Identification and Characterization of a 3’,5’-Cyclic Adenosine Monophosphate-Responsive Element in the Human CorticotropinReleasing Hormone Gene Promoter

Dietmar Florian

Spengler, Holsbier

Rainer

Rupprecht,

Lot

Phi Van*,

and

Max Planck Institute of Psychiatry Clinical Institute Department of Neuroendocrinology 8000 Munich 40, Germany

The regulation of human corticotropin-releasing hormone (hCRH) gene promoter activity by inducers of CAMP was investigated by transient transfection with a construct containing the hCRH gene promoter fused to the chloramphenicol acetyltransferase gene. Expression of hCRH-chloramphenicol acetyltransferase was strongly enhanced by forskolin in the neuroblastoma SK-N-MC and choriocarcinoma JAR cell lines. Overexpression of the catalytic subunit of protein kinase A dispensed the need for forskolin, and cotransfection of CAMP-responsive element-binding protein cDNAs enhanced forskolindependent expression of the hCRH promoter. Progressive 5’-end deletions of the hCRH promoter delineated a CAMP- responsive region between -226 and -164 base pairs. This fragment contained the sequence TGACGTCA at -221 base pairs, consistent with the consensus motif for a CRE. A homologous oligonucleotide responded to CAMP when cloned in either orientation in front of the thymidine kinase promoter. However, the level of constitutive and inductive CAMP expression was dependent on the cell line and on intrinsic properties of the promoter. Mutation of the wild type CRH-CRE sequence into an AP-1 site (TGAGTCA) completely abolished stimulation by CAMP. In contrast, coexpression of the catalytic subunit of protein kinase A dispensed the need for stimulation with forskolin, which showed that the CRH-CRE oligonucleotide served as a functional equivalent of the native CRE element. (Molecular Endocrinology 6: 1931-1941, 1992)

cellular neurons of the nucleus paraventricularis of the hypothalamus. This main mediator of the mammalian stress response is released into the hypophyseal-portal system and enhances the synthesis and release of ACTH from the anterior pituitary, which in turn stimulates adrenal steroid synthesis and release. Recent animal behavioral research (2) and increasingly sophisticated clinical research (3) has provided evidence that CRH integrates hormonal, physiological, and behavioral responses to environmental and endogenous challenges. Despite the considerable amount of information available on neuronal afferents modulating CRH secretion (2, 4), only a few studies have addressed the molecular mechanisms of CRH gene promoter regulation (5-8). Evidence from in situ studies does, however, suggest a strong neuronal (2, 9, IO) and hormonal (11-13) impact on CRH mRNA synthesis. At the level of transcription, the expression of genes is mediated through the interactions of DNA-binding proteins with specific DNA regulatory elements that are generally located in the 5’-flanking regions of the genes upstream from the transcription start sites. In 1986, Montminy et al. (14) isolated the &-acting DNA element responsible for CAMP regulation of the somatostatin promoter. The identified palindromic motif (5’TGACGTCA-3’) was designated CAMP-responsive element (CRE). A breakthrough in the understanding of CAMP-regulated gene transcription resulted from the cloning of the cDNAs encoding the DNA-binding proteins for CREs. Such studies have identified a complex superfamily of transcriptional trans-activator proteins (15). These proteins become transcriptionally active when bound to the CRE element, and both their binding and transcriptional functions are modulated by phosphorylation, predominantly catalyzed by CAMP-dependent protein kinase A (PKA) (16). Several other cellular and viral genes have been reported to contain functional CRE sequences (17-l 9). In contrast, recent studies on

INTRODUCTION Corticotropin-releasing hormone (CRH) is a 41 -amino acid neuropeptide (1) that is synthesized in the parvo068%3809/92/1931-1941$03.00/0 Molecular Endocrinology Copyright Q 1992 by The Endocrine

Soclety

1931

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MOL 1932

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Vol6No.

