Vol.
177,
No.
June
28.
1991
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
II PROTEINS CORRELATE INCREASES IN 11 AND ANNEXIN DIFFEREN F IATION IN THE PC12 PHEOCHROMOCYTOMA
1188-1193
WITH
Mary T. Fox, David A. Prentice, and James P. Hughes Department Received
April
of Life Sciences, Indiana State University, Terre Haute, IN 47809 29,
1991
Regulation of pll and annexin II by nerve growth factor, staurosporine, and epidermal growth factor was examined in PC12 rat adrenal pheochromocytoma cells using immunoblot analysis. Nerve growth factor, which is known to induce neurite outgrowth in PC12 cells, stimulated a five-fold increase in pll and the higher levels of pll were characteristic of PC12 cells exposed to nerve rowth factor for u to ten days. Nerve growth factor induced an even greater increase (13. %-fold) in annexin PI. Staurosporine, a protein kinase inhibitor that at high concentrations induces neurite formation, was as effective as nerve growth factor in increasing the intracellular levels of pll and annexin II. Epidermal growth factor was less effective than nerve growth factor and staurosporine, producing only a two-fold increase in pll and a three-fold increase in annexin II. The ineffectiveness of epidermal growth factor in increasing intracellular levels of pll and annexin II is consistent with the fact that epidermal growth factor does not stimulate neurite outgrowth in PC12 cells. Evidence presented here suggests that pll and/or annexin II may play a role in PC12 cell differentiation. Q 1991Academic Pres, Inc. SUMMARY.
Nerve growth factor (NGF) binds to specific receptors on the surface of target cells and stimulates a wide variety of the changes which lead to sympathetic neuronal differentiation (1,2). Recently it has been shown that NGF-induced differentiation of PC12 pheochromocytoma cells is associated with increased intracellular concentrations of annexin II (AnII) (3) an d increased expression of mRNA coding for the protein pll (4). pll is structurally related to the S-100 proteins, especially to S-100/3, a protein that is particularly abundant in neural tissue (5). An11 belongs to a large family of calcium- and phospholipidbinding proteins (6,7). An11 interacts with pll to form a heterotetrameric complex which is composed of two 36-kDa An11 molecules linked by their amino-terminal domains to a dimer of the ll-kDa pll protein (8). pll may be important for An11 activity, because it is in the heterotetramer form that An11 associates most readily with membrane phospholipids and with cytoskeletal actin (9,lO). Numerous functions have been proposed for the annexins (7,11,12,13), but none is as yet generally accepted. A role for pll/AnII in NGF-induced differentiation is an attractive hypothesis, especially in view of the data of Masiakowski and Shooter (14) showing that transfection of PC12 cells with pll cDNA results in constitutive NGF, nerve growth factor; AnI& annexin II; EGF, epidermal growth factor; PMSF, phenylmethylsulfonylflouride; EDTA, ethylenediamine-tetraacetic acid; SDS, sodium dodecylsulfate; TBST, Tris-buffered saline containing 0.5% Tween 20.
ABBREVIATIONS:
0006-291X/93 Copyright All rights
$1.50 Q 1991 by Academic Press. of reproduction in any form
1~. reserved.
1188
Vol.
177,
No.
3, 1991
neurite outgrowth differentiation
BIOCHEMICAL
in PC12 cells.
AND
Moreover,
BIOPHYSICAL
RESEARCH
Talian and Zelenka
COMMUNICATIONS
(15) have shown
that
of chicken lens epithelium is associated with an increase in AnII.
In this study, NGF and staurosporine
(a potent protein kinase inhibitor)
stimulated a
sustained increase in the concentrations of pll and An11 in PC12 cells. Both factors also induced neurite outgrowth, as previously reported (16,17). Epidermal growth factor (EGF), a factor which does not induce morphological
differentiation
of PC12 cells (18), stimulated
an early increase in pll and AnII, but the maximal levels attained in response to EGF were substantially lower than were those stimulated in response to either NGF or staurosporine. Taken together, these results suggest that a sustained increase in the pll/AnII heterotetramer is important in the differentiation of PC12 cells, and that pll/AnII constitute one part of the signal transduction mechanism for NGF.
