Effect of Nerve Growth Factor and Fibroblast Growth Factor on SCGlO and c-fos Expression and Neurite Outgrowth in Protein Kinase C-depleted PC 12 Cells*

The role of protein kinase C (PKC) in mediating nerve growth factor (NGF) or basic fibroblast growth factor (bFGF)-stimulated SCGlO and c-foe expression as well as neurite outgrowth was studied in PC12 cells. Activators of PKC such as phorbol 12-myristate 13- acetate (PMA) or 1-oleoyl2-acetyl glycerol mimicked the stimulatory effect of NGF and bFGF on SCGlO 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 (-90%) in total PKC activity in the continued presence of PMA. PKC NGF-or SCGlO and bFGF-stimu-lated c-fos induction. c-foe outgrowth was not abolished in PKC-depleted cells. The results imply that PKC is not involved in NGF-and bFGF-stimulated SCGlO 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 role of protein kinase C (PKC) in mediating nerve growth factor (NGF) or basic fibroblast growth factor (bFGF)-stimulated SCGlO and c-foe expression as well as neurite outgrowth was studied in PC12 cells. Activators of PKC such as phorbol 12-myristate 13acetate (PMA) or 1-oleoyl2-acetyl glycerol mimicked the stimulatory effect of NGF and bFGF on SCGlO mRNA levels. Induction involved a protein synthesisdependent 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 (-90%) in total PKC activity in the continued presence of PMA. PKC depletion did not affect NGF-or bFGFstimulated SCGlO mRNA induction and bFGF-stimulated c-fos mRNA induction. However, NGF-stimulated c-foe 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 SCGlO 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 PKCdependent and -independent pathways.
Nerve growth factor (NGF)' (for review see Ref. 1) and FGF (acidic and basic) are polypeptide hormones that play an important role in the development of certain neurons in the central and peripheral nervous system (2, 3). The rat pheochromocytoma cell line PC12 has proved to be a useful model system for studying the mechanism of action of NGF. NGF induces differentiation of PC12 cells into sympathetic neuron-like cells (for review see Refs. 4 and 5). It has recently been demonstrated that FGF induces a similar response (6-10). Although FGF binds to a different receptor than NGF (8), it is conceivable that these receptors mediate their effects through similar second messenger systems. However, the exact nature of these messenger systems has not yet been elucidated.
Recent studies have implicated protein kinase C (PKC) in mediating the effects of NGF on protein phosphorylation ( 12) and neurite outgrowth in PC12 cells (13). Consistent with this, treatment of PC12 cells with NGF induces rapid hydrolysis of phosphoinositides (14) and PKC activity (12,15). In this study we have asked whether PKC mediates the induction of gene expression by NGF and FGF. These growth factors induce at least two classes of genes in PC12 cells: an "early response" class, which includes transcriptional regulators (16), and a "middle response" class, which includes neuralspecific structural genes (17, 18).
We have examined c-fos and SCGlO mRNAs as representative members of the two classes, respectively. c-fos encodes a nuclear protein that is thought to activate transcription in response to extracellular signals (19). SCGlO is a neuralspecific gene that encodes a membrane-bound protein (20, 21), which accumulates in the growth cones of developing neurons. The induction of these genes by NGF and FGF can be mimicked by activators of PKC (16, 18). However, using depletion of PKC by chronic phorbol ester treatment, we are now able to rule out a role for PKC in the induction of SCGlO mRNA by these factors, as well as in neurite outgrowth. In the case of c-fos, induction by FGF appears not to be mediated by PKC, whereas induction by NGF is, at least in part. Thus NGF may activate some early and middle response genes through different signal transduction pathways. This method has been used previously to demonstrate that PKC plays a role in growth factor-induced responses in other systems (26)(27)(28)(29). In the present study, pretreatment of PC12 cells with PMA (750 rig/ml) led to a 90% reduction in total PKC activity, both in undifferentiated and in NGF-or bFGFdifferentiated cells (see also Ref. 15). This depletion of PKC did not affect either NGF-or bFGF-stimulated SCGlO mRNA induction, despite the fact that activators of PKC mimic this induction with respect to both kinetics and protein synthesis dependence (Figs. 2 and 3). When the drug H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) was used as an independent method of inhibiting PKC activity, NGF induction of * Portions of this paper (including "Materials and Methods," "Results," and Figs. l-9) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
SCGlO mRNA was also unaffected (data not shown). The induction of c-fos mRNA by bFGF was similarly insensitive to PKC depletion. It is possible that the 10% residual PKC activity is sufficient to support the full inductive effects of NGF and bFGF. However this seems unlikely, since control experiments demonstrated that direct activation of the residual PKC by phorbol 12,13-~butyrate failed to induce c-fos mRNA. In the case of SCGlO, phorbol 12,13-dibutyrate similarly failed to elicit an induction, although a persistent elevation of steady-state mRNA levels over control (19%) was observed due to PMA pretreatment. We therefore conclude that SCGlO induction by both bFGF and NGF is unlikely to be mediated by PKC. This conclusion is based on the present knowledge that all known subspecies of PKC are down-regulated by prolonged exposure to PMA. However, we cannot rule out the possibility that PC12 cells contain a PMA-insensitive subspecies not detected in our assays, since PKC has been shown to be a member of a multigene family (30).

