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Inhibition of protein kinase C activity promotes the neurotrophic action of epidermal and basic fibroblast growth factors
Brain Research, 473 (1988) 141-146
141
Elsevier BRE 23168
Inhibition of protein kinase C activity promotes the neurotrophic action of epidermal and basic fibroblast growth factors Richard S. Morrison 1'2, Janet L. Gross 4 and Joseph R. Moskal 1'3 Departments of tNeurosurgery, 2Anatomy and 3Neuroscience, Albert Einstein College of Medicine/Montefiore Medical Center, 111 East 210th Street, Bronx, NY (U.S.A.) and 4E.I. Du Pont De Nemours Company Inc., Wilmington, DE (U.S.A.)
(Accepted 12 July 1988) Key words: Protein kinase C; Trophic factor; Neuron; Basic fibroblast growth factor; Epidermal growth factor; Neurite outgrowth
In primary neuronal cultures the activation of protein kinase C (PKC) by tumor-promoting phorbol esters blocked growth factor-induced neuronal survival and neurite extension. Depletion of PKC markedly facilitated both epidermal growth factor and basic fibroblast growth factor-inducible neurite extension. Inhibition of PKC by H-7 also stimulated neurite extension. These results suggest that down-regulation of PKC in neurons may be required for trophic factor action.
Neurotrophic factors are hormone-like agents that act on neurons of the peripheral and central nervous system (CNS) to facilitate growth and maintain viability. A small number of these proteins have been purified to homogeneity. These include nerve growth factor (NGF) 39, brain-derived neurotrophic factor (BDNF) 3'7, acidic 33'38, and basic fibroblast growth factors (aFGF, bFGF) 26'34-37 and epidermal growth factor (EGF) 25. Although progress is being made in identifying appropriate target cells for each factor and in characterizing their receptors, very little is known about the mechanisms by which trophic factors enhance survival and promote neurite elongation. In many receptor-mediated events the turnover of phosphoinositides is coupled to receptor-ligand binding 4. Phosphoinositide hydrolysis generates inositol phosphates and diacylglycero123 which activates protein kinase C (PKC) 28 thereby altering intracellular protein phosphorylation 2°. PKC may be an important intracellular mediator of NGF action. NGF rapidly stimulates the hydrolysis of phosphoinositides in PC12 cells suggesting that it directly activates PKC 5. Furthermore, activators of PKC have been shown to
stimulate neurite outgrowth from explants of chick sensory ganglia 15 and to potentiate the action of NGF on PC12 cells 13. In contrast, inhibitors of PKC suppress NGF-directed neurite outgrowth in PC12 cells 13,21" E G F and bFGF presumably initiate their action by first binding to specific, high affinity receptors located in neuronal plasma membranes. The mechanism by which the extracellular binding of these ligands is translated into atrophic signal for neurons is unclear. In the present study the ability of E G F and b F G F to utilize the signal transduction pathway mediated by PKC was investigated in neurons derived from the CNS of neonatal rats. This was accomplished either by activating PKC following the addition of growth factor, or by depleting cells of PKC prior to growth factor treatment. Primary neuronal cultures were established as previously described 26. In brief, the brains of neonatal rat pups (0-1 day) were excised and the cortex removed. The meninges was stripped off and the tissue was trypsinized. The tissue was further dissociated by mechanical trituration and passed through Nitex mesh of varying size (130/~m and 33/~m). After pas-
Correspondence: R.S. Morrison, Department of Neurosurgery, Albert Einstein College of Medicine/Montefiore Medical Center,
111 East 210th St., Bronx, NY 10467, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
142 sage through the mesh 1.25 × 105 cells p e r cm 2 were plated in Dulbecco's modified Eagles m e d i u m / H a m ' s F-12 medium (1:1) s u p p l e m e n t e d with 10% fetal calf serum, penicillin-streptomycin and 5 tiM cytosine arabinoside. A f t e r 18 h, the cultures were converted to a chemically defined m e d i u m containing the same nutrient mixture plus hydrocortisone (50 nM), putrescine (100 ~ M ) , prostaglandin F2a (500 ng/ml), insulin (50/~g/ml), 5/~M cytosine arabinoside and the a p p r o p r i a t e factors. Cytosine arabinoside was omitted from subsequent media changes because it eventually proved toxic to the cells. All cultures were characterized i m m u n o c y t o c h e m ically using monocional antibody A2B5 as well as antisera to the middle subunit of neurofilament protein (NFP-M) (a generous gift from Dr. V. Lee, University of Pennsylvania) and glial fibrillary acidic protein ( G F A P ) (a generous gift from Dr. Larry Eng, V . A . Medical Center/Stanford University, C A ) . The puri-
12.