MKI’EIUALS
may
AND METHODS
PC12 Cell Culture. PC12 adrenal pheochromocytoma cells were urchased from the American Type Culture Collection (No. CRL 1721), and grown in fx PM1 1640 medium supplemented with 10% (v/v) heat-inactivated horse serum (HS; Sigma Chemical Co., St. Louis, MO), 5% (v/v) fetal bovine serum (CC Laboratories, Cleveland, OH), 100 units penicillin/ml, and 100 pg streptom cm/ml. The cells were maintained in a humidified atmos here of COdair (1:19) at 37” E . For differentiation studies, the medium was replaced with ii PM1 1640 containing 1% HS. Murine submaxillary 2.5s NGF (100 @ml) (Boehringer Mannheim, Indianapolis, IN), staurosporine 70 nM) (Sigma), and recombinant murine EGF (10 @ml) (a generous gift of Pittman- L oore Inc., Terre aute, IN) were used to stimulate differentiation. In 10-d neurite-outgrowth studies, 2 x 18 cells were plated into wells of 6-well lates coated with Type I rat-tail collagen (Collaborative Research, Inc., Bedford, MA). I5very second day, 50% of the medium was replaced with fresh medium containing the test agents. Protein Extraction. Proteins associated with the cytoskeleton were extracted us&g the method of Zokas and Glenney (11). Briefly, 2 x lo7 cells were washed in Ca*+/Mg +-free phosphate-buffered saline and extracted for 2 min on ice in 200 ,ul of soluble protein buffer [lo mM imidazole (pH 7.3), 75 mM KCl, 2 mM MgClJ, 0.5 mM CaC12, 1 mM NaN-j, 0.5% Triton X-100, and protease inhibitors (1 pg epstatm A/ml, 0.5 pg leupeptin/ml, 52 pg aprotininlml, 0.1 mM benzamidine and 1 m IJ PMSF)]. The mixture was centrifuged at 12,000 x g for 15 set in a microfuge and the supernatant fraction was removed. The pellets were washed with 100 ~1 of soluble protein buffer. The remaining cytoskeleton/membrane pellet was suspended in 300 ~1 of cytoskeletal protein buffer (2 mM EDTA, 0.5% SDS and the protease inhibitors indicated above), disrupted using 3, 3-set power bursts (20% of maximum) with a Branson Sonifier 250 microprobe (Branson Ultrasonics Corp., Danbury, CT), and incubated for 5 min at 23OC. The mixtures were centrifuged at 12,000 x g for 10 min in a microfuge, and the supernatant fractions were analyzed for the presence of pll and AnII. Electrophoresis and Immunostaining. For analysis of pll, samples (60 pg protein) were boiled for 10 min in the presence of 2-mercaptoethanol and subjected to electrophoresis on polyacrylamide slab gels (15.5%) prepared with 6 M urea and Tris/Tricine buffers as described by Schagger and von Jagow (19). Separated proteins were electrophoretically transferred to nitrocellulose paper which was then incubated for one hour at room tern erature with 3% gelatin in Tris-buffered saline (pH 8.0) containing 0.5% Tween 20 (TB!T). Blots were probed with an anti-rat pl 1 (1: 100 dilution in TBST). The anti-rat pll was generated in rabbits using a synthetic peptide (Multiple Peptide Systems, San Diego, CA) corresponding to amino-acid residues 17-29 of rat pll. Alkaline phosphatase-coupled goat anti-rabbit I G (Promega Corp., Madison, WI) was used as the secondary antibody. Protein A Sigma lj wats$dinated according to the lactoperoxidase method of Thorell and I (NEN Research Products, Boston, MA). [1311]Protein A having Johanson ( 1 0) using 1189
Vol.
