©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Overexpression of -Protein Kinase C Enhances Nerve Growth Factor-induced Phosphorylation of Mitogen-activated Protein Kinases and Neurite Outgrowth (*)

(Received for publication, May 25, 1995; and in revised form, September 11, 1995)

Bhupinder Hundle Thomas McMahon Jahan Dadgar Robert O. Messing (§)

From the Ernest Gallo Clinic & Research Center, Department of Neurology, University of California, San Francisco, California 94110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Protein kinase C (PKC) activation enhances neurite outgrowth in several cell lines and primary neurons. The PKC isozymes that mediate this response are unknown. One clue to their identity has come from studies using PC12 cells treated with ethanol. In these cells, ethanol increases levels of -PKC and -PKC and markedly enhances nerve growth factor (NGF)-induced neurite outgrowth and activation of mitogen-activated protein (MAP) kinases by a PKC-dependent mechanism. Since these findings suggest that -PKC or -PKC can promote neural differentiation, we studied neurite outgrowth in stably transfected PC12 cell lines that overexpress these isozymes. Overexpression of -PKC markedly increased NGF-induced neurite outgrowth. This effect was blocked by down-regulating PKC or by treating cells with the PKC inhibitor GF 109203X. In addition, overexpression of -PKC enhanced NGF-induced phosphorylation of MAP kinases. In contrast, overexpression of -PKC did not alter responses to NGF. These results demonstrate that -PKC promotes NGF-induced neurite outgrowth by enhancing NGF signal transduction. These findings suggest a role for -PKC in neural differentiation and plasticity.


INTRODUCTION

Protein kinase C (PKC) (^1)is a multigene family of phospholipid-dependent, serine-threonine kinases central to many signal transduction pathways. The endogenous activators of PKC isozymes are thought to be sn-1,2-diacylglycerols, free cis-unsaturated fatty acids, and polyphosphoinositides, produced as lipid second messengers from turnover of membrane phospholipids(1, 2, 3) . So far, 10 family members encoded by 9 different mRNAs have been described and divided into three structurally related groups: ``conventional'' cPKCs (alpha, beta(I), beta, and ) which are regulated by calcium and diacylglycerols or tumor-promoting phorbol esters; ``novel'' nPKCs which are sensitive to diacylglycerols and phorbol esters but are calcium-independent (, , , and ); and ``atypical'' aPKCs ( and /) which are insensitive to calcium, diacylglycerols, and phorbol esters(2, 4) . In addition, two recently cloned, highly related, phospholipid-dependent kinases, µ-PKC and PKD, share significant homology to nPKCs in their regulatory domains and appear to constitute a new PKC subgroup(5, 6) .

Studies with tumor-promoting phorbol esters that potently activate most PKC isozymes have implicated PKC in the differentiation of several cell types, including neurons(7) . In Xenopus embryos, phorbol esters stimulate the induction of neural cells from neuroectoderm (8) and induce neurite outgrowth from chick sensory ganglia(9) , chick ciliary ganglion neurons(10) , and several neuroblastoma cell lines (11, 12) . In the neural crest-derived cell line PC12(13) , phorbol esters enhance NGF-induced MAP kinase activation (14) and neurite outgrowth(15, 16) . Recent studies with purified isozymes, mutant cell lines, or transfected cells have implicated alpha-, beta-, -, and -PKC isozymes in the differentiation of non-neural cells(7, 17, 18, 19) . In Xenopus embryos, overexpression of alpha- or beta-PKC enhances neural induction in dorsal and ventral ectoderm(8) . However, little else is known about the role of specific PKC isozymes in neural differentiation, particularly in the regulation of neurite outgrowth.

Using PC12 cells to study mechanisms by which ethanol alters neural growth, we found that ethanol increases neurite outgrowth stimulated by NGF or basic fibroblast growth factor (bFGF)(20) . A key event in signal transduction by growth factors, including NGF and bFGF, is the activation of the closely related serine-threonine mitogen-activated protein (MAP) kinases (also known as extracellular signal-regulated kinases, or ERKs) p44 (ERK1) and p42 (ERK2) by phosphorylation on tyrosine and threonine residues(21, 22) . We recently found that ethanol increases NGF- and bFGF-induced activation of ERK1 and ERK2(14) . Enhancement of neurite outgrowth and MAP kinase activation by ethanol is blocked by down-regulation of beta-, -, and -PKC isozymes(14, 16) . Ethanol also stimulates PKC-mediated phosphorylation in PC12 cells (23) , and this is associated with increased levels of mRNA (24) and protein (23) for - and -PKC. These results suggest that ethanol enhances NGF-induced signal transduction and neurite formation by increasing levels of - and -PKC.

The isozyme of PKC is expressed predominantly in the nervous system with only trace amounts detected in non-neuronal tissues (25) while -PKC is expressed widely in neuronal and non-neuronal tissues(26) . Little is known about -PKC in regulating neural function, but recent evidence suggests a role for -PKC in neural development. In rat brain, -PKC is particularly abundant in the hippocampus, olfactory tubercle, and layers I and II of cerebral cortex(27) . Within immunoreactive neurons, it is localized to the Golgi apparatus and to axons and presynaptic nerve terminals(27) . It is activated by growth factors that stimulate neural differentiation and diacylglycerol formation such as insulin, which activates -PKC in cultured fetal chick brain neurons(28) , and nerve growth factor (NGF), which activates -PKC in PC12 cells(29) . In addition, in developing chick brain, -PKC is the major isozyme found in nondividing, differentiating neurons(30) . However, despite this suggestive evidence, no studies have yet directly demonstrated that -PKC regulates neural differentiation.

In the present study, we generated PC12 cell lines that overexpress - or -PKC to investigate the role of these isozymes in promoting neurite formation. We found that overexpression of -PKC, but not of -PKC, enhanced NGF-induced neurite outgrowth and MAP kinase activation. The findings demonstrate that -PKC is a positive regulator of NGF signal transduction and neurite growth.


