posterior neural tissue (7) and can induce both nerve growth factor (NGF)-dependent ..... measured the expression of egr-1 mRNA (56-60) and c-fos. mRNA (55).
Vol. 261, No.
OF BIOLOGICAL CHEMISTRY (c)1992 by The American Society for Biochemistry and Molecular Biology, Inc.
THEJOURNAL
Issue of September 5, pp. 17611-17616,1992 Printed in U.S.A.
Retinoic Acid Regulates Both Expressionof the Nerve Growth Factor Receptor andSensitivity to Nerve Growth Factor* (Received for publication, January 21, 1992)
Renate J. ScheibeS and John A. Wagner5 From the Department of Biological Chemistry and Molecular Pharmacology, Dana-Farber Cancer Institute and HaruardMedical School, Boston, Massachusetts 02115
Another important regulator of differentiation in the nervous system is the peptide growth factor NGF (for reviews, see Refs. 20 and 21). Two classes of NGF receptors (NGFR) have been identified (22, 23). High affinity receptors ( K d = lo-'' M ) are present at a few thousand sites per cell. There are as many as 150,000 low affinity receptors(& of IO-' M ) (22,2426). The molecular differences between the low- and highaffinity receptors are not understood. A cDNA clone of the p75NGFR, acomponent of the NGFR, has been isolated (2730). The ~ 7 5 is~a glycopolypeptide " ~ ~ (observed molecular weight of 75,000) composed of approximately 400 amino acid residues containinganextracellularNGF-bindingdomain '(31), ~ a ~membrane domain, and a cytoplasmic domain. The analysis of the high-affinity receptors and its relation to the so far by ligand-receptorcross~ 7 5 has ~ "been ~ studied ~ linking studies. There is evidence that p7fjNGFR can be converted to high-affinity receptors (24, 32-35). Moreover, the product of the trk proto-oncogene is also a component of the NGF receptor (36, 37). It is not yet clear, however, whether trk expression is sufficient for high-affinity NGF binding (38, 39). Furthermore, NGFelicits tyrosine phosphorylationof the Retinoicacid(RA),'a derivative of retinol(vitamin A) trk protein and stimulates its tyrosine kinase activity in PC12 promotes differentiation of human neuroblastoma cells ( l ) , cells (37, 38), leading to phosphorylationof other proteins on mouse teratocarcinoma cells (2), melanoma cells (21, and tyrosine. Co-expression of low-affinity receptorenhances prohuman promyelocytic leukemia cells (3). RA also effects the tein tyrosine phosphorylation (39). Thus, both ~ 7 5 and ~ " ~ ~ vertebrate limb development (4, 5) and has been implicated trk are likely to be important components of the NGF signal in a number of congenitalmalformations including brain transduction pathway. deformities (6). RA can transform anterior neural tissue to PC12 is a clonal line derived from arat pheochromocytoma posterior neural tissue (7) and can induce both nerve growth (23, 40). PC12 cell lines respond to NGF, acidic fibroblast factor (NGF)-dependent survival and theexpression of high growth factor(aFGF),and basic fibroblastgrowthfactor affinity NGFreceptors on immature sympathetic neurons(8). (bFGF), by extending long nerve-like processes and expressFour high-affinity nuclear RA receptors which belong to the ing a number of markers of neural differentiation (41, 42). family of genes encoding for nuclear steroid hormone recep- Moreover, PC12 cells deficient in protein kinase A differentors (3, 9-14) and two high-affinity cytoplasmic RA-binding tiate in response to RA (43). Thus, neural differentiation of proteins (10, 15, 16) have been identified. The RA receptors PC12 cells is regulated by both NGF andRA, suggesting that and binding proteins are expressed in neural tissues (1, 12, the influence of these two molecules must be integrated. To 17-19). investigate the interactions between the NGF- and RA-dependent signaling pathways, we have examined the effects of * This work was supported in partby National Institutesof Health RA on the expression of the NGF receptor. Grants CA 40929 and CA 22427. The costs of publication of this
In PC12cells, retinoic acid (RA) stimulates the expression of p75NGFR, a component ofthenerve growth factor (NGF) receptor, as indicated by a rapid increase in p75NGFR mRNA, an increase in the binding of '251-labeledNGF to p75NGFR, and an increase in the binding of NGF to low affinity sites.RA-treated cells are more sensitive to NGF, but not to either fibroblast growth factor or phorbol 12-myristate13-acetate, showing that RA has a specific effect on the responsiveness of PC12 cells to NGF. Exposure to RA leads neither to an increase in the expression of mRNA for trk, another component of the NGF receptor, nor to an increase in binding to high affinityreceptors, suggesting that an increase in the expression of ~ 7 5 is~ sufficient to make cells more sensitive to NGF. This work suggests that,in addition to having direct effects on gene expression,RA can indirectly modulate differentiation of neurons by modifying their expression of cell surface receptors to peptide growth factors.