10 ¸
E
6
ty of cell cultures treated with growth factor alone or p r e t r e a t e d with phorboi ester prior to growth factor treatment was similar to the results r e p o r t e d previously for cerebral cortical neuronal cultures 26. Only 4 - 7 % of all cells showed intense staining for G F A P suggesting they were astrocytes. Of all cells 9 3 - 9 7 % stained positively for N F P - M while 98% of all cells consistently stained positive with monoclonal antibody A2B5. The overlap between N F P - M and A2B5 suggests that a small percentage of the process-bearing G F A P positive cells were type 2 astrocytes 31. The addition of the P K C activator, phorbol-12myristate-13-acetate ( P M A , 80 nM) to E G F - or b F G F - t r e a t e d neurons significantly depressed the survival of neurons bearing tong processes (Fig. 1), The addition of phorbol 12,13-dibutyrate ( P D B u , 80 nM), another active t u m o r p r o m o t e r , also reduced neuronal survival. In contrast, the addition of very weak or inactive t u m o r p r o m o t e r s , such as 4fl phorbol, 4a phorbol or 4a 12,13-phorbol d i d e c a n o a t e , did not inhibit survival or neurite outgrowth (Table I). Thus, the inhibition of E G F or b F G F - m e d i a t e d trophic activity correlated with the ability of the phorboi ester derivative to stimulate PKC. The P M A - i n duced inhibition of neuronal survival was dose-dep e n d e n t ( g i = 5 nM) and optimal levels of inhibition were observed at a concentration of 40 nM (Fig. 2). The lack of inhibition observed with weak t u m o r promoters and the saturable d o s e - r e s p o n s e curve sug-
0
TABLE I The influence o f phorbol ester derivatives on the survival of EGF-treated cerebral cortical neurons
0 CDM
EGF
EGF +
PMA
bFGF
bFGF +
PMA
Fig. 1. The effect of phorbol ester treatment on trophic factorinduced neuronal survival. Neurons were plated in 35 mm dishes in DMEM/Ham's F-12 medium containing 10% fetal bovine serum (FBS) plus cytosine arabinoside (5/aM). After 18 h (day 0) the cells were converted to CDM supplemented with cytosine arabinoside (5/aM) and one of the following conditions: EGF, 10 ng/ml; EGF plus PMA, 80 nM; bFGF, 10 ng/ml; bFGF plus PMA. The cells were media changed on days 2 and 4 at which time the cytosine arabinoside was removed. Neurons bearing processes longer than 100/~m were scored in 10 random fields per dish on day 6. Three dishes were counted per treatment. The data are expressed as cells per mm 2 (mean + S.E.M.).
Primary cultures were, 18 h after plating, converted to CDM, CDM plus EGF (10 ng/mt), or CDM plus EGF with various phorbol ester derivatives. The cells were maintained and counted as described for Fig. 1. The tumor-promoting ability of each phorbol ester is listed in relative terms (+, active; -, inactive). Tumor. promoting ability also correlates with the ability to activate PKC. Condition
CDM CDM CDM CDM CDM CDM CDM
+ + + + + +
EGF EGF EGF EGF EGF EGF
Tumor promoting ability
+ + + + +
PMA PDBu fl-phorbol a-phorbol a-pDD
+++ ++ -
Cells/mm 2
2.1 + 0.22 10.1 + 0.50 3.1 + 0.30 4.6 + 0.18 11.6 + 0.57 8.1 _+ 1.21 8.6 + 1.03
143
•~- TOTAL PBC "dr PBC LP
20
TABLE II The effect of PKC activation on the survival of EGF-treated cerebral cortical neurons
Primary cultures were converted to CDM alone or CDM plus EGF (10 ng/ml) 18 h after plating. PDBu (80 nM) was added to the appropriate dishes along with EGF (0 h) or at 2, 4, 8 or 24 h following EGF addition. Control cultures received PDBu alone or EGF plus various inactive 4-a-phorbol isomers. The cells were counted 48 h after conversion to CDM as described for Fig. 1. Cells with processes ~>100~m and those with only very long processes (t>200/~m) were identified.