177,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
an average specific activity of 18 @i&g was used to label immune complexes, followed by color development using NBT/BCIP substrate (Promega). Radioactive bands were quantified directly using an automated radioanalytic imaging system (AMBIS Systems, San Diego, CA). An11 was analyzed by separation of boiled and reduced sample proteins (40 pg protein) on 12% SDS-polyacrylamide gels using the Laemmli method (21). Immunoblots were prepared as described for pll detection except for the substitution of mouse anti-An11 (Zymed Laboratories, Inc., South San Francisco, CA) and alkaline phosphatase-labeled goat anti-mouse IgG (Promega) for the primary antibody and secondary antibodies. The immunoreactive protein bands were quantified using a Gilford Model 2600 Multimedia Densitometer (Gilford Instrument Laboratories, Oberlin, OH) in the reflectance mode.
RESULTS
The levels of pll and An11 proteins in the cytoskeletal and soluble fractions of PC12 cell extracts were measured using immunoblot analysis. pll and An11 were readily detected in the cytoskeletal fraction, but these proteins were at the limits of detection in the soluble fraction. Accordingly, only the rest&s obtained from analyses of the cytoskeletal fractions are presented. Treatment of PC12 cells with NGF resulted in elevated pll levels by 4 h and a maximal five-fold increase by 12 h (Fig. 1A). Adherent PC12 cells stimulated with NGF in long-term culture on collagen-coated dishes [Fig. 2, solid bar) had pll levels similar to those detected in 24 h suspension cultures. The increase in An11 followed a time-course similar to that of pll, but An11 levels increased nine-fold by 12 h, with a maximum 13.6fold increase in 48 h (Fig. 1B). An11 levels were not measured in cells from long-term cultures. Staurosporine also stimulated a maximum five-fold increase in cytoskeletal pll (Fig. 3A), with a 7.6fold increase in An11 over the same time period (Fig. 3B). Treatment of PC12 cells with EGF resulted in only a two-fold increase in pll protein in cytoskeletal extracts (Fig. 4A). This increase was maintained over the 24 h experimental period, but maximum levels never reached those measured in NGF- or staurosporine-treated cells. The same
A
B -I -wm.r-~rroe11K
_*
3
-36
K
FIGURE 1. Time course of nerve arowth factor treatment of PC12 cells. Cells were cultured in the presence of 1OOng NGF/ml for 0, 4, 6, 8, 12 or 24h. Cytoskeletal proteins were extracted and electrophoresed. Separated proteins were probed using rabbtt anti-rat pll (A), or mouse anti-An11 (B). The procedure used was described in the text. These results are representative of three independent experiments. NDAn11 was not detectable in the 0 h sample.
1190
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FIGURE 2. Increase in ~11 protein in resuonseto lone-termculture of PC12cells with nerve growth factor and euidermal erowth factor. Cells were cultured in collagen-coated wellsfor 10d in the absenceof testagents CON), in the presenceof nerve growth factor NGF, 100@ml) or in the presenceo6 epidermalgrowth factor (EGF; 10 @ml). \ mmunostainingusing rabbit anti-rat wa~~;!g$;; descrrbedin the text. Autoradiographywasperformedusinglpodak intensifying screensat -70%. Autoradiographswere quantified using an LKB Ultroscan XL Densitometer. Values (mean f SEM) are from three separate measurements.
was true for Au11 protein levels in EGF-treated cells (maximum three-fold increase) (Fig. 4~). In long-term PC12 cell cultures, pll levels remained lower in EGF-treated cells than in NGF-treated cells (Fig. 2).
DISCUSSION NGF was the first growth factor to be described (22), and much is known about its physiological actions (23). Its mechanism of action, however, has yet to be defined. The PC12 pheochromocytoma cell line is widely used as a model for NGF-stimulated differentiation (1,2). NGF induces neurite outgrowth and other markers of differentiation in PC12 cells.
A
Hours
Hours
FIGURE 3. Time course of staurosoorine treatment of PC12 cells. Cells were cultured in the presence of 7OnM staurosporine for 0, 4, 6,8, 12 or 24h. Se arated proteins were probed using rabbit anti-rat 11 (A), or mouse anti-An11 (B5 . The procedure used was described in the text. A ese results are representative of three Independent experiments.
1191
Vol.
177,
No.