1992

the human steroid 21 -hydroxylase gene (P-450c2,) (20) and on the mouse renin gene (21, 22) revealed that CAMP-dependent transcription depends on cis-regulatory elements that are distinct from the consensus motif for CREs. In addition, the &-acting element 5’TCCCCANGGG3’, which is present in the human metallothionein,,~-, GH, and the c-myc genes, binds the unrelated activator protein 2, whose transcriptional activity is modulated via PKA and PKC pathways (23). Thus, progress in the understanding of CAMP-dependent gene transcription has led to the identification of different &-acting DNA elements that provide transactivation by transcription factors belonging to unrelated families. We recently observed stimulation of the transfected human CRH (hCRH) gene promoter by CAMP in the mouse anterior pituitary cell line AtT 20 (8) and postulated a CRE motif at position -221 base pairs (bp). In agreement with this observation, Seasholtz et al. (7) reported stimulation of the rat CRH promoter by CAMP. However, the five or six bases immediately adjacent to the core sequence of a CRE motif can be either permissive or nonpermissive for transcriptional activation by CAMP (17). To date, the results on the regulation of the human (8) and rat (7) CRH promoter by CAMP provide no conclusive evidence for a functional CRE element. In the present study we isolated the &-acting DNA element responsible for the regulation of the hCRH promoter by CAMP. To demonstrate the functional significance of the presumed CRE motif we studied the properties of a homologous oligonucleotide in the context of different heterologous promoter systems. Mutation of this CRE sequence abrogated stimulation by CAMP. In contrast, coexpression of the catalytic subunit of PKA dispensed the need for stimulation with forskolin and cotransfection of CREB cDNAs enhanced forskolindependent stimulation of the reporter gene.

11

(27) and neuropeptides (28) although CRH gene expression has not been reported in this cell line. Treatment of transfected SK-N-MC cells with 1 mM 8-Bromo-CAMP (&Br-CAMP) or 0.5 mM 3-isobutyl-lmethyl-xanthine (IMX) (a phosphodiesterase inhibitor) revealed a 4-fold increase in CAT activity compared to controls, whereas simultaneous treatment with both drugs yielded an additive 8-fold enhancement (Fig. 1, bars l-4, left). Incubation with 25 PM forskolin, a postreceptor activator of adenylate cyclase, caused an even more pronounced (13-fold) induction in CAT activity (Fig. 1, bar 5). In contrast, the promoterless parent vector pBLCAT3 exhibited only a 1.6-fold increase in CAT activity in the presence of 25 PM forskolin (data not shown), demonstrating that the induction of CAT activity in cells transfected with CRH (-666)CAT is mediated via the hCRH promoter sequence. Consistent with this observation, in the choriocarcinoma-derived placenta cell line JAR we noted a 1 O-fold induction of the hCRH promoter after treatment with 1 mM 8-Br-CAMP, 0.5 mM, IMX or 25 PM forskolin (Fig. 1, bars l-5, middle). However, no increase in CAT activity was observed after simultaneous treatment with CAMP and IMX (Fig. 1, bar 4) indicating maximal stimulation by each individual compound. To obtain preliminary information on the molecular mechanism of CAMP-dependent regulation we transfected the hCRH promoter into the F9 (teratocarcinoma, mouse) cell line. F9 cells were chosen because they are largely unresponsive to CAMP in their undifferentiated state and possess low levels of endogenous CREbinding (CREB) proteins (16). In fact, treatment of transfected cells with 1 mM 8-Br-CAMP, 0.5 mM IMX, or both, did not influence hCRH promoter activity, whereas in-

RESULTS CAMP Regulation

of the hCRH Gene Promoter

We transfected the construct CRH(-666)chloramphenicol acetyltransferase (CAT), which contains the region -666 bp to +122 bp of the hCRH promoter, and the parent vector pBLCAT3 into the neuroblastomaderived SK-N-MC cell line and the choriocarcinomaderived JAR cell line. To investigate promoter activity in the presence and absence of regulators known to activate the CAMP second messenger system we chose the JAR cell line because we had previously observed high levels of CRH mRNA in this tissue (Spengler, D., R. Rupprecht, L. Phi Van, and F. Holsboer, submitted), which is in agreement with previous reports on CRH gene expression in primary cultures of cytotrophoblast placenta tissue (25, 26). In addition, we used the SKN-MC cell line as a well characterized neuronal model that expresses aminergic neurotransmitter systems