E E 10 u.I
Condition
0
•
100 PMA (riM)
200
Fig. 2. Influence of PMA concentration on the survival of neurons from the cerebral cortex. Primary cultures were converted to CDM with EGF (10 ng/ml) and various concentrations of PMA 1.8 h after plating. Six days after conversion to CDM, the cells were counted and the data expressed as described for Fig. 1. PBL, process bearing cells; PBC LP, cells with very long processes (>200/~m).
CDM CDM CDM CDM CDM CDM CDM CDM CDM CDM
+ + + + + + + + +
EGF EGF EGF EGF EGF EGF EGF EGF PDB
Cells/mm 2
+ + + + + + +
PDB PDB PDB PDB PDB 4aPDD 4aP
0h 2h 4h 8h 24h 24 h 24 h
>lO0 gm
>200 grn
0.44 + 0.12 4.89 + 0.42 2.32 + 0.29 2.26 + 0,36 1.93+0.18 2.20 + 0.38 1.53+0.18 4.25 _+ 0.30 4.50 _+ 0.34 0.88 + 0.17
0.16 + 0.06 1.11 + 0.26 0.22 + 0.08 0.27 ___0.09 0.26_+0.12 0.20 + 0.08 0.0 0.81 +__0.16 0.70 _+0.17 0.0
gest that the p h e n o m e n o n is the result of a specific,
e x t e n d p r o c e s s e s o r t h o s e actively e l a b o r a t i n g p r o -
r e c e p t o r - m e d i a t e d e v e n t and n o t d u e to toxicity.
cesses w e r e a r r e s t e d f r o m f u r t h e r d e v e l o p m e n t after
Since t h e p h o r b o l esters w e r e a d d e d to the g r o w t h
P K C activation.
m e d i u m at the s a m e t i m e as the g r o w t h factors (day 0), and p r o c e s s - b e a r i n g n e u r o n s w e r e not c o u n t e d until day 6, it was n o t clear if t h e o b s e r v e d inhibition
TABLE III
r e s u l t e d f r o m t h e a c t i v a t i o n of P K C o r its d o w n - r e g u -
The effect of PKC depletion on the survival of EGF-treated cerebral cortical neurons
lation. T h e d e p l e t i o n of P K C is a n o r m a l e v e n t that follows its a c t i v a t i o n , a n d results f r o m the translocation o f P K C to t h e p l a s m a m e m b r a n e w h e r e it is hyd r o l y z e d 1°'22. W e t h e r e f o r e e x a m i n e d the effects of P K C a c t i v a t i o n and d e p l e t i o n on t h e t r o p h i c activities o f E G F a n d b F G F . T h e a c t i v a t i o n o f P K C at v a r i o u s times f o l l o w i n g t h e a d d i t i o n o f E G F significantly d e p r e s s e d the survival of n e u r o n s b e a r i n g short and l o n g p r o c e s s e s ( T a b l e II). N o r m a l l y , n e u r o n s t r e a t e d with E G F o r b F G F for 24 h e x h i b i t a m a r k e d i n c r e a s e in the length
Primary cultures were converted to CDM 18 h after plating. Control cultures received EGF (10 ng/ml) 1, 8 and 24 h after conversion to CDM. Experimental cultures were pretreated for intervals of 1, 8 and 24 h with PDBu (80 nM). At the end of the PDBu incubation the cultures were washed 2 x with serumfree medium and converted to CDM plus EGF (10 ng/ml). Additional control cultures were maintained for the entire period in CDM plus PDBu. Cells bearing processes ~>100/~ and those bearing only very long processes (~>200 gm) were counted 48 h after conversion to CDM as described for Table II. Condition
Cells~ram2
of n e u r i t e s c o m p a r e d to u n t r e a t e d c o n t r o l s (chemically d e f i n e d m e d i u m ( C D M ) only). In the p r e s e n t study, t h e a c t i v a t i o n o f P K C actually a r r e s t e d process o u t g r o w t h f r o m n e u r o n s that had p r e v i o u s l y b e e n e x p o s e d to E G F for 24 h. A g a i n , t r e a t m e n t with 4 a p h o r b o l and 4 a p h o r b o l 1 2 , 1 3 - d i d e c a n o a t e did n o t significantly inhibit n e u r o n a l survival o r n e u r i t e ext e n s i o n . T h e r e f o r e , n e u r o n s that h a v e not b e g u n to
CDM CDM CDM CDM CDM CDM CDM CDM
+ + + + + + +
EGF EGF EGF PDB PDB PDB PDB
1h 8h 24 h 1 h ---, EGF 8 h ~ EGF 24 h ---}EGF 48 h
> l O0t~m
>200/~m
1.20+ 0.19 4.70 + 0.36 4.13 -I- 0.32 3.27 + 0.27 4.73 + 0.28 10.07 + 0.62 17.53 _+ 0.56 3.00 +0.21
0 0.73 0.60 0.27 0.91 1.60 3.13 0
+ + + + + +
0.12 0.14 0.12 0.23 0.24 0.24