BIOCHEMICAL
3, 1991
AND
A
BlOPHYSlCAL
RESEARCH
COMMUNICATIONS
B "II -11
I.9
i:r..Ilr
CL 04
6
Hours
0
12
24
z
6
8
12
24
Hours
FIGIJRE 4. Time courseof eddermal growth factor treatment of PC12cells. Cells were cultured in the presenceof 10ng EGF/ml for 0,4,6, 8, 12or 24h. Se arated proteinswere probed usingrabbit anti-rat 11 (A), or mouseanti-An11(BP. The procedureusedwasdescribedin the text. Theseresultsare re resentativeof three Independentexperiments.*No 0 h samplewasavailablefor An PI analysis.
The extension of neurites in PC12 cells occurs no earlier than 2 d after exposure to NGF. As shown in this study and in earlier studies (4,3) neurite outgrowth is preceded by a substantial increase in pll
and An11 (Fig. 1A &B ). A similar increase in pll
precedes staurosporine-induced stimulation of neurite outgrowth (Fig. 3A&B).
and An11
Accordingly
pll and An11 may play an important role in the induction of differentiation in PC12 cells. This possibility is supported by the data of Masiakowski and Shooter (14) showing that PC12 cells transfected with pll cDNA display neurite outgrowth in the absence of NGF, and is consistent with the finding of Talian and Zelenka (15) that An11 levels increase during the differentiation of chicken lens epithelium. Moreover, PC12 cells treated with NGF or staurosporine in suspensionculture show marked and sustained increases in these proteins over the first 24 h of stimulation, before the onset of neurire outgrowth. EGF, which does not stimulate neurite outgrowth, induces sustained increasesin pll and An11 levels (Fig. 4A & B ), but EGF is much less effective than NGF or staurosporine in this regard. The sustainedincrease in pll protein is somewhat surprising becauseMasiakowski and Shooter (14) reported that EGF does not induce a sustained increase in pll mRNA. In view of the fact that NGF and staurosporine induce a larger increase in pll protein, a key factor in differentiation may be the induction of critical levels of pl 1 and AnII. The exact role of pll/AnII in differentiation is unclear at this time. However, these proteins are in a position to transmit signals through their interactions with membrane phospholipids and cytoskeletal elements. Indeed, it has been shown that the high affinity form of the NGF receptor which is essential for the differentiative response is associated with the cytoskeleton (25). Therefore it is possible that some of the actions of NGF are mediated through changes in the cytoskeleton, and that pll/AnII changes.
plays a role in such
ACKNOWLEDGMENTS: We thank Pitman-Moore, Inc. for providing recombinant murine EGF. This work was supported by the National Institutes of Health grant DK41440. 1192
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REFERENCES Greene, L.A. and Shooter, E.M. 1980 Annu. Rev. Neurosci. 3,353-402. Yanker, B.A. and Shooter, E.M. t 1982 1 Annu. Rev. Biochem. 5 1,845868. Schlaepfer, D.D. and Haigler, H.T. 1990 J. Cell Biol. 111,229-238. Masiakowski, P. and Shooter, E.M. I 1988 I Proc. Natl. Acad. Sci. USA 85, 1277-1281. Kligman, D. and Hilt, D.C. (1988) Trends Biochem. Sci. 13,437-443. Crompton, M.R., Moss, S.E., and Crumpton, M.J. (1988) Cell 55, l-3. Geisow, M.J., Fritsche, U., Hexham, J.M., Dash, B. and Johnson, T. (1986) Nature 320,636-638. Gerke, V. (1990) In Novel Calcium-Bindine Proteins (C. Heizmann, ed.), S ringer-Verlag, New York, In press. 2 erke, V. and Weber, K. (1985) J. Biol. Chem. 260,1688-1695. Glenne , J.R., Jr. (1986) Proc. Natl. Acad. Sci. USA 83,4258-4262. Zokas, E ., and Glenney, J.R. (1987) J. Cell Biol. 105,2111-2121. y;$ ler, H.T., Schlaepfer, D.D., and Burgess, W.H. (1987) J. Biol. Chem. 262, 69218 Pepihsky, R.B., and Sinclair, L.K. (1986) Nature (London) 321,81-84. Masiakowski, P. and Shooter, E.M. (1990) J. Neurosci. Res. 27,264-269. Talian, J. and Zelenka, P. (1991) Devel. Biol. 143,68-77. Greene, L.A. and Tischler, A.S. (1976) Proc. Natl. Acad. Sci. USA 73,2424-2428. Hashimoto, S. and Hagino, A. (1989) J. Neurochem. 