THE JOURNAL (c) 1992 by The American Society for Biochemistry and Molecular Biology,

OF BIOLOGICAL CHEMISTRY Inc.

Vol. 261, No. Issue of September 5, pp. 17611-17616,1992 Printed in U. S. A.

Retinoic Acid Regulates Both Expression of the Nerve Growth Factor Receptor and Sensitivity 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 Haruard Medical School, Boston, Massachusetts 02115

In PC12 cells, retinoic acid (RA) stimulates the expression of p75NGFR, a component of the nerve growth factor (NGF) receptor, as indicated by a rapid increase in p75NGFR mRNA, an increase in the binding of '251-labeled NGF 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-myristate 13-acetate, showing that RA has a specific effect on the respon- siveness 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 affinity receptors, suggest- ing 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 differ- entiation of neurons by modifying their expression of cell surface receptors to peptide growth factors.

Retinoic acid (RA),' a derivative of retinol (vitamin A) promotes differentiation of human neuroblastoma cells ( l ) , mouse teratocarcinoma cells (2), melanoma cells (21, and human promyelocytic leukemia cells (3). RA also effects the vertebrate limb development (4, 5) and has been implicated in a number of congenital malformations including brain deformities (6). RA can transform anterior neural tissue to posterior neural tissue (7) and can induce both nerve growth factor (NGF)-dependent survival and the expression of high affinity NGF receptors on immature sympathetic neurons (8). Four high-affinity nuclear RA receptors which belong to the family of genes encoding for nuclear steroid hormone recep- tors (3, 9-14) and two high-affinity cytoplasmic RA-binding proteins (10, 15, 16) have been identified. The RA receptors and binding proteins are expressed in neural tissues (1, 12, 17-19).

~

* This work was supported in part by National Institutes of Health Grants CA 40929 and CA 22427. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accord- ance 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 Harvard Medical School, Boston, MA 02115.

§ Current address: Dept. of Neurology and Neuroscience and Dept. of Cell Biology and Anatomy, Cornel1 University Medical School, 1300 York Ave., New York, NY 10021. Tel.: 212-746-6586; Fax: 212- 746-6577. To whom reprint requests and correspondence should be addressed.

The abbreviations used are: RA, retinoic acid; EDAC, ethyldi- methylaminopropyl carbodiimide; NGF, nerve growth factor; NGFR, nerve growth factor receptor; PMA, phorbol 12-myristate 13-acetate; SDS, sodium dodecyl sulfate; FGF, fibroblast growth factor.

Another important regulator of differentiation in the nerv- ous 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 ) (22,24- 26). The molecular differences between the low- and high- affinity receptors are not understood. A cDNA clone of the p75NGFR, a component of the NGFR, has been isolated (27- 30). The ~ 7 5 ~ " ~ ~ is a glycopolypeptide (observed molecular weight of 75,000) composed of approximately 400 amino acid residues containing an extracellular NGF-binding domain (31), a membrane domain, and a cytoplasmic domain. The analysis of the high-affinity receptors and its relation to the ~ 7 5 ~ " ~ ~ has been studied so far by ligand-receptor cross- linking studies. There is evidence that p7fjNGFR can be con- verted 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, NGF elicits tyrosine phosphorylation of the trk protein and stimulates its tyrosine kinase activity in PC12 cells (37, 38), leading to phosphorylation of other proteins on tyrosine. Co-expression of low-affinity receptor enhances pro- tein tyrosine phosphorylation (39). Thus, both ~ 7 5 ~ " ~ ~ and t rk are likely to be important components of the NGF signal transduction pathway.

PC12 is a clonal line derived from a rat pheochromocytoma (23, 40). PC12 cell lines respond to NGF, acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF), by extending long nerve-like processes and express- ing a number of markers of neural differentiation (41, 42). Moreover, PC12 cells deficient in protein kinase A differen- tiate in response to RA (43). Thus, neural differentiation of PC12 cells is regulated by both NGF and RA, suggesting that the influence of these two molecules must be integrated. To investigate the interactions between the NGF- and RA-de- pendent signaling pathways, we have examined the effects of RA on the expression of the NGF receptor.