144 In contrast, pretreatment of cortical neurons with PDBu alone, resulting in PKC depletion, potentiated the trophic actions of EGF and bFGF. This effect was dependent on the duration of PDBu exposure. PDBu pretreatment for 1 h did not influence EGF-mediated neuronal survival or neurite outgrowth. However, PDBu pretreatment for 8 and 24 h dramatically increased the number of surviving neurons with long processes grown in the presence of EGF (Table III). As expected, the addition of EGF to control neurons 1, 8 and 24 h after conversion to chemically defined medium enhanced the survival of total neurons and those bearing long processes. Moreover, the extent of process outgrowth was dependent on the duration of exposure to EGF. Further support for the importance of PKC downregulation during the course of EGF action was provided by the addition of a PKC inhibitor. The PKC inhibitor, H-7, promoted neurite outgrowth from cortical neurons when added alone (data not shown) or in combination with EGF (Fig. 3) or bFGF (data not shown). In contrast, the addition of H-8. an inhibitor of cyclic AMP-dependent protein kinase, to neuronal cultures antagonized the effect of EGF. In further contrast to H-7, H-8 had no effect on neuronal survival or neurite outgrowth when added alone (data not shown). Thus, depleting cerebral cortical neurons of PKC or inhibiting this enzyme appears to potentiate their response to EGF and bFGF. In contrast, the activa-
NO ADDITION
EGF
EGF+ H7
EGF + H8
0
10 CELLS/mm
2
20
Fig. 3. The influence of inhibitors of cyclic AMP-dependent protein kinase and PKC on neuronal survival. Primarycultures were, 18 h after plating, converted to CDM alone, CDM plus EGF, CDM plus EGF and the PKC inhibitor (H-7 (10/~M), or CDM plus EGF and the cyclicAMP-dependent protein kinase inhibitor H-8 (10 ~M). The cells were media changed and counted as described for Fig. 1.
tion of PKC at any time following the addition of EGF or bFGF suppresses neuronal survival and arrests neurite outgrowth. This is in contrast to NGF which appears to require the activation of PKC as part of its normal mechanism of action. It is not clear, however, if NGF activates PKC in all NGF-responsire cells or only in those cell types derived from the peripheral nervous system. NGF has been shown to have atrophic influence on cholinergic neurons in the basal forebrain j2"14'24, although the ability of PKC to mediate the action of NGF in these cells has not been determined. The results obtained with EGF and bFGF in this study are consistent with the results of other previously published studies. Thus, the mitogenic effect of a-thrombin on vascular smooth muscle cells is inhibited in the presence of PMA 16. PKC activation has also been found to inhibit the proliferation of bovine capillary endothelial cells in response to angiogenic endothelial mitogens 8. These are unusual findings since PKC activators are mitogenic or comitogenic for numerous other cell types including Swiss 3T3 cells 32, lymphocytes 19, thyroid cells ~ and chick heart mesenchymal cells 2. However, activators of PKC also inhibit the proliferation of HL-60 promyeloid leukemia cells, inducing properties found in normal macrophages 9"~7. These results together with our observations on primary cortical neurons suggest that regulation of PKC by growth factors in cells committed to differentiate, or those already fully differentiated may differ significantly from that in cells actively proliferating. PKC has been detected in rat brain at various developmental stages suggesting specific ontogenetic functions 27. In the developing fetus, high levels of PKC were detected in the ventral and dorsal marginal zones of the neural tube which are rapidly expanding zones containing numerous cell processes. In the adult brain, PKC was most highly concentrated in synaptic regions t1'27. Our results suggest that the PKC found in developing neurons is not required for process elongation, since its activation inhibits process outgrowth. In fact, down-regulation of PKC in neurons may be required for trophic factor action. The PKC that is found in developing neurons may be synthesized in preparation for synapse formation. However, this interpretation must be viewed cautiously, since multiple species of PKC exist 6As'29,
145
signal3°. Perhaps, while one PKC isozyme is being
begin differentiating. With the advent of c D N A ' s and antisera for each PKC isozyme it should be possi-
synthesized in preparation for synapse formation an-
ble to elucidate the role of PKC in the developing
other isozymic form is subject to down-regulation by trophic factors after neurons leave the cell cycle and
CNS.