53,1675-1685. Connolly, J.L., Green, S.A., and Green, L.A. 1984) J. Cell Biol. 98,457-465. Schiigger, H. and von Jagow, G. 1987 Anal. L iochem. 166,368-379. Thorell, J.I. and Johanson, B.G. t 1971 1 Biochim. Bio hys. Acta 251,363-369. Laemmli, U.K. (1970) Nature (London) 227,680~68 P. Levi-Montalcini, R. and Angeletti, P.U. (1968) Physiol. Rev. 48,534-569. Thoenen, H. and Barde, Y.-A. (1980) Physiol. Rev. 60, 1284-1327. Leonard, D.G.B., Ziff, E.B., and Greene, L.A. (1987) Mol. Cell. Biol. 7,3156-3167. Schechter, A.L. and Bothwell, M.A. (1981) Cell 24,867-874.
1193
177,
No.
June
28.
1991
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
II PROTEINS CORRELATE INCREASES IN 11 AND ANNEXIN DIFFEREN F IATION IN THE PC12 PHEOCHROMOCYTOMA
1188-1193
WITH
Mary T. Fox, David A. Prentice, and James P. Hughes Department Received
April
of Life Sciences, Indiana State University, Terre Haute, IN 47809 29,
1991
Regulation of pll and annexin II by nerve growth factor, staurosporine, and epidermal growth factor was examined in PC12 rat adrenal pheochromocytoma cells using immunoblot analysis. Nerve growth factor, which is known to induce neurite outgrowth in PC12 cells, stimulated a five-fold increase in pll and the higher levels of pll were characteristic of PC12 cells exposed to nerve rowth factor for u to ten days. Nerve growth factor induced an even greater increase (13. %-fold) in annexin PI. Staurosporine, a protein kinase inhibitor that at high concentrations induces neurite formation, was as effective as nerve growth factor in increasing the intracellular levels of pll and annexin II. Epidermal growth factor was less effective than nerve growth factor and staurosporine, producing only a two-fold increase in pll and a three-fold increase in annexin II. The ineffectiveness of epidermal growth factor in increasing intracellular levels of pll and annexin II is consistent with the fact that epidermal growth factor does not stimulate neurite outgrowth in PC12 cells. Evidence presented here suggests that pll and/or annexin II may play a role in PC12 cell differentiation. Q 1991Academic Pres, Inc. SUMMARY.
Nerve growth factor (NGF) binds to specific receptors on the surface of target cells and stimulates a wide variety of the changes which lead to sympathetic neuronal differentiation (1,2). Recently it has been shown that NGF-induced differentiation of PC12 pheochromocytoma cells is associated with increased intracellular concentrations of annexin II (AnII) (3) an d increased expression of mRNA coding for the protein pll (4). pll is structurally related to the S-100 proteins, especially to S-100/3, a protein that is particularly abundant in neural tissue (5). An11 belongs to a large family of calcium- and phospholipidbinding proteins (6,7). An11 interacts with pll to form a heterotetrameric complex which is composed of two 36-kDa An11 molecules linked by their amino-terminal domains to a dimer of the ll-kDa pll protein (8). pll may be important for An11 activity, because it is in the heterotetramer form that An11 associates most readily with membrane phospholipids and with cytoskeletal actin (9,lO). Numerous functions have been proposed for the annexins (7,11,12,13), but none is as yet generally accepted. A role for pll/AnII in NGF-induced differentiation is an attractive hypothesis, especially in view of the data of Masiakowski and Shooter (14) showing that transfection of PC12 cells with pll cDNA results in constitutive NGF, nerve growth factor; AnI& annexin II; EGF, epidermal growth factor; PMSF, phenylmethylsulfonylflouride; EDTA, ethylenediamine-tetraacetic acid; SDS, sodium dodecylsulfate; TBST, Tris-buffered saline containing 0.5% Tween 20.