EXPERIMENTAL PROCEDURES

Materials

NGF 2.5 was a gift from Dr. W. Mobley (University of California, San Francisco) or was purchased from Collaborative Research (Bedford, MA). Geneticin (G418), laminin, and poly-L-ornithine (30-70 kDa) were purchased from Sigma, and GF 109203X was from Calbiochem. H89 was from Seikagaku Cor. (Tokyo, Japan), and phorbol 12-myristate 13-acetate (PMA) was from LC Laboratories (Woburn, MA). Peroxidase-conjugated anti-rabbit IgG from goat was from Boehringer Mannheim, and fluorescein-conjugated anti-rabbit IgG from goat was from Cappel (Durham, NC). Rabbit polyclonal antibodies against -PKC and -PKC were from Santa Cruz Biotechnology, Inc. Anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology Inc. (Lake Placid, NY). The vector pMT2--PKC, which contains the full-length mouse -PKC cDNA was a gift from Dr. John Knopf (Genetics Institute). A full-length rat -PKC cDNA in pBluescript was a gift from Dr. Peter J. Parker (Imperial Cancer Research Fund, London, United Kingdom). The eukaryotic expression vector pRc/RSV was purchased from Invitrogen. Peptide (ERMRPRKRQGSVRRRV), which resembles the -PKC pseudosubstrate site with a serine for alanine substitution, was used as a substrate to assay - and -PKC activities(25) .

Cell Culture and Assay of Neurite Outgrowth

PC12 cells, originally obtained from Dr. John A. Wagner (Cornell University, New York), were cultured in plastic tissue culture flasks at 37 °C in Dulbecco's modified Eagle's medium containing 10% heat-inactivated horse serum, 5% fetal calf serum, 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin a humidified atmosphere of 90% air and 10% CO(2). Geneticin was added initially at 400 µg/ml to select clones and later was added at 200 µg/ml to maintain cultures of clones. For studies of neurite outgrowth, cells (30-35 times 10^3 cells/well) were plated on 24-well plastic culture plates pretreated for 1 h with poly-L-ornithine (100 µg/ml in 15 mM sodium borate, pH 8.4). Cells were grown in medium containing 50 ng/ml NGF, and neurites were counted every 24 h. In some experiments, cells were plated on glass coverslips pretreated with poly-L-ornithine for 1 h and then with laminin (30 µg/ml) for 24 h. A neurite was identified as a process greater than one cell body diameter in length and possessing a terminal growth cone. The percentage of cells with neurites was calculated by counting 100 cells per well in triplicate wells. Neurite length was measured from 35-mm photographs projected through a slide viewer. Measurements were converted to micrometers using a micrometer photographed and viewed at the same magnification.

Immunofluorescence Microscopy

PC12 cells (1.5 times 10^4 cells/well) were plated in 8-well tissue chamber slides (Nunc, Naperville, IL) pretreated with poly-L-ornithine and laminin. After culture for 4 days in 50 ng/ml NGF, cells were rinsed once in PBS, incubated for 3 min in 2% (v/v) paraformaldehyde in PBS and then incubated for another 3 min in 4% (v/v) paraformaldehyde in PBS. After one rinse with PBS, cells were incubated with blocking solution containing 1% (v/v) normal goat serum and 0.1% (v/v) Triton X-100 in PBS at 27 °C for 2 h. After aspiration of the blocking solution, cells were incubated at 4 °C for 24-48 h with anti--PKC or anti--PKC antibody (1 µg/ml) in PBS containing 2 mg/ml fatty acid-free bovine serum albumin and 0.1% (v/v) Triton X-100. Control wells were treated with anti-- or anti--PKC antibody that had been preincubated with a 2 µg/ml concentration of the corresponding antigen peptide for 2 h at 27 °C. Wells were aspirated and rinsed once in PBS before addition of goat anti-rabbit IgG-fluorescein-conjugated antibody (12 µg/ml) in PBS containing 2 mg/ml fatty acid-free bovine serum albumin and 0.1% (v/v) Triton X-100. After incubation at 27 °C in the dark for 2 h, wells were rinsed three times in PBS and coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA). Immunofluorescence was observed using a Leitz DMRD microscope (Leica, Wetzlar, Germany).

Generation of Overexpressing Cell Lines

A 2.7-kilobase XhoI fragment that contains the full-length mouse -PKC cDNA was excised from the vector pMT2 -PKC and cloned into the NotI site of pRc/RSV using a NotI linker to obtain pR. A 3-kilobase BamHI fragment containing the full-length -PKC cDNA was subcloned into the NotI site of pRc/RSV using a NotI linker to obtain pR. PC12 cells (10^7) were suspended in 0.5 ml of Ca- and Mg-free PBS and were electroporated with 80 µg of pR, pR, or pRc/RSV. Cells were shocked once with a Cell-Porator (Life Technologies) set at a capacitance of 800 microfarads and voltage of 200 V. At these settings, 50% of the cells survived. Cells were incubated for 15 min on ice and transferred into 40 ml of culture medium prewarmed to 37 °C. Twenty ml of this cell suspension were plated onto poly-L-ornithine-coated 150-mm tissue culture plates and incubated at 37 °C in a humidified atmosphere of 10% CO(2) and 90% air for 48 h. Cells were then treated with medium supplemented with 400 µg/ml Geneticin (G418). After selection in G418 for 2-3 weeks, surviving cells were isolated using cloning rings. Isolated colonies were transferred into 24-well culture dishes approximately 3 weeks later. For each isozyme, 46 clones were expanded and then examined for -PKC or -PKC immunoreactivity by Western blot analysis.

