Differences in gene expression profile among SH-SY5Y neuroblastoma subclones with different neurite outgrowth responses to nerve growth factor

Nerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF-induced neurite-positive (NIN+) subclones that showed high responsiveness to NGF-induced neurite outgrowth and three NGF-induced neurite-negative (NIN-) subclones that abolished NGF-induced neurite outgrowth from parental SH-SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506-mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF-induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF-induced increases in mRNAs for the immediate-early genes (IEGs) c-fos and NGF inducible (NGFI) genes NGFI-A, NGFI-B and NGFI-C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN- cell line, particularly in terms of axonal vesicle-related genes and growth cone guidance-related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN- cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH-SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.     

Differential expression of immediate early genes in the hippocampus and spinal cord

We have demonstrated that immediate early genes can be differentially activated within the central nervous system. We examined the effects of tetanic stimulation in the hippocampus and of noxious sensory stimulation of the spinal cord on the expression of eight immediate early genes. Induction of long-term potentiation (LTP) in the dentate gyrus resulted in an increase in mRNA and protein for NGFI-A (also termed Zif/268, Egr-1, or Krox 24), and less consistently for jun-B mRNA. No increase was seen for c-fos, NGFI-B, c-jun, jun-D, SRF, or PC4 mRNAs. Blockade of the NMDA receptor prevented the induction of both LTP and NGFI-A mRNA in the dentate gyrus. However, commissural stimulation, which prevented the induction of LTP, resulted in bilateral activation of all the genes examined, including NGFI-A. No change was seen in animals trained in a water maze. These results suggest that no simple relationship exists between LTP, spatial learning, and immediate early gene induction. Stimulation of sensory fibers resulted in an increase in mRNA for NGFI-A, c-fos, SRF, NGFI-B, and c-jun in spinal cord neurons. Blockade of the NMDA receptor had no effect on immediate early gene induction in the spinal cord.     

A branched signaling pathway for nerve growth factor is revealed by Src-, Ras-, and Raf-mediated gene inductions.

A myriad of gene induction events underlie nerve growth factor (NGF)-induced differentiation of PC12 cells. To dissect the signal transduction pathways which lead to NGF actions, we have assessed the relative roles of NGF receptor, Src, Ras, and Raf activities in mediating specific gene inductions. We have used the PC12 cell line as well as sublines which inducibly express activated forms of either Src, Ras, or Raf or a dominant inhibitory form of Ras (p21N17 Ras) to study the expression of multiple NGF-inducible mRNAs. The NGF induction of NGFI-A, transin, and VGF mRNAs was mimicked by activated forms of Src, Ras, or Raf and was blocked by p21N17 Ras. The NGF induction of SCG10 mRNA was mimicked only by activated Src and Ras and was blocked by p21N17 Ras, while the induction of Thy-1 mRNA was mimicked only by activated Src and was not blocked by p21N17 Ras. The NGF induction of mRNAs for two sodium channel types was neither mimicked by any activated oncoprotein nor blocked by p21N17 Ras. From these and previous results, we suggest a model in which a linear order of NGF receptor, Src, Ras, and Raf activities is used by NGF to elicit gene inductions. These signaling components define branchpoints in the pathway to specific gene induction events, providing a mechanism for generating a host of diverse NGF actions.     

Oncogene N-ras mediates selective inhibition of c-fos induction by nerve growth factor and basic fibroblast growth factor in a PC12 cell line.

A cell line was generated from U7 cells (a subline of PC12 rat pheochromocytoma cells) that contains a stably integrated transforming mouse N-ras (Lys-61) gene under the control of the long terminal repeat from mouse mammary tumor virus. Such cells, designated UR61, undergo neuronal differentiation upon exposure to nanomolar concentrations of dexamethasone, as a consequence of expression of the activated N-ras gene (I. Guerrero, A. Pellicer, and D.E. Burstein, Biochem, Biophys. Res. Commun. 150:1185-1192, 1988). Exposure of UR61 cells to either nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) results in a marked induction of c-fos RNA, with kinetics paralleling those of NGF- or bFGF-induced expression of c-fos RNA in PC12 cells. Dexamethasone-induced expression of activated N-ras p21 results in blocking of c-fos RNA induction by NGF or bFGF in a time-dependent manner. Activated N-ras p21-mediated inhibition of c-fos RNA induction in UR61 cells is selective for NGF and bFGF and is not due to selective degradation of c-fos RNA. Normal and transforming N-ras can trans activate the chloramphenicol acetyltransferase gene linked to mouse c-fos regulatory sequences when transient expression assays are performed. Our observations suggest that N-ras p21 selectively interacts with pathways involved in induction of c-fos expression which initiate at the receptors for NGF and bFGF.     