ABBREVIATIONS:
0006-291X/93 Copyright All rights
$1.50 Q 1991 by Academic Press. of reproduction in any form
1~. reserved.
1188
Vol.
177,
No.
3, 1991
neurite outgrowth differentiation
BIOCHEMICAL
in PC12 cells.
AND
Moreover,
BIOPHYSICAL
RESEARCH
Talian and Zelenka
COMMUNICATIONS
(15) have shown
that
of chicken lens epithelium is associated with an increase in AnII.
In this study, NGF and staurosporine
(a potent protein kinase inhibitor)
stimulated a
sustained increase in the concentrations of pll and An11 in PC12 cells. Both factors also induced neurite outgrowth, as previously reported (16,17). Epidermal growth factor (EGF), a factor which does not induce morphological
differentiation
of PC12 cells (18), stimulated
an early increase in pll and AnII, but the maximal levels attained in response to EGF were substantially lower than were those stimulated in response to either NGF or staurosporine. Taken together, these results suggest that a sustained increase in the pll/AnII heterotetramer is important in the differentiation of PC12 cells, and that pll/AnII constitute one part of the signal transduction mechanism for NGF.
MKI’EIUALS
may
AND METHODS
PC12 Cell Culture. PC12 adrenal pheochromocytoma cells were urchased from the American Type Culture Collection (No. CRL 1721), and grown in fx PM1 1640 medium supplemented with 10% (v/v) heat-inactivated horse serum (HS; Sigma Chemical Co., St. Louis, MO), 5% (v/v) fetal bovine serum (CC Laboratories, Cleveland, OH), 100 units penicillin/ml, and 100 pg streptom cm/ml. The cells were maintained in a humidified atmos here of COdair (1:19) at 37” E . For differentiation studies, the medium was replaced with ii PM1 1640 containing 1% HS. Murine submaxillary 2.5s NGF (100 @ml) (Boehringer Mannheim, Indianapolis, IN), staurosporine 70 nM) (Sigma), and recombinant murine EGF (10 @ml) (a generous gift of Pittman- L oore Inc., Terre aute, IN) were used to stimulate differentiation. In 10-d neurite-outgrowth studies, 2 x 18 cells were plated into wells of 6-well lates coated with Type I rat-tail collagen (Collaborative Research, Inc., Bedford, MA). I5very second day, 50% of the medium was replaced with fresh medium containing the test agents. Protein Extraction. Proteins associated with the cytoskeleton were extracted us&g the method of Zokas and Glenney (11). Briefly, 2 x lo7 cells were washed in Ca*+/Mg +-free phosphate-buffered saline and extracted for 2 min on ice in 200 ,ul of soluble protein buffer [lo mM imidazole (pH 7.3), 75 mM KCl, 2 mM MgClJ, 0.5 mM CaC12, 1 mM NaN-j, 0.5% Triton X-100, and protease inhibitors (1 pg epstatm A/ml, 0.5 pg leupeptin/ml, 52 pg aprotininlml, 0.1 mM benzamidine and 1 m IJ PMSF)]. The mixture was centrifuged at 12,000 x g for 15 set in a microfuge and the supernatant fraction was removed. The pellets were washed with 100 ~1 of soluble protein buffer. The remaining cytoskeleton/membrane pellet was suspended in 300 ~1 of cytoskeletal protein buffer (2 mM EDTA, 0.5% SDS and the protease inhibitors indicated above), disrupted using 3, 3-set power bursts (20% of maximum) with a Branson Sonifier 250 microprobe (Branson Ultrasonics Corp., Danbury, CT), and incubated for 5 min at 23OC. The mixtures were centrifuged at 12,000 x g for 10 min in a microfuge, and the supernatant fractions were analyzed for the presence of pll and AnII. Electrophoresis and Immunostaining. For analysis of pll, samples (60 pg protein) were boiled for 10 min in the presence of 2-mercaptoethanol and subjected to electrophoresis on polyacrylamide slab gels (15.5%) prepared with 6 M urea and Tris/Tricine buffers as described by Schagger and von Jagow (19). Separated proteins were electrophoretically transferred to nitrocellulose paper which was then incubated for one hour at room tern erature with 3% gelatin in Tris-buffered saline (pH 8.0) containing 0.5% Tween 20 (TB!T). Blots were probed with an anti-rat pl 1 (1: 100 dilution in TBST). The anti-rat pll was generated in rabbits using a synthetic peptide (Multiple Peptide Systems, San Diego, CA) corresponding to amino-acid residues 17-29 of rat pll. Alkaline phosphatase-coupled goat anti-rabbit I G (Promega Corp., Madison, WI) was used as the secondary antibody. Protein A Sigma lj wats$dinated according to the lactoperoxidase method of Thorell and I (NEN Research Products, Boston, MA). [1311]Protein A having Johanson ( 1 0) using 1189
Vol.