Western Analysis

To detect PKC immunoreactivity, cells were cultured on poly-L-ornithine-coated 100-mm tissue culture dishes at a density of 6 times 10^6 cells/dish. Media were removed and cells were rinsed twice at 37 °C with buffer A containing 120 mM NaCl, 1.4 mM CaCl(2), 0.8 mM MgSO(4), 1 mM NaH(2)PO(4), 10 mM glucose, and 25 mM Hepes (pH 7.4). Cells were scraped into 1 ml of Buffer A containing 40 µg/ml leupeptin, 40 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride, and 400-µl aliquots were frozen quickly in liquid nitrogen. Concentrated sample buffer was added to frozen samples to yield a final solution containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol. Samples were heated at 90 °C for 10 min, passed five times through a 26-gauge needle, and then centrifuged at 10,000 times g for 10 min. Samples (80 µg per lane) were separated by SDS-PAGE using 10% gels. Proteins were electrophoretically transferred for 2 h at 4 °C to Hybond-C extra membranes (Amersham). Membranes were blocked for 1 h with 5% non-fat dry milk dissolved in Tris-buffered saline (20 mM Tris-HCl, pH 7.4, 137 mM NaCl; TBS) containing 0.05% Tween 20 (TBS-T). Blots were then incubated with rabbit polyclonal antibody to -PKC (0.5 µg/ml) or -PKC (0.5 µg/ml) for 45 min at room temperature. Blots were washed three times with TBS-T for 7 min and then were incubated with goat anti-rabbit IgG-peroxidase-conjugated antibody (1:1000 dilution) in blocking solution for 30 min. Blots were washed three times for 5 min in TBS-T and once for 5 min in TBS. Immunoreactive bands were detected with the ECL kit (Amersham). ERK tyrosine phosphorylation was detected as described previously(14) .

Protein Kinase C Assay

PC12 clones were grown to 70% confluence in T-75 flasks. Cells were harvested in 20 ml of fresh culture medium and centrifuged for 5 min at 250 times g. The cell pellet was resuspended in 20 ml of buffer A at 4 °C and centrifuged again. Cells were lysed in 1 ml of extraction buffer containing 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 10 mM EGTA, 50 µg/ml leupeptin, 20 µg/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride. Cells were homogenized with 20 strokes in an ice-cold Teflon-glass homogenizer. Sucrose and Triton X-100 were added to final concentrations of 250 mM and 1%, respectively, and final volume was adjusted to 2 ml with extraction buffer. The cell lysate was mixed for 20 min at 4 °C, and cell debris was removed by centrifugation at 12,000 times g for 20 min at 4 °C. The supernatant fraction was further centrifuged in an air centrifuge at 180,000 times g for 20 min at 4 °C. Proteins were separated by fast protein liquid chromatography (Pharmacia Biotech Inc.). Samples containing 1.5 mg of protein were loaded onto a Mono Q column equilibrated with buffer B (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 mM EGTA, 10% (v/v) glycerol, and 0.1% (v/v) beta-mercaptoethanol) at a flow rate of 0.3 ml/min. The column was washed with 10 ml of buffer B, and proteins were eluted with a 15-ml 0-400 mM linear NaCl gradient in buffer B at 0.2 ml/min. Fractions of 0.5 ml were collected. Calcium-independent PKC activity in 60 µl of each fraction was measured in triplicate. The reaction mixture (0.1 ml) contained 2 µmol of Tris-HCl, pH 7.5, 24 µg of phosphatidylserine, 0.8 µg of sn-1,2-dioleoylglycerol, 2 µmol of magnesium acetate, 25 nmol of peptide-, and 2 nmol of [-P]ATP (0.5-1 times 10^6 cpm/nmol). Background activity was measured in the absence of lipids. The reaction was carried out at 27 °C for 6 min and terminated by the addition of 30 µl of stop solution (200 mM EDTA and 200 mM ATP). Samples (0.1 ml) were spotted onto phosphocellulose papers, which were washed three times in water and rinsed once with 95% ethanol. Filters were dried and radioactivity was determined by scintillation counting. PKC activity in each fraction was calculated as the difference between counts/min measured in the presence and absence of phosphatidylserine and diacylglycerol. The area beneath the curve of PKC activity in fractions containing peak - or -PKC immunoreactivity was calculated with the computer program SigmaPlot (Jandel Scientific, Corte Madera, CA). Values obtained were divided by values measured in corresponding fractions from control cells to determine the fold increase in - or -PKC activity in transfected clones.

Miscellaneous Procedures

Protein concentrations were measured by the Bradford method (31) with bovine IgG standards. Unless stated otherwise, results are expressed as mean ± S.E. values. Except where noted, differences between means were analyzed by ANOVA and where p < 0.05, the significance of differences between means was evaluated by the Scheffe F-test.


RESULTS

Localization of -PKC and -PKC in Differentiating PC12 Cells

We examined the subcellular localization of - and -PKC in PC12 cells by immunofluorescence microscopy. In undifferentiated cells, -PKC immunoreactivity was found throughout the cytoplasm in a granular pattern (Fig. 1A). -PKC immunoreactivity was also present in the cytoplasm with localized areas of intense cytoplasmic staining seen in several cells (Fig. 1D). In cells differentiated for 4 days with NGF, immunoreactivity to -PKC (Fig. 1C) was observed within nuclei and in a thin perinuclear band. Intense -PKC immunoreactivity was also present asymmetrically next to the nucleus, possibly in the region of the Golgi apparatus. Very low levels were present elsewhere in the soma, neurites, and growth cones. In contrast, intense -PKC immunoreactivity was observed in growth cones and neurite shafts and in the cytoplasm of the cell soma (Fig. 1F).


Figure 1: Immunofluorescence detection of - and -PKC in differentiating PC12 cells. PC12 cells were treated without (A, B, D, and E) or with (C and F) 50 ng/ml NGF for 4 days prior to fixation and staining with antibodies against -PKC (A-C) or -PKC (D-F). Immunoreactivity was inhibited when antibodies were preincubated with their corresponding peptide antigens (B and E). Bar = 50 µm.