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36
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39
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141
Elsevier BRE 23168
Inhibition of protein kinase C activity promotes the neurotrophic action of epidermal and basic fibroblast growth factors Richard S. Morrison 1'2, Janet L. Gross 4 and Joseph R. Moskal 1'3 Departments of tNeurosurgery, 2Anatomy and 3Neuroscience, Albert Einstein College of Medicine/Montefiore Medical Center, 111 East 210th Street, Bronx, NY (U.S.A.) and 4E.I. Du Pont De Nemours Company Inc., Wilmington, DE (U.S.A.)
(Accepted 12 July 1988) Key words: Protein kinase C; Trophic factor; Neuron; Basic fibroblast growth factor; Epidermal growth factor; Neurite outgrowth
In primary neuronal cultures the activation of protein kinase C (PKC) by tumor-promoting phorbol esters blocked growth factor-induced neuronal survival and neurite extension. Depletion of PKC markedly facilitated both epidermal growth factor and basic fibroblast growth factor-inducible neurite extension. Inhibition of PKC by H-7 also stimulated neurite extension. These results suggest that down-regulation of PKC in neurons may be required for trophic factor action.
Neurotrophic factors are hormone-like agents that act on neurons of the peripheral and central nervous system (CNS) to facilitate growth and maintain viability. A small number of these proteins have been purified to homogeneity. These include nerve growth factor (NGF) 39, brain-derived neurotrophic factor (BDNF) 3'7, acidic 33'38, and basic fibroblast growth factors (aFGF, bFGF) 26'34-37 and epidermal growth factor (EGF) 25. Although progress is being made in identifying appropriate target cells for each factor and in characterizing their receptors, very little is known about the mechanisms by which trophic factors enhance survival and promote neurite elongation. In many receptor-mediated events the turnover of phosphoinositides is coupled to receptor-ligand binding 4. Phosphoinositide hydrolysis generates inositol phosphates and diacylglycero123 which activates protein kinase C (PKC) 28 thereby altering intracellular protein phosphorylation 2°. PKC may be an important intracellular mediator of NGF action. NGF rapidly stimulates the hydrolysis of phosphoinositides in PC12 cells suggesting that it directly activates PKC 5. Furthermore, activators of PKC have been shown to
stimulate neurite outgrowth from explants of chick sensory ganglia 15 and to potentiate the action of NGF on PC12 cells 13. In contrast, inhibitors of PKC suppress NGF-directed neurite outgrowth in PC12 cells 13,21" E G F and bFGF presumably initiate their action by first binding to specific, high affinity receptors located in neuronal plasma membranes. The mechanism by which the extracellular binding of these ligands is translated into atrophic signal for neurons is unclear. In the present study the ability of E G F and b F G F to utilize the signal transduction pathway mediated by PKC was investigated in neurons derived from the CNS of neonatal rats. This was accomplished either by activating PKC following the addition of growth factor, or by depleting cells of PKC prior to growth factor treatment. Primary neuronal cultures were established as previously described 26. In brief, the brains of neonatal rat pups (0-1 day) were excised and the cortex removed. The meninges was stripped off and the tissue was trypsinized. The tissue was further dissociated by mechanical trituration and passed through Nitex mesh of varying size (130/~m and 33/~m). After pas-
Correspondence: R.S. Morrison, Department of Neurosurgery, Albert Einstein College of Medicine/Montefiore Medical Center,
111 East 210th St., Bronx, NY 10467, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
142 sage through the mesh 1.25 × 105 cells p e r cm 2 were plated in Dulbecco's modified Eagles m e d i u m / H a m ' s F-12 medium (1:1) s u p p l e m e n t e d with 10% fetal calf serum, penicillin-streptomycin and 5 tiM cytosine arabinoside. A f t e r 18 h, the cultures were converted to a chemically defined m e d i u m containing the same nutrient mixture plus hydrocortisone (50 nM), putrescine (100 ~ M ) , prostaglandin F2a (500 ng/ml), insulin (50/~g/ml), 5/~M cytosine arabinoside and the a p p r o p r i a t e factors. Cytosine arabinoside was omitted from subsequent media changes because it eventually proved toxic to the cells. All cultures were characterized i m m u n o c y t o c h e m ically using monocional antibody A2B5 as well as antisera to the middle subunit of neurofilament protein (NFP-M) (a generous gift from Dr. V. Lee, University of Pennsylvania) and glial fibrillary acidic protein ( G F A P ) (a generous gift from Dr. Larry Eng, V . A . Medical Center/Stanford University, C A ) . The puri-
12.