~
article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by the Deutsche Forschungsgemeinschaft (Sche 309/ 1 - 2 ) during part of this work. Current address: Dept. of Medicine, Beth Israel Hospital and HarvardMedical School,Boston, MA 02115. ยง Current address: Dept. of Neurology and Neuroscience and Dept. of Cell Biology and Anatomy, Cornel1 University MedicalSchool, 1300 York Ave., New York, NY 10021. Tel.: 212-746-6586; Fax: 212746-6577. To whom reprint requests and correspondence should be addressed. The abbreviations used are: RA,retinoicacid;EDAC, ethyldimethylaminopropyl carbodiimide; NGF, nerve growth factor; NGFR, nerve growth factor receptor; PMA, phorbol 12-myristate 13-acetate; SDS, sodium dodecyl sulfate; FGF, fibroblastgrowth factor.
EXPERIMENTALPROCEDURES
Materials-[["'PIdCTP (3000 Ci/mmol = 370 MBq/ml) and [""PI CTP (300 Ci/ml = 370 MBq/ml) were obtained from Du Pont-New England Nuclear. Ribonucleotide triphosphates were from Promega (Madison, WI). Poly-D-lysine ( M , 130,000) and all-trans-retinoicacid were obtained from Sigma. 2.5 S NGF (PNGF) was purified from mouse submaxilary gland as described (44). '"1-Labeled PNGF was obtained from Amersham (1500 Ci/mmol). Cell Culture-PC12 cells (45) were cultured a t 37 "C in 10% CO, in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 5% horse serum. PC12 cells were plated directly on untreated tissue culture plastic (Falcon). LA-N-1 human neuroblastoma cells (46) and the SH-SY5Ycells, a subclone of the SK-NS H cells were a gift of J. R. Perez-Polo (Universityof Texas Medical
17611
17612
Regulates Acid Retinoic
Branch, Galvaston, TX). The culturemedium was Dulbecco's modified Eagle's medium containing 10% fetal calf serum. LA-N-1 cells were plated on poly-D-lysine-coated (50 pg/ml) dishes and SH-SY5Y cells were cultured on untreated tissue culture dishes. Medium was changed every 2-3 days and cells were passaged once/week. RNA Isolation and Analysis-For RNA isolation, cells were plated in 150-mm dishes. The medium was changed 2 days after the cells were seeded and 100 ng/ml NGF or 1 P M RA was added. Media and supplements were added as described in the figure legends. The cell density used for Northern (RNA) blot analysis was approximately lo',' cells/l50-mm dish. Total cellular RNA was purified from PC12 cells, according to the methodof C h i r p i n e t al. (47). The RNA was size fractionated on 1.2% agarose-formaldehyde gels and transferred to nitrocellulosefilter. ThecDNAs werelabeledwith [R'P]dCTP using a random hexamer as a primer (48). Fikerswere prehybridized at 42 "C for at least 5 h in a solution containing 50%formamide, 5 X SSC (1 X SSC = 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7 ) , 0.02% polyvinylpyrrolidone,0.02% Ficoll, 0.02% bovine serum albumin, 1%sodium dodecyl sulfate (SDS), and 100 pg/rnl denatured calf thymus DNA. Hybridization was performed for 12 h a t 42 "C in the samesolutioncontaining 1-5 X 10" cpm/ml of labeledDNA probes. Blots were washed with 0.2 X SSC and 0.5% SDS at 65 "C twice, each for 30 min. Autoradiography was performed with intensifying screensat -TO "C. NCF Receptor-binding Analysis and Cross-linkinx of NCF to p7Fj.v';FR-For Scatchard analysis, cultures were seeded a t a n initial density of 1 X 10" cells/ml in 150-mm dishes, and treated with RA (IO-" M). Three days later, hinding of ""I-labeled NGF (3 PM to 3 nM) to intactcells (0.5 X 10" cells/ml) was measured using the sucrose sedimentation protocol (49). Nonspecific binding of 1Y511-labeled NGF was determined by the addition of 1 mM unlabeled NGF. The levels of nonspecific binding variedfrom5 to 25% of total binding. All determinations