Western Analysis of PKC Isozymes and PKC Activity in Transfected Clones

To overexpress PKC isozymes, we generated stably transfected PC12 cells that overexpress - or -PKC (see ``Experimental Procedures''). Two clones overexpressing -PKC, 1 and 2, and two clones overexpressing -PKC, 1 and 2, were identified by Western analysis (Fig. 2) and used for additional studies.


Figure 2: Western analysis of -PKC and -PKC immunoreactivity in transfected PC12 cells. PC12 cells were transfected by electroporation with the parent vector pRc/RSV (C1, C2), a vector containing the cDNA sequence for -PKC (1, 2) or a vector containing the sequence for -PKC (1, 2). Stable transfectants were selected in G418 and expanded. A, Western blots showing increased -PKC immunoreactivity in clones 1 and 2 and increased -PKC immunoreactivity in 1 and 2 in comparison with empty vector control clones C1 and C2. Immunoreactivity was similar in control clones and parental PC12 cells. B, bar graph showing percent increase in - or -PKC immunoreactivity above controls C1 or C2 in - and -PKC-transfected clones. Data are from 3-6 experiments. p < 0.04 for all PKC-transfected clones compared to control (two-tailed t-test).



To determine whether the increase in immunoreactivity reflected increased expression of an active PKC isozyme, PKC activity was measured in Triton X-100-solubilized lysates of cloned cell lines. To remove endogenous PKC inhibitors that prevent assay of PKC activity (32) and to separate -PKC from -PKC, the cell lysates were subjected to ion exchange chromatography using a Mono Q column. Western analysis of fractions eluted from the column revealed that -PKC immunoreactivity was present in fractions 16-27 with a peak in fraction 17 (Fig. 3A). In contrast, -PKC was present only in fractions 23-27 (Fig. 3A) with the strongest immunoreactivity in fractions 25-27.


Figure 3: Calcium-independent PKC activity in transfected clones. PC12 clones were homogenized and solubilized in Triton X-100, and proteins were separated after centrifugation by Mono Q chromatography. A, immunoblots of column fractions from 2 cells stained with antibody to -PKC or -PKC as indicated. B, Ca-independent PKC activity in Mono Q column fractions from -PKC-transfected clones assayed in parallel with fractions from C1 control cells. C, Ca-independent PKC activity in Mono Q column fractions from -PKC-transfected clones assayed in parallel with fractions from parental PC12 or C1 control cells. Activity in fractions 15-20 which lack -PKC immunoreactivity were similar in -PKC transfected and control cell lines (data not shown).



Ca-independent PKC activity in the - and -PKC-transfected clones was compared with activity in wild-type PC12 cells or with activity in the empty vector control clone C1 (Fig. 3, B and C). In fractions containing the highest -PKC immunoreactivity, Ca-independent PKC activity was increased by 2.2-fold in clone 1 (fractions 16-21) and by 1.4-fold in clone 2 (fractions 14-18) compared to activity in corresponding fractions from C1 cells. In fractions 23-27, which contained -PKC immunoreactivity, Ca-independent PKC activity was increased by 1.6-fold in clone 1 and by 2.6-fold in clone 2 compared to control cells. Thus, 1 and 2 cells contained elevated Ca-independent PKC activity in fractions with the greatest -PKC immunoreactivity and 1 and 2 cells showed increased PKC activity only in fractions with -PKC immunoreactivity.

Growth and Morphology of PKC-transfected PC12 Cells

During the logarithmic phase of cell growth, the rates of growth in clones 1, 2, 1, and 2 were not significantly different from the rates of growth observed in the parent PC12 cell line or in C1 control cells (data not shown). Prior to NGF treatment, the morphology of 1, 2, 1, and 2 cells was similar to that of parental PC12 cells, except that 2 cells tended to be more flattened and polygonal in shape. NGF stimulated neurite formation in all clones, but neurite outgrowth was markedly increased in clones 1 and 2 compared to parental PC12 cells or control clones C1 and C2 ( Fig. 4and Table 1). As shown in Table 1, many cells in 1 and 2 cultures expressed 4 or more neurites per cell. On average, 1 cells expressed 2.9 ± 0.2 neurites per cell, and 2 cells expressed 2.8 ± 0.1 neurites per cell. This was significantly greater than the average number of neurites per cell expressed by parental PC12 (1.6 ± 0.1) or C1 control (1.7 ± 0.1) cells (ANOVA, Scheffe F-test). In contrast, the number of cells expressing neurites, the length of neurites, and the number of neurites per cell were similar in clones 1 and 2, C1 and C2 control cells, and parental PC12 cells. ( Fig. 4and Table 1).


Figure 4: Morphology of wild-type and PKC-transfected PC12 cells after exposure to NGF. Cells were cultured for 4 days on glass coverslips treated with poly-L-ornithine and laminin in the presence of 50 ng/ml NGF. Parental PC12 cells (PC) and clones transfected with the empty pRc/RSV vector (C1), -PKC (1 and 2), or -PKC (1 and 2) were visualized by transmitted light interference contrast microscopy. Bar = 25 µm.





The time course and concentration dependence of NGF-induced neurite outgrowth was examined in clones that overexpressed -PKC. Differentiation occurred more rapidly in clones 1 and 2 than in control cells (Fig. 5A). In addition, as shown in Fig. 5B, the percentage of cells with neurites in cultures treated for 4 days with a maximally effective concentration of NGF (100 ng/ml) was significantly greater in 1 (56 ± 2%) and 2 (64 ± 1%) cells than in C1 (45 ± 2%) or parental PC12 (47 ± 2%) cells (ANOVA, Scheffe F-test). In contrast, the EC for NGF-induced neurite outgrowth was similar in PC12 (125 ± 28 pM), C1 control (124 ± 44 pM), 1 (191 ± 63), and 2 (182 ± 11 pM) cells (p = 0.54, n = 3).