2 SK-N-MC

345 JAR

12345 F9

Fig. 1. Stimulation of hCRH Gene Promoter Activity by Regulators of CAMP SK-N-MC (neuroblastoma), JAR (choriocarcinoma), and F9 (teratocarcinoma) cells were transfected with 5 pg of the plasmid CRH(-666)CAT. Induction of CAT activity for the indicated treatments was referred to the basal expression of CRH(-666)CAT in each cell line. Electroporated cells were incubated for 24 h with: bar 1, no regulator; bar 2, 1 mM 8BrCAMP; bar 3, 0.5 mM IMX; bar 4, 1 mM 8-Br-CAMP + 0.5 mM IMX; and bar 5, 25 PM forskolin. Results represent the mean + SEM from four independent experiments expressed in terms of induction.

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hCRH Promoter Regulation by CAMP

1933

cubation with 2.5 PM forskolin caused a 2.5fold in this parameter (Fig. 1, bar 5, right).

increase

Regulation of hCRH Promoter Activity by Overexpression of PKA and CREB cDNAs The CAMP-dependent PKA regulates the expression of genes containing a CRE element by enhanced phosphorylation of CREB proteins, resulting in potentiated transcriptional activity. To evaluate the impact of PKA on CAMP-mediated induction of CRH(-666)CAT we cotransfected an expression vector (MtC) containing the coding sequence for the catalytic subunit of PKA under the control of the mouse metallothionein (Mt-1) promoter. Cotransfection of 5 pg and 10 pg MtC expression plasmid into the SK-N-MC cell line resulted in a significant elevation of CRH(-666)CAT activity compared to controls (4.5-fold and 5.4-fold, respectively; Fig. 2, bars 1, 3, and 5). Induction of the metallothionein promoter by 100 PM ZnCI, caused a further enhancement (8.6-fold and 9.6-fold) of CRH(-666)CAT activity (Fig. 2, bars 4 and 6). In contrast, in control experiments no change in basal CRH(-666)CAT expression was observed after cotransfection with 5 pg or 10 wg of the analog Mt-1 plasmid (data not shown). In addition, treatment of transfected cells with ZnCI, revealed no differences in basal or forskolinstimulated expression of CRH(-666)CAT compared to untreated controls (data not shown). Various proteins unrelated to the CREB family could serve as substrates for the catalytic subunit of PKA and thus mimic regulation via a CRE element. To rule out this possibility the impact of cotransfected CREB proteins on CRH(-666)CAT expression was studied (Fig. 3). Introduction of 2.5 pg or 5.0 Fg CREB-A or -B expression vector distinctly enhanced forskolin-de-

I

SENSE

FOR

+

1 Zn

++

+

I Zn

ANTiSENSE

Fig. 3. Regulation

of hCRH Gene Promoter Activity by CREB Expression Vectors SK-N-MC cells were transfected with 5 pg CRH(-666)CAT and treated with 25 PM forskolin (bars l-9). Cotransfection of 2.5 Kg (bars 2 and 3) or 5.0 pg (bars 4 and 5) CREB-A and -B cDNA expressed in sense orientation enhanced CRH(-666)CAT activity. In contrast, cotransfection of 2.5 pg (bars 6 and 7) or 5.0 kg (bars 8 and 9) CREB-A or-B cDNA in antisense orientation inhibited CRH(-666)CAT stimulation. Results represent the mean + SEM from four independent experiments expressed in terms of induction.

pendent CRH(-666)CAT activity (Fig. 1, bars l-5). The two isoforms of hCREB protein were equally effective in inducing the hCRH promoter. In control experiments no changes were observed in forskolin-stimulated hCRH promoter activity when 2.5 pg or 5.0 fig of the parent vector pSG5 were used (data not shown). Enhanced transcription of the hCRH promoter required simultaneous treatment with forskolin, because cotransfection with 2.5 gg or 5.0 fig CREB-A or -B expression vector alone did not stimulate the hCRH promoter (data not shown). To obtain further evidence for the regulation of hCRH promoter activity by CREB proteins, expression plasmids containing the CREB-A and -B cDNAs in antisense orientation were introduced into the SK-N-MC cell line. Cotransfection with 2.5 pg CREB-A or -B antisense expression vector revealed a significant reduction (1.7fold and 1.5-fold, respectively) in forskolin-stimulated CRH(-666)CAT activity (Fig. 3, bars 6 and 7) with a further decline (2.6-fold and 2.4-fold, respectively) when 5 pg of the antisense constructs were used (Fig. 3, bars 8 and 9). Localization