were done in triplicates and data are presented as mean -C S.E. T o cross-link iZ'II-labeledNGF to the low-affinity receptor, PC12 cells were removed from the dishes 72 h after treatment with either NGF (100 ng/ml) or RA(10"' M) using phosphate-buffered saline containing 1 mM EDTA, pelleted by centrifugation (2,000 rpm for 7 min a t room temperature), and resuspended in 4 ml of NGF binding huffer containing phosphate-buffered saline, 1 mg/ml glucose, and 1 mg/ml bovine serum alhumin. Cells were incubated in 0.5 nM ""Ilabeled NGF for 2 h a t 4 "C. NGF was cross-linked using ethyldimethylaminopropyl carbodiimide (EDAC; Pierce) a t a concentration of 90 mg with 1.5 ml of phosphate-buffered saline for 15 min a t room temperature. Unbound ""I-labeled NGF was removed by washing the cells3 times with NGF-binding buffer. The cellswere lysed by resuspending them in lysis buffer containing 100 mM Tris, 1% Nonidet P-40, 66 mM EDTA, 1 mM leupeptin, 1 mM aprotonin, 1 mM phenylmethylsulfonylfluoride.Insoluble material was removed by centrifugation a t 1,500 rpm for 7 min, and the supernatant fraction was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiographed.
NGF Receptors A. RA
(hr)
trk
0 3 12 24 48
.
*
rc)oO
p75NGFR
tubulin
-
-
3.1 kb
-
2.8 kb
lkLpyl
0.
0
6
12
18
24
30
36
4482
time(h)
FIG. 1. Northern blot analysis of the ~ 7 5 ~ and " ~ trk ' genes. A, total cellular RNA (30 pg) was isolated at the indicated times after the addition ofRA(10"' M ) and fractionated ona1.2%agarose/ formaldehyde gel. The RNA was transferred to nitrocellulose and probed with a random primer-labeled cDNA probe for p75s""', trh. or n-tubulin (2 X 10" cpm/ml, 1 X 10" cpm/ml, and 1 X 10'' cpm/ml, respectively). H , the relative mRNA levels are expressed in arbitrary units determinedby densitometry scanningfollowed hy normalization of the p7SS';"' and trh mRNA level to the n-tubulin mRNA level for each sample. The experiment was performed 3 times; the result is expressed in relative RNA units. Time Course (hr)
0 3 6 8
RA
+ + + + + + + + -
Cycloheximide
-
- -
0 3 6 88
- + + + + +
P75NGFR
RESULTS
RA Induces the Expression of the NGF Receptor-RA may have a direct effect on the expression of neural markers, but it could also influence neural differentiationby regulating the expression of signal transduction systems that are responsive t o peptidehormones.Wechose to determine whether RA might influence the responsiveness of PC12 cells to NGF by modulating the expression of the NGF receptors. By 3 h, RA ( M) increased the expression of ~'75""~'' mRNA about2fold over the basal level, and by 12 h the expression of the receptor increased slightly more than 3-fold over the basal level. In time course studies we found that the mRNA level remained elevated for more than 3 days after the additionof RA did RA (Fig. 1,additional data not shown). Interestingly, not increase the expression of the human trkproto-oncogene mRNA within the time frameof the experiment (Fig. 1). Induction of ~75''''~~Gene Expression Reflects Transcriptional Activation-RA is known to directly regulate the transcription of a number of genes such as the homeobox gene ERA-I/Hoxl.G (50,51) and theRAR-P gene (14, 52) and the induction of these early response genes is rapid and inde-
tubulin
FIG. 2. Effectofcycloheximide on the expression of cells were exposed to RA (lo-" M ) for 0, 3, 6, or 8 h, ~ 7 5 " " ~ 'PC12 . in the presence and ahsence of cycloheximide (10 pg/ml), or exposed to cycloheximide alone. Total RNA was isolated and examined by Northern blot analysis asdescribed in the legend to Fig. 1. Morphological signs of toxicity were apparent only 24 h after cycloheximide addition.