Figure 5: Concentration dependence and time course of NGF-induced neurite outgrowth in -PKC-transfected cells. Cells were cultured on plastic tissue culture dishes treated with poly-L-ornithine and laminin in 0.08-100 ng/ml NGF for 4 days (A) or 50 ng/ml NGF for 0-10 days (B). Data shown are mean ± S.D. values from representative experiments repeated twice with similar results. Means ± S.E. for EC values and maximal responses in cells treated with NGF for 4 days are given in the text.



Inhibition or Down-regulation of PKC Prevents -PKC-mediated Enhancement of Neurite Outgrowth

If enhanced neurite outgrowth observed in clones 1 and 2 is due to overexpression of -PKC, then inhibition of PKC should prevent the increase in NGF-induced neurite outgrowth observed in these clones. Two approaches were taken to inhibit PKC. First, cells were treated with 50 ng/ml NGF in the presence or absence of 1 µM GF 109203X, a relatively selective PKC inhibitor that inhibits PKC isozymes with IC values of 15-20 nM(33) . Second, cells were treated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h prior to addition of 50 ng/ml of NGF. We have previously shown that treatment with this concentration of PMA down-regulates beta-, -, and -PKC in NGF-treated PC12 cells(16) . As shown in Fig. 6, GF 109203X decreased and PMA pretreatment completely prevented enhancement of neurite outgrowth due to overexpression of -PKC. Neither treatment reduced neurite outgrowth in parental PC12 cells, empty vector-transfected cells, or clones that overexpressed -PKC. GF 109203X also inhibits cAMP-dependent protein kinase with an IC value of 2 µM(33) . However, treatment with 1 µM H-89, a selective inhibitor of cAMP-dependent protein kinase(34) , did not reduce neurite outgrowth in control, parental, or PKC-transfected cells (not shown). Thus, inhibiting or down-regulating PKC prevented enhancement of neurite outgrowth due to overexpression of -PKC.


Figure 6: Inhibition and down-regulation of PKC reduce neurite outgrowth in PKC-transfected clones. Clones were cultured on poly-L-ornithine-treated tissue culture dishes for 4 days in the presence of 50 ng/ml NGF, with (striped bars) or without (black bars) 1 µM GF 109203X. In addition, some cells were treated with 100 nM PMA for 24 h before NGF was added to the cultures in the continued presence of PMA for 4 days (gray bars). Data are from 3-6 experiments. *, significantly different compared with 1 cells treated with NGF alone;**, significantly different compared with 2 cells treated with NGF alone (ANOVA, Scheffe F-test).



MAP Kinase Phosphorylation in PKC-transfected Cells

In PC12 cells, NGF and bFGF cause sustained activation of ERK1 and ERK2 MAP kinases, which appears to be important for stimulation of neural differentiation(35) . Recently, we found that ethanol, like PMA, markedly increases NGF-induced phosphorylation and activation of ERK1 and ERK2(14) . Since ethanol increases expression of - and -PKC (23) , we predicted that one of these isozymes modulates MAP kinase activation.

To test this hypothesis, we measured NGF-stimulated tyrosine phosphorylation of ERK1 and ERK2 in clones that overexpress - or -PKC. In cells transfected with the empty vector pRc/RSV, NGF stimulated phosphorylation of 44- and 42-kDa proteins (Fig. 7), which correspond to ERK1 and ERK2(36) . Phosphorylation was maximal after 5 min and then persisted at a lower level for at least 1 h. This pattern of phosphorylation is identical with that observed in the parent PC12 cell line(14) . In the -PKC-transfected clone 2, ERK1 and ERK2 phosphorylation was maximal after 5 min of NGF treatment and remained elevated near maximal levels for at least 60 min (Fig. 7, A and B). This pattern of increased ERK phosphorylation resembles that seen in ethanol-treated PC12 cells(14) . A similar persistent increase in phosphorylation was observed in 1 cells (Fig. 7C). In the -PKC-transfected clones 1 and 2, the time course (data not shown) and extent (Fig. 7C) of NGF-stimulated phosphorylation of ERK1 and ERK2 was similar to phosphorylation in control cells. Thus, -PKC overexpression increased NGF-stimulated ERK phosphorylation while overexpression of -PKC did not.


Figure 7: NGF-stimulated MAP kinase phosphorylation in PKC-transfected PC12 cells. Clones were cultured for 24 h on poly-L-ornithine-treated dishes and then treated with 50 ng/ml NGF. ERK1 and ERK2 tyrosine phosphorylation was detected by Western analysis of cell lysates using anti-phosphotyrosine antibody. A, time course of ERK1 and ERK2 tyrosine phosphorylation in clones C1 and 2. B, quantitation of ERK1 and ERK2 tyrosine phosphorylation by scanning densitometry in 2 cells (bullet) and C1 cells (circle). C, tyrosine phosphorylation of ERK1 and ERK2 in control cells and PKC-transfected clones after 20 min of treatment with 50 ng/ml NGF. Data are expressed as a percentage of maximal phosphotyrosine immunoreactivity in the parental PC12 cell line (PC) measured after 5 min of NGF treatment and are from 4 experiments. *, significantly different compared with phosphotyrosine immunoreactivity present in PC12 or C1 cells after 20 min of NGF treatment (ANOVA, Scheffe F-test).




DISCUSSION

Our findings provide the first evidence that -PKC regulates NGF signaling and neurite outgrowth. Previously, we demonstrated that chronic exposure to ethanol enhances NGF responses in PC12 cells, and this enhancement is abolished by down-regulation of beta-, -, and -PKC(16) . Since we also found that ethanol increases PKC activity and levels of - and -PKC(23) , we hypothesized that one of these two PKC isozymes mediates ethanol's effects on NGF-induced neurite outgrowth. In the present study, we found that -PKC was abundant in growth cones and neurites of differentiating PC12 cells, while -PKC was not. The localization of -PKC in growth cones and neurites is consistent with a role in regulating neurite outgrowth(37) . Moreover, overexpression of -PKC enhanced NGF-induced activation of MAP kinases and NGF-induced neurite outgrowth, whereas overexpression of -PKC did not. Thus, our results clearly identify -PKC as a positive modulator of NGF signal transduction and NGF-induced neurite outgrowth.