has

CREB

of the hCRH CRE

,

++

Fig. 2. Stimulation of hCRH Gene Promoter Activity by MtC SK-N-MC cells were transfected with 5 fig CRH(-666)CAT. Cotransfection of 5 kg or 10 wg of the plasmid MtC (bars 36) dispensed the need for stimulation by 25 FM forskolin (bar 2). Induction of the metallothionein promoter by 100 PM ZnCI, (bars 4 and 6) caused a further increase in CRH(-666)CAT expression compared with untreated controls (bars 3 and 5). Results represent the mean + SEM from four independent experiments expressed in terms of induction.

To delineate hCRH promoter regions required for regulation by CAMP, sequences from the 5’-end of the plasmid CRH(-666)CAT were progressively deleted. The set of constructs so obtained was transfected into the JAR and SK-N-MC cell lines. To avoid differences conferred by the length of the promoter fragment rather than by treatment with forskolin, we compared induction of each deletion construct with its corresponding basal value. These forskolin-mediated induction ratios (induced vs. noninduced CAT expression) for each 5’-

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MOL 1934

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end-deleted hCRH promoter plasmid are given in Fig. 4. In the JAR cell line, plasmids with the promoter end points -917, -666, -571, -295, -251, -245, and -226 bp clearly responded to forskolin. In addition, there was a gradual increase in forskolin induction ratios (11.6-fold to 15.9-fold) as the size of deletions in the hCRH promoter increased (Fig. 4). In contrast, plasmids with the promoter end points -163, -145, -55, and -24 bp were only slightly stimulated (2-fold) similar to the findings for the parent vector pBLCAT3 (Fig. 4). The 1.6-fold induction of the pBLCAT3 vector served as a negative control and was considered to be nonspecific. The above observations were extended by transfection of the SK-N-MC neuroblastoma cell line with the 5’-end-deleted hCRH promoter constructs (Fig. 4). Consistent with the findings for the JAR cell line, constructs with the truncation points -917, -666, -571, -285, -252, -245, and -226 bp revealed a strong induction with forskolin, about the same magnitude as for CRH(-666)CAT (Fig. 4). In contrast, we noted a less pronounced (2.5-fold) drop in the induction with forskolin of the plasmid CRH(-163)CAT compared to the 7.2-fold decrease in the JAR cell line. Therefore, additional binding sites for transcription factors modulated or induced by CAMP appear to reside between positions -143 and -55 of the hCRH promoter. However, no increase in induction with forskolin for the plasmids CRH(-163)CAT and CRH(-143)CAT was observed after cotransfection of 5 pg CREB-A or -B expression vector (data not shown). There were no significant differences in the basal levels of expression of CRH(-971)CAT and CRH(-163)CAT, whereas basal activity downstream of -143 bp appeared to be reduced. Further downstream deletions in the constructs CRH(-53)CAT and

Vo16No.11

CRH(-23)CAT skolin.