pendent of proteinsynthesis.The induction of ~ 7 5 " " ~ ~ mRNA also occurred normally in the presence of sufficient cycloheximide to decrease protein synthesis more than 90% (Fig. 2), indicating that inductionof the ~ 7 5 gene ~ "is also ~ ~ independent of concurrent protein synthesis. Cycloheximide alone does not significantly increase the expression of the ~ 7 5 " ~ "gene at either 3 (data not shown) or8 h (Fig. 2). RA may regulate the abundance of ~ 7 5 " " ~ mRNA " either by effecting transcription or by influencing mRNA stability. To help determinethe mechanism by which RA induced ~ 7 5 " " ~ mRNA, " the mRNA half-life was measured in cells
Regulates Acid Retinoic that had been treated with RA for 8 h as well as in untreated PC12 cells. Actinomycin D (2 pg/ml) was added to prevent de novo synthesis of ~ 7 5 mRNA, ~ " ~and ~ the stability of the RNA was measured in both control and RA-treated PC12 cells. The half-life of the ~ 7 5 (4~ h)"was ~ not ~ significantly different in the presence and absence of RA, although the absolute level of the mRNA was higher in cells that hadbeen exposed to RA (Fig. 3, A and B ) . RA Increases the Concentrationof NGF Receptor on theCell Surface-To determine whether RA increases the binding of NGF to PC12 cells, we performed Scatchard plot analysisof the binding of 12sI-labeledNGF to control PC12 cells and cells treated with RA. The binding studieswere conducted at 4 "C t o minimize the effect of other biological phenomena,in particular, internalization of receptors. PC12 cells contained high-affinity receptors (& = 3.8 f 1.6 X lo-" M) and lowaffinity receptors (& = 1.2 0.1 X lo-' M; Fig. 4). Pretreatment with RA M) resulted inanapproximate 3-fold increase in the number of low-affinity binding sites(from 4.3 f 1.2 X lo4 to 12.6 f 1.8 X lo4 sites/cell; Fig. 4) without causing a significant change in the high-affinity sites (3.6 f 0.5 X lo3 to 3.6 f 0.4 X lo3 sites/cell; Fig. 4). Exposure of cells to RA for 24 h did not cause a significant change inlowaffinity binding (1.2 f 0.1 X lo-' to 1.5 f 0.2 X lo-' M) or high-affinity binding (3.8 f 1.6 x lo-" to 3.7 f 1.4 x lo-" M ) . Thus, there is an increase in the number of low-affinity sites for NGF that corresponds to theincrease in theexpression of the mRNA for ~ 7 5 ~ The " ~ failure ~ . to observe any
*
6 hr "
0
1
"
+ + + +
2
0
4
2
1
4 .