Overexpression of -PKC in 3T3 fibroblasts stimulates cell division and induces anchorage-independent growth(38) . In contrast, in PC12 cells, overexpression of -PKC facilitated neuronal differentiation without affecting the growth rate. Similar results have been observed with expression of oncogenic Ras mutants that stimulate cell division and transformation in fibroblasts, but induce neuronal differentiation of PC12 cells(39) . These parallel observations suggest that -PKC modulates signaling pathways regulated by Ras. This is supported by our finding that overexpression of -PKC increased NGF-induced phosphorylation of MAP kinases, a process known to be Ras-dependent in PC12 cells(40) . The mechanisms responsible for translating these events into signals for cell division or differentiation appear cell-specific and are currently unknown, but are likely to involve signaling pathways downstream of Ras and -PKC.

We do not know the mechanism by which -PKC enhances NGF-induced activation of MAP kinases. Ras is active when GTP is bound and is inactivated by the GTPase activating protein Ras-GAP which promotes the formation of inactive Ras-GDP(41) . In lymphocytes, activation of PKC inhibits Ras-GAP and increases the formation of Ras-GTP(42) . Thus, -PKC could enhance MAP kinase activation by decreasing Ras-GAP activity. Alternatively, -PKC may promote activation of kinases downstream of Ras that lead to activation of MAP kinases. MAP kinases are activated by phosphorylation on tyrosine and threonine residues by MAPK (or ERK) kinases (MAPKKs or MEKs). In PC12 cells, NGF stimulation activates MEK1 (MAPKK-1)(43, 44) . Recent evidence in PC12 cells indicates that the Ras-regulated kinase B-Raf is activated by NGF and in turn activates MEK1 by phosphorylation(45) . Recombinant alpha-, beta-, and -PKC expressed in insect cells (46) and alpha-PKC in fibroblasts (47) activate Raf-1, a kinase related to B-Raf, and studies in fibroblasts indicate that alpha-PKC activates Raf-1 by direct phosphorylation(47) . Further studies are needed to determine whether -PKC phosphorylates and promotes activation of B-Raf in PC12 cells.

We found that the initial peak phase of NGF-stimulated MAP kinase phosphorylation was not altered by overexpression of -PKC. Instead, -PKC overexpression prevented the subsequent decline in MAP kinase phosphorylation. This suggests that -PKC may negatively regulate mechanisms that deactivate MAP kinases. At least two phosphatases have been identified that dephosphorylate MAP kinases(48) . In PC12 cells, NGF stimulates expression of MKP1, a dual specificity threonine-tyrosine phosphatase that dephosphorylates and deactivates MAP kinases. Moreover, protein phosphatase 2A (PP2A) can dephosphorylate the activating phosphoserine in MEK1 and phosphothreonines in ERK1 and ERK2 in vitro. In addition, ERK1 has recently been shown to retrophosphorylate MEK and reduce MEK activity, thereby reducing ERK activation through negative feedback control(49) . Thus, inhibition of phosphatases or MEK1 retrophosphorylation may be additional mechanisms by which -PKC sustains MAP kinase activation.

Other PKC isozymes besides -PKC might promote neurite formation. NGF has recently been reported to stimulate sustained translocation of -PKC to the particulate fraction of PC12 cells, suggesting a role for this isozyme in NGF-induced neurite outgrowth(50) . However, we found that overexpression of -PKC did not increase NGF-induced neurite outgrowth, suggesting that such a role for -PKC is unlikely. In neurons, the growth cone-associated protein GAP-43 (neuromodulin, B-50) is regulated by PKC-mediated phosphorylation (51) and is a particularly good substrate for beta-PKC(52) . GAP-43 appears to be important for neurite outgrowth since antisense oligonucleotides against GAP-43 reduce neurite formation in PC12 cells (53) and suppress growth cone development and neurite branching in chick dorsal root ganglion cells(54) . NGF treatment also increases levels of beta-PKC, which accumulates as neurites elongate(55) . Although these findings are suggestive, overexpression studies or studies with isozyme-selective inhibitors will be needed to determine whether beta-PKC contributes to NGF-induced differentiation.

Although -PKC is activated by NGF(29) , it does not appear to be required for NGF-induced differentiation of PC12 cells since down-regulation of multiple PKC isozymes, including -PKC, does not inhibit NGF-induced neurite outgrowth(16, 56) . Our findings indicate instead that -PKC plays a modulatory role in neurite outgrowth. This may provide a mechanism whereby neurotransmitters that stimulate diacylglycerol formation and activate PKC could promote neurite outgrowth. Such a mechanism could contribute to activity-dependent remodeling of synaptic connections during normal development of the nervous system(57) .

Our findings also suggest that excessive activation of -PKC contributes to abnormal neurite outgrowth observed in certain disease states. Excessive consumption of ethanol can damage the nervous system by interfering with growth and remodeling of neurites. Several reports indicate that ethanol enhances the growth of dendrites and axons in certain brain regions(58, 59, 60, 61, 62) . Our studies using PC12 cells suggest that -PKC could mediate this process, since ethanol increases levels of -PKC(23) , and NGF-induced neural differentiation is similarly enhanced in ethanol-treated cells (20) and in clones that overexpress -PKC. Particularly striking changes in growth occur in the hippocampus of rats exposed to ethanol in utero, where axons of dentate granule cells (mossy fibers) grow excessively and invade the stratum oriens of CA3(59) . Since -PKC is expressed in mossy fibers and their terminals(27) , increased expression of -PKC induced by ethanol may mediate this overgrowth. In addition, abnormal mossy fiber projections are found in the supragranular layer of the hippocampal dentate gyrus in humans with epilepsy and in animals following chemical or repetitive electrical stimuli that induce epilepsy(63) . It is possible that increased activation of -PKC during excessive neuronal activity promotes mossy fiber sprouting associated with the kindling of epileptic foci. Further investigations into the role of -PKC in neural differentiation and in these pathological states awaits the development of isozyme-specific inhibitors that can be used in animal models of development, epilepsy, and the fetal alcohol syndrome.


