenuated after RA treatment, possibly as a result of decreased NPY receptor expression.. Treatment of SK-N-MC cells with RA, a known differentiating agent, leads to decreased expression of functional NPY receptors and a concomitant decrease in the mitogenic effect of NPY. This supports the role for NPY in the pathogenesis of NE.

Topics: Cell Division; Cell Transformation, Neoplastic; Down-Regulation; Humans; Mitogens; Neuroectodermal Tumors, Primitive, Peripheral; Neuropeptide Y; Receptors, Neuropeptide Y; Tretinoin; Tumor Cells, Cultured

The specific binding of 125I-labeled neuropeptide Y (NPY) and the biological response to NPY receptor activation were measured in cultured human neuroepithelioma (SK-N-MC) cells. A single class of high-affinity binding sites [dissociation constant (KD) = 0.2 nM] was characterized both by equilibrium binding of 125I-NPY concentrations less than 1 nM and kinetically by the initial rates of 125I-NPY association and dissociation. Specific 125I-NPY binding was decreased in a concentration-dependent manner by inclusion of guanine nucleotides in the incubation medium. The existence of multiple affinity states or NPY receptor subtypes was suggested by 1) a Hill coefficient of less than 1.0 obtained when analyzing equilibrium binding with 125I-NPY concentrations greater than 1 nM, 2) biphasic dissociation of 125I-NPY, 3) an increase in the component of rapid dissociation and decrease in the component of slow dissociation when guanine nucleotides were present during dissociation of 125I-NPY, and 4) displacement of 125I-NPY by unlabeled peptide with a slope factor of 0.6. Exposure of intact cells to NPY caused a concentration-dependent pertussis toxin-sensitive inhibition of forskolin-stimulated cellular adenosine 3',5'-cyclic monophosphate (cAMP) accumulation [50% effective concentration (EC50) = 0.4 nM]. In contrast, NPY had no effect on cellular inositol phosphate content or protein kinase C activation. These results demonstrate that NPY binds specifically to a G protein-linked receptor that inhibits adenylate cyclase in SK-N-MC cells.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Animals; Binding, Competitive; Carbachol; Cell Line; Cell Membrane; Cells, Cultured; Colforsin; Cyclic AMP; Humans; Inositol Phosphates; Isoproterenol; Kinetics; Neuroectodermal Tumors, Primitive, Peripheral; Neuropeptide Y; Pertussis Toxin; Receptors, Neuropeptide Y; Receptors, Neurotransmitter; Tumor Cells, Cultured; Virulence Factors, Bordetella

The cholinergic human neuroepithelioma cell line SK-N-MCIXC expressed mRNAs for the neuropeptides cholecystokinin (CCK), neuropeptide Y, gastrin-releasing peptide (GRP) and enkephalin. The CCK transcript of about 800 nt was present at very high levels and CCK-like peptides immunoreactive to a C-terminal CCK octapeptide antiserum were present in the cell line and its medium. This clonal neuronal cell line provides a unique model system to identify cis- and trans-acting factors responsible for neuron-specific expression and regulation of the CCK gene. Furthermore, the pluripotent properties of the undifferentiated cell line may open studies on neuronal differentiation at the level of co-expression of neuropeptides and transmitters.

Topics: Blotting, Northern; Cholecystokinin; Enkephalins; Gastrin-Releasing Peptide; Gene Expression; Humans; Neuroectodermal Tumors, Primitive, Peripheral; Neuropeptide Y; Neuropeptides; Peptides; RNA, Messenger; Tumor Cells, Cultured