177,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
an average specific activity of 18 @i&g was used to label immune complexes, followed by color development using NBT/BCIP substrate (Promega). Radioactive bands were quantified directly using an automated radioanalytic imaging system (AMBIS Systems, San Diego, CA). An11 was analyzed by separation of boiled and reduced sample proteins (40 pg protein) on 12% SDS-polyacrylamide gels using the Laemmli method (21). Immunoblots were prepared as described for pll detection except for the substitution of mouse anti-An11 (Zymed Laboratories, Inc., South San Francisco, CA) and alkaline phosphatase-labeled goat anti-mouse IgG (Promega) for the primary antibody and secondary antibodies. The immunoreactive protein bands were quantified using a Gilford Model 2600 Multimedia Densitometer (Gilford Instrument Laboratories, Oberlin, OH) in the reflectance mode.
RESULTS
The levels of pll and An11 proteins in the cytoskeletal and soluble fractions of PC12 cell extracts were measured using immunoblot analysis. pll and An11 were readily detected in the cytoskeletal fraction, but these proteins were at the limits of detection in the soluble fraction. Accordingly, only the rest&s obtained from analyses of the cytoskeletal fractions are presented. Treatment of PC12 cells with NGF resulted in elevated pll levels by 4 h and a maximal five-fold increase by 12 h (Fig. 1A). Adherent PC12 cells stimulated with NGF in long-term culture on collagen-coated dishes [Fig. 2, solid bar) had pll levels similar to those detected in 24 h suspension cultures. The increase in An11 followed a time-course similar to that of pll, but An11 levels increased nine-fold by 12 h, with a maximum 13.6fold increase in 48 h (Fig. 1B). An11 levels were not measured in cells from long-term cultures. Staurosporine also stimulated a maximum five-fold increase in cytoskeletal pll (Fig. 3A), with a 7.6fold increase in An11 over the same time period (Fig. 3B). Treatment of PC12 cells with EGF resulted in only a two-fold increase in pll protein in cytoskeletal extracts (Fig. 4A). This increase was maintained over the 24 h experimental period, but maximum levels never reached those measured in NGF- or staurosporine-treated cells. The same
A
B -I -wm.r-~rroe11K
_*
3
-36
K
FIGURE 1. Time course of nerve arowth factor treatment of PC12 cells. Cells were cultured in the presence of 1OOng NGF/ml for 0, 4, 6, 8, 12 or 24h. Cytoskeletal proteins were extracted and electrophoresed. Separated proteins were probed using rabbtt anti-rat pll (A), or mouse anti-An11 (B). The procedure used was described in the text. These results are representative of three independent experiments. NDAn11 was not detectable in the 0 h sample.
1190
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No.