-
NGF Receptors
17613
0.5 1
0.4
a
-I\
RA
control 0.0
0.0
t
2.5
5.0
7.5
10.0
12.5
15.0
bound fmoles
FIG.4. Scatchard plot of specific NGF binding to RAtreated PC12 cells. PC12 cells were incubated in the presence and absence of RA M). Threedayslaterthe binding of "'I-labeled NGF (3 p~ to 3 nM) to cells (0.5 x lo6 cells/ml) was measured a t 4 "C for 90 min as described under "Experimental Procedures." r
N
0
-
220
-
110
-
70
FIG. 5. Affinity cross-linking of '251-labeledNGF to PC12 cells. PC12 cells (2 X 10'/ml) were incubated with 0.5 nM '2SI-labeled NGF for 2 h a t 4 "C and then cross-linked using EDAC. Lane I , untreated PC12 cells; lane 2, PC12 cells treated with 100 ng/ml NGF M RA for 3 days. for 3 days; lane 3, PC12cells treated with Exposure, 24 h a t -70 "C.
RA
time after Ihe addition of aclinomycin D (hr)
FIG.3. Effect of RA on the stability of ~ 7 5 mRNA. ~ ' ~PC12 ~ cells were cultured in either the presence (+) and absence (-) of RA M). After 8 h, actinomycin D (2 pg/ml) was added and RNA was isolated after 1, 2, or 4 h. Total RNA (30 pg) was fractionated, transferred to nitrocellulose, and hybridized with cDNA probe for ~ 7 5 ~(1' X; lo6 ~ ~cpm/ml). The exposure time was 4 days. The level of ~ 7 5 ~ ' ;mRNA ~' was determined by densitometry, normalized to the total amountof 28 S RNA, and plotted as a percentage of ~ 7 5 mRNA remaining for each time after addition of the transcription inhibitors. The experiment was repeated three times with similar results.
increase in the numberof high-affinity sitesfor NGF suggests that there was no increase in the expression of trk which is consistent with the lack of effect of RA on trk mRNA levels (Fig. 1). To confirm that RA increases the concentration of the lowaffinity NGF receptor, we cross-linked l"I-labeled NGF to the low-affinity NGF receptor using the hydrophilic reagent EDAC which generates a 75-kDa complex consisting of NGF andthe low-affinity NGF receptor (53). EDAC does not efficiently cross-link NGF to the high-affinity receptor. Exposure of PC12 cells to RA for 3daysresults in a %fold increase in the concentration of ~ 7 5 (Fig. ~ "5).~Thus ~ RA increases the expression of p75NGFR at both the mRNA and protein levels. if RA influenced ~ "As~an ~ alternative approach to determining the expression of the p75N"FR,we studied the effect of RA on the neuroblastoma lines LA-N-1, which expresses both lowand high-affinity NGF receptors, and SH-SY5Y, which ex-
Regulates NGF Receptors
Acid Retinoic
17614
presses only high-affinity receptors (54). In these experiments '"I-labeled NGF was present at a concentration sufficient to saturate binding to both low- and high-affinity sites. Under these conditions,exposure to RA produced a 2.9-fold increase in the binding of NGF to LA-N-1, but did not effect the binding of NGF to SH-SY5Y (Table I), supporting the suggestion that RA can increase NGF bindingby increasing the number of low-affinity receptors. RA Effects the Induction of Early Response Genes-One of the most rapid responses of PC12 cells to NGFis the induction of a number of genes (e.g. egr-IINGFlA, c-fos, NGFlB, etc.) whose expression is frequently considered to be important for subsequent steps in differentiation (55). T o determine whether the expression of these early response genes may be changed when cells were exposed to RA prior to NGF, we