FOOTNOTES

*
This work was supported by grants from the National Institute on Alcohol Abuse and Alcoholism and from the Alcoholic Beverage Medical Research Foundation (to R. O. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Bldg. 1, Rm. 101, 1001 Potrero Ave., San Francisco, CA 94110. Tel.: 415-648-7111 (Ext. 329); Fax: 415-648-7116; romes@itsa.ucsf.edu.

(^1)
The abbreviations used are: PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; NGF, nerve growth factor; bFGF, basic fibroblast growth factor; MAP, mitogen-associated protein; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; ANOVA, analysis of variance.


REFERENCES

  1. Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M., and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367 [Abstract/Free Full Text]
  2. Nishizuka, Y. (1992) Science 258, 607-614 [Medline] [Order article via Infotrieve]
  3. Nakanishi, H., Brewer, K. A., and Exton, J. H. (1993) J. Biol. Chem. 268, 13-16 [Abstract/Free Full Text]
  4. Selbie, L. A., Schmitz-Peiffer, C., Sheng, Y., and Biden, T. J. (1993) J. Biol. Chem. 268, 24296-24302 [Abstract/Free Full Text]
  5. Johannes, F.-J., Prestle, J., Eis, S., Oberhagemann, P., and Pfizenmaier, K. (1994) J. Biol. Chem. 269, 6140-6148 [Abstract/Free Full Text]
  6. Valverde, A. M., Sinnett-Smith, J., Van Lint, J., and Rozengurt, E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8572-8576 [Abstract]
  7. Goodnight, J., Mischak, H., and Mushinski, J. F. (1994) Adv. Cancer Res. 64, 159-209 [Medline] [Order article via Infotrieve]
  8. Otte, A. P., and Moon, R. T. (1992) Cell 68, 1021-1029 [Medline] [Order article via Infotrieve]
  9. Hsu, L., Natyzak, D., and Laskin, J. D. (1984) Cancer Res. 44, 4607-4614 [Abstract]
  10. Bixby, J. L. (1989) Neuron 3, 287-297 [Medline] [Order article via Infotrieve]
  11. Pahlman, S., Ruusala, A.-I., Abrahamsson, L., Odelstad, L., and Nilsson, K. (1983) Cell Differ. 12, 165-170 [CrossRef][Medline] [Order article via Infotrieve]
  12. Spinelli, W., Sonnenfeld, K. H., and Ishii, D. N. (1982) Cancer Res. 42, 5067-5073 [Abstract]
  13. Greene, L. A., and Tischler, A. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2424-2428 [Abstract]
  14. Roivainen, R., McMahon, T., and Messing, R. O. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1891-1895 [Abstract]
  15. Hall, F. L., Fernyhough, P., Ishii, D. N., and Vulliet, P. R. (1988) J. Biol. Chem. 263, 4460-4466 [Abstract/Free Full Text]
  16. Roivainen, R., McMahon, T., and Messing, R. O. (1993) Brain Res. 624, 85-93 [CrossRef][Medline] [Order article via Infotrieve]
  17. Gruber, J. R., Ohno, S., and Niles, R. M. (1992) J. Biol. Chem. 267, 13356-13360 [Abstract/Free Full Text]
  18. Macfarlane, D. E., and Manzel, L. (1994) J. Biol. Chem. 269, 4327-4331 [Abstract/Free Full Text]
  19. Powell, C. T., Leng, L., Dong, L., Kiyokawa, H., Busquets, X., O'Driscoll, K., Marks, P. A., and Rifkind, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 147-151 [Abstract]
  20. Messing, R. O., Henteleff, M., and Park, J. J. (1991) Brain Res. 565, 301-311 [CrossRef][Medline] [Order article via Infotrieve]
  21. Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556 [Free Full Text]
  22. Blenis, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5889-5892 [Abstract]
  23. Messing, R. O., Petersen, P. J., and Henrich, C. J. (1991) J. Biol. Chem. 266, 23428-23432 [Abstract/Free Full Text]
  24. Roivainen, R., Hundle, B., and Messing, R. O. (1994) in Toward a Molecular Basis of Alcohol Use and Abuse (Jansson, B., J ö rvall, H., Rydberg, U., Terenius, L., and Vallee, B. L., eds) pp. 29-38, Birkh ä user Verlag, Basel
  25. Koide, H., Ogita, K., Kikkawa, U., and Nishizuka, Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1149-1153 [Abstract]
  26. Leibersperger, H., Gschwendt, M., Gernold, M., and Marks, F. (1991) J. Biol. Chem. 266, 14778-14784 [Abstract/Free Full Text]
  27. Saito, N., Itouji, A., Totani, Y., Osawa, I., Koide, H., Fujisawa, N., Ogita, K., and Tanaka, C. (1993) Brain Res. 607, 241-248 [CrossRef][Medline] [Order article via Infotrieve]
  28. Heidenreich, K. A., Toledo, S. P., Brunton, L. L., Watson, M. J., Daniel-Issakani, S., and Strulovici, B. (1990) J. Biol. Chem. 265, 15076-15082 [Abstract/Free Full Text]
  29. Ohmichi, M., Zhu, G., and Saltiel, A. R. (1993) Biochem. J. 295, 767-772 [Medline] [Order article via Infotrieve]
  30. Mangoura, D., Sogos, V., and Dawson, G. (1993) J. Neurosci. Res. 35, 488-98 [Medline] [Order article via Infotrieve]