EXPERIMENTAL PROCEDURES

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-retinoic acid 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 neuro- blastoma cells (46) and the SH-SY5Y cells, a subclone of the SK-N- SH cells were a gift of J. R. Perez-Polo (University of Texas Medical

17611

17612 Retinoic Acid Regulates NGF Receptors

Branch, Galvaston, TX). The culture medium was Dulbecco's modi- fied 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 PM 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 method of Chi rp in e t al. (47). The RNA was size fractionated on 1.2% agarose-formaldehyde gels and transferred to nitrocellulose filter. The cDNAs were labeled with [R'P]dCTP using a random hexamer as a primer (48). Fikers were 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 same solution containing 1-5 X 10" cpm/ml of labeled DNA 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 inten- sifying screens at -TO "C.

NCF Receptor-binding Analysis and Cross-linkinx of NCF to p7Fj.v';FR-For Scatchard analysis, cultures were seeded a t an 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 intact cells (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 varied from 5 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-labeled NGF 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 ""I- labeled NGF for 2 h a t 4 "C. NGF was cross-linked using ethyldime- thylaminopropyl 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 cells 3 times with NGF-binding buffer. The cells were lysed by resuspending them in lysis buffer containing 100 mM Tris, 1% Non- idet P-40, 66 mM EDTA, 1 mM leupeptin, 1 mM aprotonin, 1 mM phenylmethylsulfonyl fluoride. 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 auto- radiographed.

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 differentiation by regulating the expression of signal transduction systems that are responsive to peptide hormones. We chose 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 about 2- fold 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 addition of RA (Fig. 1, additional data not shown). Interestingly, RA did not increase the expression of the human trk proto-oncogene mRNA within the time frame of the experiment (Fig. 1).

Induction of ~75' ' ' '~~ Gene Expression Reflects Transcrip- tional Activation-RA is known to directly regulate the tran- scription of a number of genes such as the homeobox gene ERA-I/Hoxl.G (50,51) and the RAR-P gene (14, 52) and the induction of these early response genes is rapid and inde-

A.

R A ( h r ) 0 3 12 24 48

t r k * . - - 3.1 kb

p75NGFR rc)oO - 2.8 kb

tubulin lkLpyl

0 .

0 6 1 2 18 2 4 3 0 3 6 4 2 4 8

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 of RA (10"' M ) and fractionated on a 1.2% 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 determined by densitometry scanning followed 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 0 3 6 8 8

R A + + + + + + + + -

Cycloheximide - - - - + + + + +

P75NGFR

tubulin

FIG. 2. Effect of cycloheximide on the expression of ~75""~' . PC12 cells were exposed to RA (lo-" M ) for 0, 3, 6, or 8 h, 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 as described in the legend to Fig. 1. Morpho- logical signs of toxicity were apparent only 24 h after cycloheximide addition.

pendent of protein synthesis. 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 induction of 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) or 8 h (Fig. 2).

RA may regulate the abundance of ~ 7 5 " " ~ " mRNA either by effecting transcription or by influencing mRNA stability. To help determine the mechanism by which RA induced ~ 7 5 " " ~ " mRNA, the mRNA half-life was measured in cells

Retinoic Acid Regulates NGF Receptors 17613

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 had been exposed to RA (Fig. 3, A and B ) .

RA Increases the Concentration of NGF Receptor on the Cell Surface-To determine whether RA increases the binding of NGF to PC12 cells, we performed Scatchard plot analysis of the binding of 12sI-labeled NGF to control PC12 cells and cells treated with RA. The binding studies were conducted at 4 "C to 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 low- affinity receptors (& = 1.2 * 0.1 X lo-' M; Fig. 4). Pretreat- ment with RA M) resulted in an approximate 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 in low- affinity 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 the increase in the expres- sion of the mRNA for ~ 7 5 ~ " ~ ~ . The failure to observe any

6 hr

"" + + + +

0 1 2 4 0 1 2 4

. -

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 amount of 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.

0.5 1 a 0.4 -I \

R A

control

0.0 t

0.0 2.5 5.0 7 .5 10.0 12.5 15.0

bound fmoles FIG. 4. Scatchard plot of specific NGF binding to RA-

treated PC12 cells. PC12 cells were incubated in the presence and absence of RA M). Three days later the 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-labeled NGF 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 for 3 days; lane 3, PC12 cells treated with M RA for 3 days. Exposure, 24 h a t -70 "C.

increase in the number of high-affinity sites for 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 low- affinity 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 and the low-affinity NGF receptor (53). EDAC does not efficiently cross-link NGF to the high-affinity receptor. Ex- posure of PC12 cells to RA for 3 days results 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.

