[CANCER RESEARCH 62, 4867– 4875, September 1, 2002] Advances in Brief TrkA as a Life and Death Receptor: Receptor Dose as a Mediator of Function 1 Chaohua Yan, Ye Liang, Karen D. Nylander, and Nina Felice Schor 2 The Pediatric Center for Neuroscience, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15213 Abstract Nerve growth factor (NGF) has been implicated as both an inhibitor and an inducer of apoptosis. Binding of NGF to its TrkA receptor is generally considered to have an antiapoptotic effect. However, neuroblas- tomas that overexpress TrkA have a good prognosis and frequently regress by apoptosis either spontaneously or after chemotherapeutic treat- ment, whereas those that express little or no TrkA are lethal in 80 –95% of patients, despite maximal therapy. We now report that NGF treatment of PC12 neural crest tumor cells trkA-transfected to express TrkA at levels analogous to those seen in “good prognosis” neuroblastomas results in cell death; similar treatment of native cells that express TrkA at levels similar to those of “poor prognosis” neuroblastomas has no effect on cell survival. Morphological and biochemical studies indicate that NGF induces atypi- cal apoptosis that is abrogated by the tyrosine kinase inhibitor K252, but not by an inhibitor of NGF-p75 binding, in trkA-transfected PC12 cells. Differential activation of the TrkA-phosphorylated mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase 1-phos- phorylated ERK-phosphorylated cAMP-responsive element-binding pro- tein and TrkA-phosphorylated MAP/ERK kinase 3/6-phosphorylated p38 MAP signal transduction pathways, also suppressible by K252, occurs in the two cell types. This difference may mediate their differential survival after exposure to NGF. Introduction NGF 3 is the most well-studied representative of a family of trophic factors that includes brain-derived neurotrophic factor, neurotro- phin-3, and neurotrophin 4/5. It has been known to have pleiotropic effects on different cell types (1–5). Initially, NGF was shown to promote the proliferation, survival, and maturation of neural target cells (6 –9). Cell death is generally thought to be a consequence of NGF withdrawal, rather than NGF exposure (10 –12). In recent years, however, it has been demonstrated that the biolog- ical effects of NGF are mediated by two classes of receptors: (a) p75 glycoprotein, which belongs to the superfamily of tumor necrosis factor receptors; and (b) TrkA glycoprotein, a transmembrane tyrosine kinase of 140 kDa with a cytoplasmic tyrosine kinase domain (13). The binding of NGF to each of these receptors triggers particular cascades of cellular signaling events. The binding of NGF to TrkA, the high-affinity NGF receptor, results in TrkA phosphorylation that will in turn lead to a scaffolding role for TrkA and recruitment of several adapter proteins and enzymes that ultimately propagate the NGF signal (14, 15). Among these proteins, the adapter protein Shc and phospholipase C have been involved in the activation of ERKs (16, 17). In contrast, the binding of NGF to its low-affinity receptor, p75, has variably been found to induce or prevent apoptosis. p75 has been shown to activate programmed cell death through a mechanism involving the stress kinase JNK (18 –20). However, this receptor also activates NF-B, which is thought to promote survival and counter- balance the proapoptotic signal through up-regulation of TRAF1, TRAF2, and the inhibitor of apoptosis proteins (e.g., c-IAP1 and c-IAP2) and blocking of activation of the caspase pathway (21–25). Although the roles of NGF receptors in transducing signals are beginning to be elucidated, because the expression of TrkA and p75 is cell type specific, cell line specific, and even cell cycle specific, the precise effects and signaling mechanisms of ligand activation of NGF receptors are still the subject of considerable investigation and con- troversy. For example, the role of NGF in the induction and progres- sion or maturation and regression of neural tumors remains specula- tive. In present study, we used native and trkA-transfected PC12 pheochromocytoma cells to examine the effects on cell survival of TrkA and p75 ligand activation by NGF in cells of varied TrkA content and to explore the signaling mechanisms triggered by binding of NGF to its receptors in these cells. Materials and Methods Chemicals and Reagents. NGF was obtained from Boehringer Mannheim. Preparation and characterization of the monoclonal antibody mAbNGF30 have been described previously (26). This antibody binds to the p75 binding site of NGF, blocking the binding of NGF to p75 and converting NGF to a TrkA- specific ligand. K252, a TrkA tyrosine kinase inhibitor, was purchased from Calbiochem (San Diego, CA) and prepared in DMSO as a 100 M stock solution. Anti-TrkA and anti-phosphotyrosine (clone 4G10) antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal anti-MEK kinase1 IgG, rabbit polyclonal anti-MEK1, rabbit polyclonal anti- ERK1, mouse monoclonal anti-phospho-ERK, rabbit polyclonal anti-MEK4, goat polyclonal anti-p-CREB, rabbit polyclonal anti-MEK3, mouse mono- clonal anti-phospho-MEK3/6 IgG, mouse monoclonal anti-p38, rabbit poly- clonal anti-NF-B p65, rabbit polyclonal anti-c-IAP2, and rabbit antimouse IgG-horseradish peroxidase antibodies were obtained from Santa Cruz Bio- technology (Santa Cruz, CA). Rabbit polyclonal anti-phospho-p38 MAPK, rabbit polyclonal anti-phospho-MEK1/2, and rabbit polyclonal anti-phospho- SEK1/MKK4 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Cy2-conjugated AffiniPure rabbit antimouse IgG(H+L) and Cy3-conjugated AffiniPure goat antirabbit IgG(H+L) antibodies were ob- tained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA) and used for fluorescent immunostaining of cells. 7-AAD, PI, Hoechst dye 33342, saponin, and LDH assay kits were purchased from Sigma (St. Louis, MO). N2 supplement (100) was obtained from Invitrogen (Carlsbad, CA). Cell Cultures. Native [40,000 p75 receptors/cell and 400 TrkA recep- tors/cell (p75+++ TrkA+)] and trkA-transfected [40,000 of each receptor/ cell (p75+++ TrkA+++)] PC12 cells were the kind gift of Dr. H. Uri Saragovi (26). All cells used in these studies were demonstrated to be Myco- plasma free using a MycoTect Kit (Life Technologies, Inc.). Both native and trkA-transfected PC12 cells were maintained with regular medium [DMEM supplemented with 10% horse serum, 5% fetal bovine serum (Atlanta Biologi- cals, Norcross, GA), and 1.1% penicillin/streptomycin (Invitrogen)] for at least 2 days, followed by replacement of regular medium with serum-free medium Received 5/3/02; accepted 7/17/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the National Institute of Neurological Disease and Stroke and the National Cancer Institute (Grants R01-NS38569 and R01-CA74289, respectively) of the NIH and the Carol Ann Craumer Endowment Fund of Children’s Hospital of Pittsburgh. 2 To whom requests for reprints should be addressed, at Children’s Hospital of Pittsburgh, Rangos Research Center, 3460 Fifth Avenue, Pittsburgh, PA 15213. 3 The abbreviations used are: NGF, nerve growth factor; ERK, extracellular signal- regulated kinase; CREB, cAMP-responsive element-binding protein; p-CREB, phospho- rylated CREB; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAP/ERK kinase; JNK, c-Jun-NH 2 -terminal kinase; NF-B, nuclear factor B; PI, propidium iodide; 7-AAD, 7-amino-actinomycin D; LDH, lactate dehydrogenase; TUNEL, terminal de- oxynucleotidyl transferase-mediated nick end labeling; IAP, inhibitor of apoptosis; TRAF, tumor necrosis factor receptor-associated factor. 4867 on May 6, 2017. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from