10 ¸
E
6
ty of cell cultures treated with growth factor alone or p r e t r e a t e d with phorboi ester prior to growth factor treatment was similar to the results r e p o r t e d previously for cerebral cortical neuronal cultures 26. Only 4 - 7 % of all cells showed intense staining for G F A P suggesting they were astrocytes. Of all cells 9 3 - 9 7 % stained positively for N F P - M while 98% of all cells consistently stained positive with monoclonal antibody A2B5. The overlap between N F P - M and A2B5 suggests that a small percentage of the process-bearing G F A P positive cells were type 2 astrocytes 31. The addition of the P K C activator, phorbol-12myristate-13-acetate ( P M A , 80 nM) to E G F - or b F G F - t r e a t e d neurons significantly depressed the survival of neurons bearing tong processes (Fig. 1), The addition of phorbol 12,13-dibutyrate ( P D B u , 80 nM), another active t u m o r p r o m o t e r , also reduced neuronal survival. In contrast, the addition of very weak or inactive t u m o r p r o m o t e r s , such as 4fl phorbol, 4a phorbol or 4a 12,13-phorbol d i d e c a n o a t e , did not inhibit survival or neurite outgrowth (Table I). Thus, the inhibition of E G F or b F G F - m e d i a t e d trophic activity correlated with the ability of the phorboi ester derivative to stimulate PKC. The P M A - i n duced inhibition of neuronal survival was dose-dep e n d e n t ( g i = 5 nM) and optimal levels of inhibition were observed at a concentration of 40 nM (Fig. 2). The lack of inhibition observed with weak t u m o r promoters and the saturable d o s e - r e s p o n s e curve sug-
0
TABLE I The influence o f phorbol ester derivatives on the survival of EGF-treated cerebral cortical neurons
0 CDM
EGF
EGF +
PMA
bFGF
bFGF +
PMA
Fig. 1. The effect of phorbol ester treatment on trophic factorinduced neuronal survival. Neurons were plated in 35 mm dishes in DMEM/Ham's F-12 medium containing 10% fetal bovine serum (FBS) plus cytosine arabinoside (5/aM). After 18 h (day 0) the cells were converted to CDM supplemented with cytosine arabinoside (5/aM) and one of the following conditions: EGF, 10 ng/ml; EGF plus PMA, 80 nM; bFGF, 10 ng/ml; bFGF plus PMA. The cells were media changed on days 2 and 4 at which time the cytosine arabinoside was removed. Neurons bearing processes longer than 100/~m were scored in 10 random fields per dish on day 6. Three dishes were counted per treatment. The data are expressed as cells per mm 2 (mean + S.E.M.).
Primary cultures were, 18 h after plating, converted to CDM, CDM plus EGF (10 ng/mt), or CDM plus EGF with various phorbol ester derivatives. The cells were maintained and counted as described for Fig. 1. The tumor-promoting ability of each phorbol ester is listed in relative terms (+, active; -, inactive). Tumor. promoting ability also correlates with the ability to activate PKC. Condition
CDM CDM CDM CDM CDM CDM CDM
+ + + + + +
EGF EGF EGF EGF EGF EGF
Tumor promoting ability
+ + + + +
PMA PDBu fl-phorbol a-phorbol a-pDD
+++ ++ -
Cells/mm 2
2.1 + 0.22 10.1 + 0.50 3.1 + 0.30 4.6 + 0.18 11.6 + 0.57 8.1 _+ 1.21 8.6 + 1.03
143
•~- TOTAL PBC "dr PBC LP
20
TABLE II The effect of PKC activation on the survival of EGF-treated cerebral cortical neurons
Primary cultures were converted to CDM alone or CDM plus EGF (10 ng/ml) 18 h after plating. PDBu (80 nM) was added to the appropriate dishes along with EGF (0 h) or at 2, 4, 8 or 24 h following EGF addition. Control cultures received PDBu alone or EGF plus various inactive 4-a-phorbol isomers. The cells were counted 48 h after conversion to CDM as described for Fig. 1. Cells with processes ~>100~m and those with only very long processes (t>200/~m) were identified.
E E 10 u.I
Condition
0
•
100 PMA (riM)
200
Fig. 2. Influence of PMA concentration on the survival of neurons from the cerebral cortex. Primary cultures were converted to CDM with EGF (10 ng/ml) and various concentrations of PMA 1.8 h after plating. Six days after conversion to CDM, the cells were counted and the data expressed as described for Fig. 1. PBL, process bearing cells; PBC LP, cells with very long processes (>200/~m).