measured the expression of egr-1 mRNA (56-60) and c-fos mRNA (55). RA increased the potency of NGF in inducing egr-1 and c-fos by 8- and 3-fold, respectively (Fig. 6). Thus, the RA-induced increase in the expression of the ~ 7 5 is~ correlated with anincreased sensitivity to NGF. This increase in sensitivity to NGFcould be a reflection of TABLEI Effects of RA on total 12'ZI-NGFbinding inPC12, SH-SYSY, and LA-N-1 cells Summary of 3 differentexperiments performed a t 4 "C asdescribed under "Experimental Procedures." Nonspecific binding, which was less than 10%of total binding, has been subtracted from all values. Specifically bound '"1-NGF Cell line
RA
None cprnllO6 cells
PC12 LA-N-1
SH-SY5Y
9,749 k 143 6,268 f 53 505 f 64
28,731 f 124 17,245 f 138 498 & 87
the increase in the expression of p75NGFR, but it could also reflect a more general increase in the sensitivity of the cells to agents thatinduce immediate earlygenes. To test thisidea we measured the sensitivity of the cells to FGF and PMA. FGF isa growthfactor that acts through a cell surface receptor to induce immediate early gene expression as well as neural differentiation (41, 42), while PMA directlyinduces gene expression by activating protein kinase C. Exposure of PC12 cells to RA did not increase the sensitivity of PC12 cells to either FGF or PMA, showing that there has been no generalized increase in the sensitivity of the cells to agents that induce gene expression. This supports the idea that the increase in ~ 7 5 seen ~ after " ~ exposure ~ to RA is responsible for the increased sensitivityof these cells to NGF. DISCUSSION
We have demonstrated that RA induces the expression of the p75NGFR, but not trk, and increases the sensitivityof PC12 " ~ to~ NGF. The simplest interpretation of these observacells tionsisthatan increase in the expression of ~ 7 5 is~ " ~ ~ sufficient to make thecells more sensitive to NGF when the induction of immediate early genes is studied. Alternatively, it is possible that the increased sensitivity of PC12 cells to NGF results in an effect of RA on a different constituent of the signal transduction pathway. This alternative explanation is less likely because the sensitivityof cells to FGF and PMA does not change after RA exposure. The interaction of NGF with PC12 cells is mediated by two classes of receptors that can be distinguished on thebasis of their affinities (22), but the molecular composition of these receptors and thespecific roles played by the low- and high-affinity receptors are not yet completely understood (seeRef. 61 for a discussion). While some have suggested that expression of both ~ 7 5 and ~ trk " ~ ~ are required to form high-affinity receptors (38, 53), others
A
1'
a
-
E 30
20
R*
FIG.6. Induction of egr-I and cfos expression by NGF. PC12cells were exposed to RA M ) for 48 h. Cells were treated for 60 min a t 37 "C with increasing concentrations of NGF ( A and E),FGF (C), or PMA ( D ) .Total RNA (20 Kg) was isolated and Northern blot analysis performed as described in the legend to Fig 1. The random primerlabeled probes usedin the hybridizations B wereegr-1 ( A , C, and D ) or c-fos ( B ) cDNAs (1 X lo6 cpm/ml). The levels of egr-1 and c-fos mRNAs were normalized t o that of ru-tubulin mRNA. Densitometry results areexpressed in relative RNA units.
,0001 ,001
.01
1
1 1 0 01 0
I
r conlrol
.