As an alternative approach to determining if RA influenced the expression of the p75N"FR, we studied the effect of RA on the neuroblastoma lines LA-N-1, which expresses both low- and high-affinity NGF receptors, and SH-SY5Y, which ex-

17614 Retinoic Acid Regulates NGF Receptors

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 sug- gestion that RA can increase NGF binding by 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 NGF is 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). To 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 an increased sensitivity to NGF.

This increase in sensitivity to NGF could be a reflection of

TABLE I Effects of RA on total 12'ZI-NGF binding in PC12, SH-SYSY,

and LA-N-1 cells Summary of 3 different experiments performed a t 4 "C as described

under "Experimental Procedures." Nonspecific binding, which was less than 10% of total binding, has been subtracted from all values.

Cell line Specifically bound '"1-NGF

None RA cprnllO6 cells

PC12 9,749 k 143 28,731 f 124 LA-N-1 6,268 f 53 SH-SY5Y

17,245 f 138 505 f 64 498 & 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 that induce immediate early genes. To test this idea we measured the sensitivity of the cells to FGF and PMA. FGF is a growth factor that acts through a cell surface receptor to induce immediate early gene expression as well as neural differentiation (41, 42), while PMA directly induces 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 gener- alized increase in the sensitivity of the cells to agents that induce gene expression. This supports the idea that the in- crease in ~ 7 5 ~ " ~ ~ seen after exposure to RA is responsible for the increased sensitivity of these cells to NGF.

DISCUSSION

We have demonstrated that RA induces the expression of the p75NGFR, but not trk, and increases the sensitivity of PC12 cells to NGF. The simplest interpretation of these observa- tions is that an increase in the expression of ~ 7 5 ~ " ~ ~ is sufficient to make the cells 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 sensitivity of 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 the basis of their affinities (22), but the molecular composition of these receptors and the specific roles played by the low- and high-affinity receptors are not yet completely understood (see Ref. 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

FIG. 6. Induction of egr-I and c- fos expression by NGF. PC12 cells 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 primer- labeled probes used in the hybridizations were egr-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 to that of ru-tubulin mRNA. Densitome- try results are expressed in relative RNA units.

A

a E - 30

,0001 ,001 .01 1 1 1 0 1 0 0

NGF (nglnll)

B

20

10

0 0001 ,001 .01 .1 1 1 0 1 0 0

'1 20 -

R* r conl ro l

I .

.01 .1 1 0 100

FGF (nglml)

'1 20-

10 -

0 I 1 1 1 0 1 0 0

NGF (nglml) PYA (nu)

Retinoic Acid Regulates NGF Receptors 17615

have suggested that trk alone may constitute the high-affinity receptor (37). Our data supports the observation that the expression of p7?jNGFR, even if it is not absolutely required for the biological effects of NGF, can exert an important modu- latory effect on the potency of NGF. Thus, the expression of ~ 7 5 ~ " ~ ~ which can be increased by RA, results in an increased sensitivity to NGF as measured by induction of the immediate early genes or as measured by enhancement of protein tyro- sine phosphorylation (39).

Although the low-affinity receptor appears to be important for regulating protein tyrosine kinase activity and induction of immediate early genes, blockade of 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 binding of NGF to the low- and high-affinity NGF receptors may regulate different aspects of the response cells to NGF ( i e . induction of immediate early genes and stimulation of neurite forma- tion, respectively). Consistent with this hypothesis, we have not been able to demonstrate any increase in the potency of NGF in regulating morphological differentiation in RA- treated 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 syn- thesis. 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 pres- ence of cross-hybridizing species or instability of the tran- scription 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 control of p75NGFR expression by RA require studies of the promoter elements of this gene. On the basis of the study of reporter constructs between the promoter of the ~ 7 . 5 ~ ' ~ ~ and chloramphenicol acetyltransfer- ase (66), we have tentatively concluded that the RA 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 morpholog- ical differentiation (55, 67). We have not observed any in- crease in the potency of NGF in stimulating morphological differentiation after exposure to RA (data not shown), sup- porting the idea that the levels of these immediate early genes are not rate-limiting for 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. I t 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 mechanisms used by RA to mod- ulate the response of the cells to NGF. The responses of both PC12 cells and immature chick sympathetic neurons to RA

(8) suggest that it will be important to consider both the direct effects of RA on gene expression and the ability of RA to modulate the responsiveness of neurons to endogenous 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- sions and the gift of ~ 7 5 ~ ' ~ ~ promoter constructs. We appreciate the 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.

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32. 33. 34. 35.

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