  31. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  32. Kraft, A. S., and Anderson, W. B. (1983) Nature 301, 621-623 [Medline] [Order article via Infotrieve]
  33. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., and Kirilovsky, J. (1991) J. Biol. Chem. 266, 15771-15781 [Abstract/Free Full Text]
  34. Chijiwa, T., Mishima, A., Hagiwara, M., Sano, M., Hayashi, K., Inoue, T., Naito, K., Toshioka, T., and Hidaka, H. (1990) J. Biol. Chem. 265, 5267-5272 [Abstract/Free Full Text]
  35. Marshall, C. J. (1995) Cell 80, 179-185 [Medline] [Order article via Infotrieve]
  36. Ahn, N., Robbins, D. J., Haycock, J. W., Seger, R., Cobb, M., and Krebs, E. G. (1992) J. Neurochem. 59, 147-156 [Medline] [Order article via Infotrieve]
  37. Campenot, R. B., Draker, D. D., and Senger, D. L. (1994) J. Neurochem. 63, 868-878 [Medline] [Order article via Infotrieve]
  38. Mischak, H., Goodnight, J., Kolch, W., Martiny-Barons, G., Schaechtle, C., Kazanietz, M. G., Blumberg, P. M., Pierce, J. H., and Mushinski, J. F. (1993) J. Biol. Chem. 268, 6090-6096 [Abstract/Free Full Text]
  39. Barbacid, M. (1987) Annu. Rev. Biochem. 56, 779-827 [CrossRef][Medline] [Order article via Infotrieve]
  40. Thomas, S. M., DeMarco, M., D'Arcangelo, G., Halegoua, S., and Brugge, J. S. (1992) Cell 68, 1031-1040 [Medline] [Order article via Infotrieve]
  41. Satoh, T., Nakafuku, M., and Kaziro, Y. (1992) J. Biol. Chem. 267, 24149-24152 [Free Full Text]
  42. Downward, J., Graves, J. D., Warne, P. H., Rayter, S., and Cantrell, D. A. (1990) Nature 346, 719-723 [CrossRef][Medline] [Order article via Infotrieve]
  43. Alessi, D. R., Saito, Y., Campbell, D. G., Cohen, P., Sithanandam, G., Rapp, U., Ashworth, A., Marshall, C. J., and Cowley, S. (1994) EMBO J. 13, 1610-1619 [Abstract]
  44. Rosen, L. B., Ginty, D. D., Weber, M. J., and Greenberg, M. E. (1994) Neuron 12, 1207-1221 [Medline] [Order article via Infotrieve]
  45. Jaiswal, R. K., Moodie, S. A., Wolfman, A., and Landreth, G. E. (1994) Mol. Cell. Biol. 14, 6944-6953 [Abstract]
  46. Sözeri, O., Vollmer, K., Liyanage, M., Frith, D., Kour, G., Mark, G. E., III, and Stabel, S. (1992) Oncogene 7, 2259-2262 [Medline] [Order article via Infotrieve]
  47. Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenzeller, G., Marmé, D., and Rapp, U. R. (1993) Nature 364, 249-252 [CrossRef][Medline] [Order article via Infotrieve]
  48. Hunter, T. (1995) Cell 80, 225-236 [Medline] [Order article via Infotrieve]
  49. Brunet, A., Pagès, G., and Pouysségur, J. (1994) FEBS Lett. 346, 299-303 [CrossRef][Medline] [Order article via Infotrieve]
  50. O'Driscoll, K. R., Teng, K. K., Fabbro, D., Greene, L. A., and Weinstein, I. B. (1995) Mol. Biol. Cell 6, 449-458 [Abstract]
  51. Widmer, F., and Caroni, P. (1993) J. Cell Biol. 120, 503-512 [Abstract]
  52. Sheu, F.-S., Marais, R. M., Parker, P. J., Bazan, N. G., and Routtenberg, A. (1990) Biochem. Biophys. Res. Commun. 171, 1236-1243 [Medline] [Order article via Infotrieve]
  53. Jap Tjoen San, E. R. A., Schnidt-Michels, M., Oestreicher, A. B., Gispen, W. H., and Schotman, P. (1992) Biochem. Biophys. Res. Commun. 187, 839-846 [Medline] [Order article via Infotrieve]
  54. Aigner, L., and Caroni, P. (1993) J. Cell Biol. 123, 417-429 [Abstract]
  55. Wooten, M. W., Seibenhener, M. L., Soh, Y., Ewald, S. J., White, K. R., Lloyd, E. D., Olivier, A., and Parker, P. J. (1992) FEBS Lett. 298, 74-78 [CrossRef][Medline] [Order article via Infotrieve]
  56. Reinhold, D. S., and Neet, K. E. (1989) J. Biol. Chem. 264, 3538-3544 [Abstract/Free Full Text]
  57. Goodman, C. S., and Shatz, C. J. (1993) Neuron 72, 77-98
  58. Miller, M. W., Nicholas, N. L., and Rhoades, R. W. (1990) J. Comp. Neurol. 297, 91-105 [Medline] [Order article via Infotrieve]
  59. West, J. R., Hodges, C. A., and Black, A. C. J. (1981) Science 211, 957-959 [Medline] [Order article via Infotrieve]
  60. Volk, B. (1984) in Neurobehavioral Teratology (Yanai, J., ed) pp. 163-193, Elsevier Science Publishers B.V., Amsterdam
  61. Pentney, R. J., and Quackenbush, L. J. (1990) Alcohol. Clin. Exp. Res. 14, 878-886 [Medline] [Order article via Infotrieve]
  62. Cadete-Leite, A., Tavares, M. A., Uylings, H. B. M., and Paula-Barbosa, M. (1988) Brain Res. 473, 1-14 [CrossRef][Medline] [Order article via Infotrieve]
  63. Sutula, T. P. (1993) in Epilepsy: Models, Mechanisms, and Concepts (Schwartzkroin, P. A., ed) pp. 304-322, Cambridge University Press, New York

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