abolished

Transfer of CAMP-Dependent Heterologous Promoter

induction

Regulation

by for-

to a

To test whether sequences in the 5’-flanking region of the hCRH gene could confer CAMP responsiveness to a heterologous promoter we created several fusion constructs containing hCRH promoter DNA restriction fragments in front of the thymidine kinase (tk) promoter of the reporter pBLCAT2. A schematic diagram of these fusion constructs is given in Fig. 5 (left panel). We calculated basal expression of these heterologous constructs by comparison with the unmodified parent vector pBLCAT2, while referring induction by forskolin to the corresponding basal CAT values of each plasmid investigated. Interestingly, insertion of the depicted hCRH promoter fragments caused an enhanced basal expression of the transfected tk-CAT promoter in the JAR cell line (Fig. 5). This increase occurred in the absence of forskolin and was therefore taken to repreSent constitutive expression. Treatment with 25 KM forskolin, however, produced an additional increase in reporter activity, which was most pronounced for the construct CRH(-666/-36)CAT (4.8-fold). Hereafter, the difference between constitutive and forskolin-dependent induction is referred to as inductive expression. The parent vector pBLCAT2 was only slightly induced by forskolin compared with its untreated control. In contrast, cotransfection of these heterologous constructs into the SK-N-MC cell line revealed a different pattern of expression. In this cell line, enhancement of basal promoter activity was considerably reduced, whereas induction by 25 PM forskolin was more pronounced for all constructs tested (Fig. 5). Dual Enhancer Oligonucleotide

Fig. 4. Effect of Progressive 5’-End Deletions of the hCRH Promoter on Regulation by Forskolin JAR and SK-N-MC cells were transfected with 5 pg of each hCRH-CAT construct containing the indicated amount of 5’flanking DNA. Open boxes represent hCRH promoter sequences. The CRE motif at -221 bp is represented by black boxes. Induction by 25 @M forskolin for each deletion construct was referred to its basal expression. Results represent the mean + SEM from six independent experiments expressed in terms of induction.

completely

Activities

of the hCRH CRE

Inspection of the hCRH promoter fragment revealed the sequence 5’-TGACGTCA-3’ between -228 and -221 bp homologous with a CRE consensus motif (TGACGTCA). To provide evidence for the functional significance of this motif we designed oligonucleotides homologous to -238 to -216 bp and tested their properties in a heterologous promoter system. For this purpose we cloned single or multiple copies of this 27bp oligonucleotide [wild type CRE (wtCRE)] in either the forward [tk-CAT(wtCRE),_,,] or reverse [tkCAT(wtCRE),m3,,] orientation, in front of the tk promoter of pBLCAT2 (Fig. 6). We calculated constitutive enhancer function of these reporter plasmids by comparing CAT activity from untreated JAR cells with the parent vector pBLCAT2 (tk-CAT), whereas we referred induction by 25 PM forskolin to the basal CAT value of each construct tested. Insertion of a single wtCRE oligonucleotide in either the forward or reverse orientation increased basal expression of the tk promoter approximately lo-fold (Fig. 6). Addition of a second or third

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hCRH

Promoter

Regulation

by CAMP

1935

Fold Induction

in CAT Activity

JAR

tkCA T

CR/-/

(-917 / -36)

CRH

(-666

-36)

tkCAT

CRH

(-666 /-127)

tkCAT

CRH

(-332

(417, 1

I

CAT

-666)

/

SK-N-MC

constttutive

inductiv.9

consttutive

inductive

7.3 * 0.4

2.5 f 0.1

2.5 It 0.1

5.0 f 0.4

w6

w

I

f-666)

/- 126) tkCA T

CAT

13.7

* 0.5

4.8

CAT

10.8

f 0.4

CAT

8.5

f

&T]

tkCAT

1 .o

0.2

f

3.1 f0.3

15.3f0.9

4.2 f 0.2

2.4f0.2

11.8 f0.9

3.2*

2.1 f 0.1

8.7

1.0

2.5 * 0.1

0.3

0.2

1.1 fO.l

f

0.3

Fig. 5. Restriction Fragments of the hCRH Promoter Confer CAMP Responsiveness to the Heterologous tk Promoter Restriction fragments of the hCRH promoter were cloned in front of the heterologous tk promoter. The hCRH promoter sequences are represented by open boxes, and their truncation points are indicated in parentheses. The CRE motifs are shown as black boxes and the tk promoters as hatched boxes. JAR and SK-N-MC ceils were transfected with 5 pg of each hCRH-tk-CAT construct. Constitutive expression of the constructs was referred to the parent vector (tk-CAT), which was included in each transfection series. Induction by 25 PM forskolin was compared to the basal expression of each construct. Results represent the mean f SEM from four independent experiments expressed in terms of induction.