CDM CDM CDM CDM CDM CDM CDM CDM CDM CDM
+ + + + + + + + +
EGF EGF EGF EGF EGF EGF EGF EGF PDB
Cells/mm 2
+ + + + + + +
PDB PDB PDB PDB PDB 4aPDD 4aP
0h 2h 4h 8h 24h 24 h 24 h
>lO0 gm
>200 grn
0.44 + 0.12 4.89 + 0.42 2.32 + 0.29 2.26 + 0,36 1.93+0.18 2.20 + 0.38 1.53+0.18 4.25 _+ 0.30 4.50 _+ 0.34 0.88 + 0.17
0.16 + 0.06 1.11 + 0.26 0.22 + 0.08 0.27 ___0.09 0.26_+0.12 0.20 + 0.08 0.0 0.81 +__0.16 0.70 _+0.17 0.0
gest that the p h e n o m e n o n is the result of a specific,
e x t e n d p r o c e s s e s o r t h o s e actively e l a b o r a t i n g p r o -
r e c e p t o r - m e d i a t e d e v e n t and n o t d u e to toxicity.
cesses w e r e a r r e s t e d f r o m f u r t h e r d e v e l o p m e n t after
Since t h e p h o r b o l esters w e r e a d d e d to the g r o w t h
P K C activation.
m e d i u m at the s a m e t i m e as the g r o w t h factors (day 0), and p r o c e s s - b e a r i n g n e u r o n s w e r e not c o u n t e d until day 6, it was n o t clear if t h e o b s e r v e d inhibition
TABLE III
r e s u l t e d f r o m t h e a c t i v a t i o n of P K C o r its d o w n - r e g u -
The effect of PKC depletion on the survival of EGF-treated cerebral cortical neurons
lation. T h e d e p l e t i o n of P K C is a n o r m a l e v e n t that follows its a c t i v a t i o n , a n d results f r o m the translocation o f P K C to t h e p l a s m a m e m b r a n e w h e r e it is hyd r o l y z e d 1°'22. W e t h e r e f o r e e x a m i n e d the effects of P K C a c t i v a t i o n and d e p l e t i o n on t h e t r o p h i c activities o f E G F a n d b F G F . T h e a c t i v a t i o n o f P K C at v a r i o u s times f o l l o w i n g t h e a d d i t i o n o f E G F significantly d e p r e s s e d the survival of n e u r o n s b e a r i n g short and l o n g p r o c e s s e s ( T a b l e II). N o r m a l l y , n e u r o n s t r e a t e d with E G F o r b F G F for 24 h e x h i b i t a m a r k e d i n c r e a s e in the length
Primary cultures were converted to CDM 18 h after plating. Control cultures received EGF (10 ng/ml) 1, 8 and 24 h after conversion to CDM. Experimental cultures were pretreated for intervals of 1, 8 and 24 h with PDBu (80 nM). At the end of the PDBu incubation the cultures were washed 2 x with serumfree medium and converted to CDM plus EGF (10 ng/ml). Additional control cultures were maintained for the entire period in CDM plus PDBu. Cells bearing processes ~>100/~ and those bearing only very long processes (~>200 gm) were counted 48 h after conversion to CDM as described for Table II. Condition
Cells~ram2
of n e u r i t e s c o m p a r e d to u n t r e a t e d c o n t r o l s (chemically d e f i n e d m e d i u m ( C D M ) only). In the p r e s e n t study, t h e a c t i v a t i o n o f P K C actually a r r e s t e d process o u t g r o w t h f r o m n e u r o n s that had p r e v i o u s l y b e e n e x p o s e d to E G F for 24 h. A g a i n , t r e a t m e n t with 4 a p h o r b o l and 4 a p h o r b o l 1 2 , 1 3 - d i d e c a n o a t e did n o t significantly inhibit n e u r o n a l survival o r n e u r i t e ext e n s i o n . T h e r e f o r e , n e u r o n s that h a v e not b e g u n to
CDM CDM CDM CDM CDM CDM CDM CDM
+ + + + + + +
EGF EGF EGF PDB PDB PDB PDB
1h 8h 24 h 1 h ---, EGF 8 h ~ EGF 24 h ---}EGF 48 h
> l O0t~m
>200/~m
1.20+ 0.19 4.70 + 0.36 4.13 -I- 0.32 3.27 + 0.27 4.73 + 0.28 10.07 + 0.62 17.53 _+ 0.56 3.00 +0.21
0 0.73 0.60 0.27 0.91 1.60 3.13 0
+ + + + + +
0.12 0.14 0.12 0.23 0.24 0.24
144 In contrast, pretreatment of cortical neurons with PDBu alone, resulting in PKC depletion, potentiated the trophic actions of EGF and bFGF. This effect was dependent on the duration of PDBu exposure. PDBu pretreatment for 1 h did not influence EGF-mediated neuronal survival or neurite outgrowth. However, PDBu pretreatment for 8 and 24 h dramatically increased the number of surviving neurons with long processes grown in the presence of EGF (Table III). As expected, the addition of EGF to control neurons 1, 8 and 24 h after conversion to chemically defined medium enhanced the survival of total neurons and those bearing long