c-fos Induction by NGF May Occur via Both PKC-dependent and -independent
Pathways-PKC depletion produced a significant inhibition of c-fos induction by NGF, but not by bFGF (Fig. 6). This result resembles the observation that cfos mRNA induction by several other growth factors was decreased but not abolished in PKC-depleted 3T3-Ll flbroblasts and adipocytes (29) as well as in Balb/c/3TQ cells (31). This suggests that while bFGF stimulates c-fos mRNA induction entirely via a PKC-independent pathway, NGF induction of c-fos may occur via both PKC-dependent and -independent pathways, a conclusion similar to that reached in these other systems. (Although such a partial inhibition could also reflect simply the incomplete depletion of PKC, the phorbol 12,13dibutyrate controls argue against this (Fig. 7B)J Curiously, although c-fos induction by bFGF in PC12 cells appears to occur exclusively via a PKC-independent pathway, in 3T3-Ll cells bFGF induces this same gene via both PKC-dependent and -independent pathways (29). This difference could reflect differences in the cellular environment in which the FGF receptor functions.

NGF-and bFGF-induced
Neurite Outgrowth in PKC-depleted PC12 Cells-Previous reports suggested that PKC may be involved in NGF-induced neurite outgrowth in PC12 cells (31,32). Recently Reinhold and Kenneth (33) reported that NGF can promote neurite outgrowth in PKC-depleted PC12 cells. We have confirmed their observations and extended them to show that bFGF-induced neurite outgrowth is similarly unaffected by PKC depletion. These data suggest that both NGF and bFGF mediate their effects on neurite outgrowth via a PKC-independent pathway. In further support of this, addition of the PKC inhibitor H-7 to NGF-treated cells also did not abolish neurite outgrowth (data not shown). Although depletion of PKC did not prevent NGF or bFGFinduced neurite outgrowth, the neurites in PKC-depleted cells showed some morphological changes, and the rate of their initiation increased compared with that of NGF-or bFGFinduced neurites. This effect was seen most strikingly in the case of bFGF. A similar result was observed in H-7-treated cells exposed to NGF (not shown). Such an enhancement of neurite extension in the absence of PKC activity may reflect an inhibition, by phosphorylation, of some mechanism underlying the process of outgrowth (e.g. assembly of microtubules (34)). mechanist Involved in NGF and bFGF Signal Transduction-The results presented in this paper, taken together with those in an earlier study (18), seem to rule out PKC, CAMPdependent, and calcium/calmodulin-dependent protein kinases as mediating the induction of neuronal differentiation by NGF and FGF (at least as measured by SCGlO mRNA induction). Other reports have similarly failed to identify a second messenger system for NGF responses (for reviews see Ref. 5). We suggest, therefore, that NGF mediates its effects via a specific effector system which is expressed in NGFresponsive cells. In support of this idea, cloned NGF receptors expressed in cell lines lacking endogenous receptors fail to mediate any NGF responses. However, when expressed in NR18 cells (a PCl2-derived mutant which lacks NGF receptors), such exogenously introduced receptors are able to promote c-fos mRNA induction by NGF (35). NGF and FGF receptors are different (B), and FGF but not NGF receptors appear to have tyrosine kinase activity (36). Although the similarity in the response of PC12 cells to both NGF and FGF is striking (6-lo), the kinase inhibitor K-252a selectively blocked NGF-but not FGF-induced neurite outgrowth and NGF receptor mRNA induction (37,38). This suggests that both factors may share part but not all of the signal transduction pathway. Whatever the case, our data appear to rule out a role for PKC in mediating the induction, by either NGF or FGF, of both SCGlO mRNA and neurite outgrowth. It is therefore likely that a unique second messenger is involved in the signal transduction pathway of at least NGF in PC12 cells.