The Urokinase Plasminogen Activator Receptor (UPAR) Is Preferentially Induced by Nerve Growth Factor in PC12 Pheochromocytoma Cells and Is Required for NGF-Driven Differentiation

Nerve growth factor (NGF)-driven differentiation of PC12 pheo-chromocytoma cells is a well studied model used both to identify molecular, biochemical, and physiological correlates of neurotrophin-driven neuronal differentiation and to determine the causal nature of specific events in this differentiation process. Although epidermal growth factor (EGF) elicits many of the same early biochemical and molecular changes in PC12 cells observed in response to NGF, EGF does not induce molecular or morphological differentiation of PC12 cells. The identification of genes whose expression is differentially regulated by NGF versus EGF in PC12 cells has, therefore, been considered a source of potential insight into the molecular specificity of neurotrophin-driven neuronal differentiation. A " second generation " representational difference analysis procedure now identifies the urokinase plasminogen activator receptor (UPAR) as a gene that is much more extensively induced by NGF than by EGF in PC12 cells. Both an antisense oligonucleotide for the UPAR mRNA and an antibody directed against UPAR protein block NGF-induced morphological and biochemical differentiation of PC12 cells; NGF-induced UPAR expression is required for subsequent NGF-driven differentiation. In the PC12 pheochromocytoma cell model system, nerve growth factor (NGF) acts as a neurogenic agent, inducing a differentiation program. In contrast, epidermal growth factor (EGF) acts as NGF and EGF both rapidly induce the transcription of primary response or immediate-early genes, genes whose transcription requires only the activation of preexisting signaling molecules and transcription factors (Hersch-man, 1991). We showed previously that representational difference analysis (RDA) (Lisitsyn et al., 1993) is an effective way to clone genes that are differentially induced by NGF versus EGF in PC12 cells (Vician et al., 1997). One advantage of RDA is that it can be iterated; cDNAs for genes known to be preferentially expressed in a given cell population can be added to the " driver " cDNA population, and RDA can be repeated. The previously identified genes enriched in the " tester " cDNA population should be eliminated, and additional amplicons that are elevated in the tester versus driver population should be enriched in the reiterated RDA procedure. We have used a " second generation " RDA procedure to identify additional genes that are preferentially induced in NGF-versus EGF-treated PC12 cells. One of the genes identified in this iterated RDA analysis of NGF-driven neuronal differentiation is the urokinase plasminogen activator receptor (UPAR). UPAR is a glycosyl-phosphatidylinositol (GPI)-linked membrane protein lacking transmembrane and cytosolic domains (Ploug et al., 1991; Wang …