.01
NGF (nglnll)
10
.1
100
FGF (nglml)
'1
20-
20
10
10
0
-
0 0001 ,001
.01
.1
NGF (nglml)
1
100 10
I
1
100
1
10
PYA
(nu)
17615
Retinoic Acid RegulatesNGF Receptors have suggested that trk alone may constitute thehigh-affinity receptor(37). Our data supports the observation that the expression of p7?jNGFR, even if it is notabsolutely required for the biological effects of NGF, can exert an importantmodulatory effect on the potencyof NGF. Thus, the expression of ~ 7 5 which ~ " can ~ be~ increased by RA, results ina n increased sensitivity to NGF as measured by induction of the immediate early genes or as measured by enhancement of protein tyrosine phosphorylation (39). Although the low-affinity receptor appears tobe important for regulating protein tyrosine kinase activity and induction of immediate early genes, blockadeof the low-affinity receptor with a specific antibody does not inhibit the morphological differentiation of either PC12 cells or primary dorsal root ganglion cultures to NGF (62, 63). Thus the bindingof NGF to the low- and high-affinity NGF receptorsmayregulate different aspects of the response cells to NGF ( i e . induction of immediate early genes and stimulation of neurite formation, respectively). Consistent with this hypothesis, we have not been able to demonstrate any increase in thepotency of NGFin regulatingmorphological differentiationin RAtreated PC12 cells (data not shown). Furthermore, induction of high-affinity NGF receptors in immature chick sympathetic neurons resulted in an increased morphological response to NGF (8). In some neuroblastoma cell lines, RA can induce the expression of both high- and low-affinity receptors for NGF (64) and increase the morphological response to NGF. The regulation of ~ 7 . 5 ~expression ' ~ ~ occurs rapidly after exposure to RA, and is independent of current protein synthesis. The induction of p7!jNGFR expression does not appear t o reflect a difference in mRNA stability. This supports the idea that there is a direct effect of RA on the expression of the p71jNGFR, an effect that is presumably mediated by the RARs. Increases in transcription were not demonstrated by nuclear run-on transcription assays, but,because of the presence of cross-hybridizing species or instability of the transcription complex, this type of assay does not always reveal a n effect on transcription. For example, the promoter of the gene ERA-1 contains RA responsive elements (65), but the transcriptional effects of RA are not clearly demonstrated in nuclear run-on transcription assays. Further analysis of the molecular mechanism of the controlof p75NGFR expression by RA require studies of the promoter elementsof this gene. On the basis of the study of reporter constructs between the ' ~chloramphenicol ~ acetyltransferpromoter of the ~ 7 . 5 ~and ase (66),we have tentatively concluded that theRA responsive sequence does not lie within the first 1.2 kilobases upstream of the transcription initiation site (data not shown). RA itself can induce the expression of c-fos (43),and exposure to RA increases the potency of NGF in inducing both c-fos and egr-1; but it has been difficult to determine whether induction of these genes is necessary for morphological differentiation (55, 67). We have not observed any increase in the potency of NGF in stimulating morphological differentiation after exposure to RA (data not shown), supporting theidea that thelevels of these immediate earlygenes are not rate-limitingfor neurite formation. Alternatively, the effects of RA may be limited to immediate early genes and not have an impact on downstream genes that are required for differentiation.It is possible that a metabolite of RA, and not RA itself, induces the expression of ~ 7 5 ~ ~ ~ The PC12 line is a useful system in which to study both the biochemical mechanism used by RA to regulate neural differentiation (43) and the mechanismsused by RA to modulate theresponse of the cells to NGF. Theresponses of both PC12 cells and immature chick sympathetic neurons to RA
(8)suggest that itwill be important toconsider both the direct effects ofRA on gene expression and the ability of RA to modulate theresponsiveness of neurons toendogenous growth factors, like NGF when evaluating the effects of RA on the regulation of neural development and differentiation. Acknowledgments-We thank Dr. Moses Chao for helpful discus~ ' ~ constructs. ~ We appreciate the sions and thegift of ~ 7 5 promoter assistance of Leni Kaplan in performing cross-linking experiments. W e thank Drs. David Ginty and Richard Asher for discussion and comments on the manuscript. REFERENCES 1. Haussler, M., Sidell, N., Kelly, M., Donaldson, C., Altman, A,, and Mangelsdorf, D. (1983) Proc. Natl. Acad. Sci. U. S. A 80, 5525-5529 2. Strickland, Smith, K. K., and Marotti, K. R. (1980) Cell 2 1 , 3 4 7 - 3 5 5 3. Breitman. T. R.. Selonick. S.. and Collins. S.J. (1980) Proc. Natl. Acad. S c t " 6 ~ S .A. 77, 2936-2940' 4. Thaller, C., and Eichele, G. (1987) Nature 327,625-628 5. Tickle, C., Alberts, B., Wolpert, L., and Lee, J. (1982) Nature 296, 564-
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