Normal stimulation of the synthesis,phosphorylation and DNA binding activity of c-Fos and Zif268 proteins by nerve growth factor is not sufficient to mediate neuronal differentiation of PC12 cells

Early-response genes (ERGs) are rapidly induced by nerve growth factor (NGF) in the PC12 rat pheochromocytoma cell line. To analyze the possible role of Ras and ERGs in neuronal differentiation, experiments were carried out to study the involvement of Ras proteins in the NGF-stimulated expression of two ERG-coded proteins (c-Fos and Zif268) implicated in NGF signaling. Using PC12 subclones expressing the dominant negative Ha-Ras Asn-17 protein, NGF-induced expression, phosphorylation and DNA-binding of these ERG products were found to be not sufficient to convey the biological response of PC12 cells to NGF.     

Effect of nerve growth factor and fibroblast growth factor on SCG10 and c-fos expression and neurite outgrowth in protein kinase C-depleted PC12 cells

The role of protein kinase C (PKC) in mediating nerve growth factor (NGF) or basic fibroblast growth factor (bFGF)-stimulated SCG10 and c-fos expression as well as neurite outgrowth was studied in PC12 cells. Activators of PKC such as phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl 2-acetyl glycerol mimicked the stimulatory effect of NGF and bFGF on SCG10 mRNA levels. Induction involved a protein synthesis-dependent mechanism and was maximal within 12-24 h of exposure. Chronic treatment of the cells with PMA for up to 8 days resulted in a substantial decrease (approximately 90%) in total PKC activity in the continued presence of PMA. PKC depletion did not affect NGF- or bFGF-stimulated SCG10 mRNA induction and bFGF-stimulated c-fos mRNA induction. However, NGF-stimulated c-fos mRNA induction was attenuated. In addition, induction of neurite outgrowth was not abolished in PKC-depleted cells. The results imply that PKC is not involved in NGF- and bFGF-stimulated SCG10 mRNA induction and neurite outgrowth. Furthermore, while the effect of bFGF on c-fos mRNA induction is PKC-independent, that of NGF is mediated by PKC-dependent and -independent pathways.     

The ras suppressor, RSU-1, enhances nerve growth factor-induced differentiation of PC12 cells and induces p21CIP expression.

The Rsu-1 Ras suppressor gene was isolated based on its ability to inhibit v-Ras transformation. Using Rsu-1 transfectants of the pheochromocytoma cell line PC12, we demonstrated previously that Rsu-1 expression inhibited Jun kinase activation but enhanced Erk2 activation in response to epidermal growth factor. In the present study, the Rsu-1 PC12 transfectants were used to investigate the role of Rsu-1 in nerve growth factor (NGF)- and v-Ki-ras-mediated neuronal differentiation. NGF-induced neurite extension was enhanced, not inhibited, by the expression of Rsu-1 in PC12 cells. The activation of Erk kinase activity in response to NGF was sustained longer in the Rsu-1 transfectants compared with the vector control cells. During NGF-mediated differentiation, an increase in the expression of specific mRNAs for the early response genes Fos, cJun, and NGF1a was detected in both the vector control and Rsu-1 transfectants. The expression of the differentiation-specific genes VGF8 and SCG10 was similar in Rsu-1 transfectants compared with the vector control cells. The induction of Rsu-1 expression in these cell lines did not inhibit v-Ki-ras-induced differentiation, as measured by neurite extension. These data suggest that although Rsu-1 blocked some Ras-dependent response(s), these responses were not required for differentiation. Moreover, the induction of Rsu-1 expression in the PC12 clones resulted in growth inhibition and p21(WAF/CIP) expression. Hence, Rsu-1 expression enhances NGF-induced differentiation while inhibiting the growth of cells.     

Differential Ras-Dependence of Gene Induction by Nerve Growth Factor and Second Messenger Analogs in PC12 Cells

Induction of neurite formation by nerve growth factor (NGF) in PC12 pheochromocytoma cells can be efficiently inhibited by expressing a dominant negative mutant form of the small guanine nucleotide binding Ha-Ras protein in these cells. The block in NGF-induced neuritogenesis caused by inhibition of endogenous Ras proteins was found to be partially relieved by simultaneous stimulation of cAMP- or Ca⁺⁺-dependent signaling pathways. Since expression of certain genes is believed to be involved in NGF-signaling leading to morphological differentiation, we decided to study the combined effects of NGF and second messenger analogs on gene expression in PC12 cell lines expressing different levels of the interfering Ras protein. We found NGF-second messenger combinations that induced normal c-fos, zif268 and nur77 early-response gene expression without neuritogenesis, and, conversely, cell lines in which certain combination treatments caused partial neuronal differentiation in the absence of substantial activation of these genes. Similarly, neurite outgrowth induced by combination treatments does not seem to require the activation of the late-response transin gene. Our results thus suggest a lack of strong correlation between NGF-stimulated early- and secondary-response gene induction and morphological differentiation.