Differentiation of PC12 cells with v‐src: Comparison with nerve growth factor

Abstract

The PC12 rat pheochromocytoma cell line is used extensively as a model to study neuronal differentiation. These cells resemble adrenal chromaffin cells, differentiating both morphologically and biochemically when cultured in the presence of dexamethasone, but develop a sympathetic neuron-like phenotype when cultured in the presence of nerve growth factor. Expression of the protein product of the v-src oncogene in PC12 cells also induces neurite outgrowth similar to that resulting from nerve growth factor treatment (Alema et al: Nature 316:557-559, 1985). It is thus possible that c-src or a src-like tyrosine kinase participates in the signal transduction pathway by which nerve growth factor acts on PC12 cells. In this study a temperature-sensitive v-src gene has been introduced into PC12 cells. When cultures of these src-transformed cells are switched from the nonpermissive (40 degrees C) to the permissive (37 degrees C) temperature they elaborate neurites. The differentiation induced by src has been compared with that induced by nerve growth factor by determining whether src-transformed PC12 cells at 37 degrees C exhibit the same biochemical alterations as those induced in PC12 cells treated with nerve growth factor. Neurite extension at 37 degrees C in v-src-transformed cells, like NGF-induced differentiation, is accompanied by an increase in the nerve growth factor-inducible large external (NILE) protein. However, neurite extension in v-src-transformed cells is not blocked by the protein kinase inhibitor K-252a, which completely blocks NGF-induced neurite extension. Likewise, EGF receptor down-regulation and the development of saxitoxin and tetanus toxin binding sites are either much reduced or completely absent in src-differentiated compared with NGF-differentiated PC12 cells.

Enhanced expression of Ca2+ channels by nerve growth factor and the v‐src oncogene in rat phaeochromocytoma cells.

“…Alternatively, v-src could mimic the action of trk and thus induce phosphorylation of certain substrates that feed into the differentiation pathway. This could account for the differences seen between NGF and v-src-induced differentiation (Rausch et al 1989;Thomas et al 1991).…”

“…Another tyrosine kinase, the product of the v-src oncogene, mimics certain characteristics of NGF-induced neuronal differentiation in PC12 cells (Alema, Casalbore, Agostini & Tato, 1985;Maher 1988;Rausch et al 1989;Thomas et al 1991). The following evidence suggests that the src tyrosine kinase may play a role in neuronal differentiation: (i) the existence of a neurone-specific, alternatively spliced c-src mRNA (Levy, Dorai, Wang & Brugge, 1987;Martinez, Mathey-Prevot, Bernards & Baltimore, 1987), (ii) the localization of this form of the src protein to specific neurones (Brugge.…”

“…What are the similarities and differences in the cell signalling pathways activated by each of these tyrosine kinases? K252a, a protein kinase C inhibitor closely related to staurosporine, inhibited NGF-induced differentiation and c-fos transcription (Hashimoto, 1988;Koizumi, Contreras, Matsuda, Hama, Lazarovici & Guroff, 1988;Lazarovici et al 1989), but did not inhibit v-src-induced differentiation in PC12 cells (Rausch et al 1989). Staurosporine blocked both differentiation and enhancement of wo-CgTX binding sites in PC12 cells treated with NGF (Usowicz et al 1990).…”

Testing the in vivo role of protein kinase C and c-fos in neurite outgrowth by microinjection of antibodies into PC12 cells.

“…The rat pheochromocytoma cell line PC12 has been used widely to study neuronal differentiation (Greene and Tischler, 1982) and is a useful system in which to distinguish those intracellular signals that may be specific for inducing differentiation on the one hand from those that induce proliferation on the other. PC12 cells proliferate as round chromaffin-like cells when grown in standard culture conditions, but on addition of nerve growth factor (NGF) or basic fibroblast growth factor (bFGF), the cells stop dividing, extend numerous processes, and display characteristics of fully differentiated sympathetic neurons (Green and Tischler, 1982;Togari et al, 1985;Rydel and Green, 1987;Schubert et al, 1987 Technology, Pasadena, CA 91125. or src transforming proteins (Alema et al, 1985;BarSagi and Feramisco, 1985;Satoh et al, 1987;Eveleth et al, 1989;Rausch et al, 1989). Microinjection of antibodies that block the function of either the ras or the src proteins inhibits the induction of neurite outgrowth by NGF or by bFGF in both fused (Hagag et al, 1986) and native PC12 cells (Altin et al, 1991b), suggesting that these proteins are essential components of signal transduction pathways leading to differentiation in response to NGF and bFGF in PC12 cells.…”

Activation of Codependent Transcription Factors Is Required for Transcriptional Induction of the vgf Gene by Nerve Growth Factor and Ras

“…The addition of NGF to these cells triggers the activation of a tyrosine kinase-containing receptor, including the proto-oncoprotein Trk (27,29,31), and the activation of a variety of signal transduction pathways, leading to the expression of a multiplicity of genes (reviewed in reference 25). Several cytoplasmic protooncoproteins, including c-Src (1,32,48,63), c-Shc (50), c-Ras (2,23,43,60), and Raf kinases (44,64,69), have been implicated in NGF action. A host of studies in various cell types indicate that Ras is a pivotal mediator of receptor and nonreceptor tyrosine kinase signaling.…”