processes. Moreover, the extent of process outgrowth was dependent on the duration of exposure to EGF. Further support for the importance of PKC downregulation during the course of EGF action was provided by the addition of a PKC inhibitor. The PKC inhibitor, H-7, promoted neurite outgrowth from cortical neurons when added alone (data not shown) or in combination with EGF (Fig. 3) or bFGF (data not shown). In contrast, the addition of H-8. an inhibitor of cyclic AMP-dependent protein kinase, to neuronal cultures antagonized the effect of EGF. In further contrast to H-7, H-8 had no effect on neuronal survival or neurite outgrowth when added alone (data not shown). Thus, depleting cerebral cortical neurons of PKC or inhibiting this enzyme appears to potentiate their response to EGF and bFGF. In contrast, the activa-
NO ADDITION
EGF
EGF+ H7
EGF + H8
0
10 CELLS/mm
2
20
Fig. 3. The influence of inhibitors of cyclic AMP-dependent protein kinase and PKC on neuronal survival. Primarycultures were, 18 h after plating, converted to CDM alone, CDM plus EGF, CDM plus EGF and the PKC inhibitor (H-7 (10/~M), or CDM plus EGF and the cyclicAMP-dependent protein kinase inhibitor H-8 (10 ~M). The cells were media changed and counted as described for Fig. 1.
tion of PKC at any time following the addition of EGF or bFGF suppresses neuronal survival and arrests neurite outgrowth. This is in contrast to NGF which appears to require the activation of PKC as part of its normal mechanism of action. It is not clear, however, if NGF activates PKC in all NGF-responsire cells or only in those cell types derived from the peripheral nervous system. NGF has been shown to have atrophic influence on cholinergic neurons in the basal forebrain j2"14'24, although the ability of PKC to mediate the action of NGF in these cells has not been determined. The results obtained with EGF and bFGF in this study are consistent with the results of other previously published studies. Thus, the mitogenic effect of a-thrombin on vascular smooth muscle cells is inhibited in the presence of PMA 16. PKC activation has also been found to inhibit the proliferation of bovine capillary endothelial cells in response to angiogenic endothelial mitogens 8. These are unusual findings since PKC activators are mitogenic or comitogenic for numerous other cell types including Swiss 3T3 cells 32, lymphocytes 19, thyroid cells ~ and chick heart mesenchymal cells 2. However, activators of PKC also inhibit the proliferation of HL-60 promyeloid leukemia cells, inducing properties found in normal macrophages 9"~7. These results together with our observations on primary cortical neurons suggest that regulation of PKC by growth factors in cells committed to differentiate, or those already fully differentiated may differ significantly from that in cells actively proliferating. PKC has been detected in rat brain at various developmental stages suggesting specific ontogenetic functions 27. In the developing fetus, high levels of PKC were detected in the ventral and dorsal marginal zones of the neural tube which are rapidly expanding zones containing numerous cell processes. In the adult brain, PKC was most highly concentrated in synaptic regions t1'27. Our results suggest that the PKC found in developing neurons is not required for process elongation, since its activation inhibits process outgrowth. In fact, down-regulation of PKC in neurons may be required for trophic factor action. The PKC that is found in developing neurons may be synthesized in preparation for synapse formation. However, this interpretation must be viewed cautiously, since multiple species of PKC exist 6As'29,
145
signal3°. Perhaps, while one PKC isozyme is being
begin differentiating. With the advent of c D N A ' s and antisera for each PKC isozyme it should be possi-
synthesized in preparation for synapse formation an-
ble to elucidate the role of PKC in the developing
other isozymic form is subject to down-regulation by trophic factors after neurons leave the cell cycle and
CNS.
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36
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