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AND
BIOPHYSICAL
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COMMUNICATIONS
FIGURE 2. Increase in ~11 protein in resuonseto lone-termculture of PC12cells with nerve growth factor and euidermal erowth factor. Cells were cultured in collagen-coated wellsfor 10d in the absenceof testagents CON), in the presenceof nerve growth factor NGF, 100@ml) or in the presenceo6 epidermalgrowth factor (EGF; 10 @ml). \ mmunostainingusing rabbit anti-rat wa~~;!g$;; descrrbedin the text. Autoradiographywasperformedusinglpodak intensifying screensat -70%. Autoradiographswere quantified using an LKB Ultroscan XL Densitometer. Values (mean f SEM) are from three separate measurements.
was true for Au11 protein levels in EGF-treated cells (maximum three-fold increase) (Fig. 4~). In long-term PC12 cell cultures, pll levels remained lower in EGF-treated cells than in NGF-treated cells (Fig. 2).
DISCUSSION NGF was the first growth factor to be described (22), and much is known about its physiological actions (23). Its mechanism of action, however, has yet to be defined. The PC12 pheochromocytoma cell line is widely used as a model for NGF-stimulated differentiation (1,2). NGF induces neurite outgrowth and other markers of differentiation in PC12 cells.
A
Hours
Hours
FIGURE 3. Time course of staurosoorine treatment of PC12 cells. Cells were cultured in the presence of 7OnM staurosporine for 0, 4, 6,8, 12 or 24h. Se arated proteins were probed using rabbit anti-rat 11 (A), or mouse anti-An11 (B5 . The procedure used was described in the text. A ese results are representative of three Independent experiments.
1191
Vol.
177,
No.
BIOCHEMICAL
3, 1991
AND
A
BlOPHYSlCAL
RESEARCH
COMMUNICATIONS
B "II -11
I.9
i:r..Ilr
CL 04
6
Hours
0
12
24
z
6
8
12
24
Hours
FIGIJRE 4. Time courseof eddermal growth factor treatment of PC12cells. Cells were cultured in the presenceof 10ng EGF/ml for 0,4,6, 8, 12or 24h. Se arated proteinswere probed usingrabbit anti-rat 11 (A), or mouseanti-An11(BP. The procedureusedwasdescribedin the text. Theseresultsare re resentativeof three Independentexperiments.*No 0 h samplewasavailablefor An PI analysis.
The extension of neurites in PC12 cells occurs no earlier than 2 d after exposure to NGF. As shown in this study and in earlier studies (4,3) neurite outgrowth is preceded by a substantial increase in pll
and An11 (Fig. 1A &B ). A similar increase in pll
precedes staurosporine-induced stimulation of neurite outgrowth (Fig. 3A&B).
and An11
Accordingly
pll and An11 may play an important role in the induction of differentiation in PC12 cells. This possibility is supported by the data of Masiakowski and Shooter (14) showing that PC12 cells transfected with pll cDNA display neurite outgrowth in the absence of NGF, and is consistent with the finding of Talian and Zelenka (15) that An11 levels increase during the differentiation of chicken lens epithelium. Moreover, PC12 cells treated with NGF or staurosporine in suspensionculture show marked and sustained increases in these proteins over the first 24 h of stimulation, before the onset of neurire outgrowth. EGF, which does not stimulate neurite outgrowth, induces sustained increasesin pll and An11 levels (Fig. 4A & B ), but EGF is much less effective than NGF or staurosporine in this regard. The sustainedincrease in pll protein is somewhat surprising becauseMasiakowski and Shooter (14) reported that EGF does not induce a sustained increase in pll mRNA. In view of the fact that NGF and staurosporine induce a larger increase in pll protein, a key factor in differentiation may be the induction of critical levels of pl 1 and AnII. The exact role of pll/AnII in differentiation is unclear at this time. However, these proteins are in a position to transmit signals through their interactions with membrane phospholipids and cytoskeletal elements. Indeed, it has been shown that the high affinity form of the NGF receptor which is essential for the differentiative response is associated with the cytoskeleton (25). Therefore it is possible that some of the actions of NGF are mediated through changes in the cytoskeleton, and that pll/AnII changes.
plays a role in such
ACKNOWLEDGMENTS: We thank Pitman-Moore, Inc. for providing recombinant murine EGF. This work was supported by the National Institutes of Health grant DK41440. 1192
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