Fold

Induction

in CAT

Activity

JAR fk-CA T tk-CAT (wtCRE)

&-Z-J ,s

+

tk-CAT (wfCRE) 2s tk-CA T (wiCRE)

3s

fk-CA T (wtCRE)

, as

CAT

1

CAT

I-+I-,~ -bI+wI-,

+

tk-CAT (wtCRE) 2as fk-CAT (WtCRE) 3as

w

1 +

CAT

1 f-

inductive

1 .o

0.9 f 0.1

10.0

I!L 0.4

2.2 f 0.1

13.6f

1.5

2.1 + 0.2

24.0 f 2.6

2.6 k 0.2

8.7 ZiT0.2

2.7 + 0.2

CAT

+I+ 1 c-

constitu five

w

CAT

1

1.1

2.5 f 0.2

+ I .3

3.4 f 0.3

10.6f 14.4

Fig. 6. Oligonucleotides Homologous with the hCRH CRE Motif Confer Dual CAMP Responsiveness An oligonucleotide (wtCRE) homologous with the sequence -238 to -216 bp of the hCRH promoter was cloned multiple copies (open boxes) in forward or reverse orientation (arrows) in front of the tk promoter (hatched boxes). expression of these constructs was referred to the parent vector (tk-CAT). Expression by treatment with 25 PM compared with the basal CAT activity of each construct tested. Results represent the mean + SEM from four experiments expressed in terms of induction.

copy of the CRE oligonucleotide caused a further increase in basal activity, which tended to be more pronounced in the forward than the reverse orientation (24fold VS. 1Cfold) (Fig. 6). In contrast, inducible enhancer function of these oligonucleotides was much less de-

pendent on copy number increase in CAT induction, the forward orientation and the reverse orientation (Fig. Because previous authors

and from from 6). (17,

in single or Constitutive forskolin is independent

revealed a moderate 2.2-fold to 2.6-fold in 2.7-fold to 3.4-fold in 29) had reported

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that

MOL 1936

ENDO.

Vo16No.11

serted in forward orientation into the reporter plasmid AMTV-LUC (AMTV-LUC(mtCRE),-,,) and tested for basal and forskolin-induced luciferase activity. The mutated CRE motif almost completely abolished induction by elevated levels of CAMP in both the SK-N-MC (Fig. 8) and JAR (data not shown) cell lines and revealed a marked difference (about 13-fold) in induction between three copies of the wild type and the mutated CRE motif (Fig. 7). Basal levels of the AMTV-LUC reporter plasmids increased slightly for multiple copies of mtCRE in the SK-N-MC cell line (Fig. 8). Similar data were obtained for the JAR cell line under unstimulated conditions (data not shown). This enhancement of basal AMTV-LUC(mtCRE),_, promoter activity appeared to be unrelated to the constitutive enhancer function of the hCRH wtCRE element because it was not observed when transfected cells where grown in media supplemented with 1% fetal calf serum (Fig. 8, *).

inducible and constitutive enhancer activities of a CRE element depend on the promoter tested, the properties of the wtCRE oligonucleotide in the context of a second unrelated promoter were investigated by inserting single or multiple copies in forward orientation into a modified mammary mouse tumor virus promoter (AMTV). This promoter is devoid of the glucocorticoidresponsive region between -180 and -88 bp and drives the expression of the luciferase (LUC) reporter gene. We introduced the construct into the SK-N-MC and JAR cell lines and evaluated them for basal and forskolin-induced luciferase activity. We compared basal levels of expression with the parent AMTV-LUC vector and referred induced levels to the corresponding basal activity of each construct (Fig. 7). These experiments revealed a strong CAMP-inducible enhancer potential of the AMTV constructs in the SK-N-MC cell line, which was dependent on the copy number of the inserted wtCRE oligonucleotides (6.6-fold to 52.1 -fold) (Fig. 7) whereas basal promoter activity was not modulated. Consistent with this observation, in the JAR cell line we noted a predominant inducible enhancer activity of the AMTV-LUC(wtCRE)1-3 constructs, with a 36.2fold stimulation of luciferase activity for four copies of the hCRH wtCRE element. In contrast to the tk constructs, AMTV-LUC activity increased only moderately under basal conditions (2.1 -fold to 5.3-fold) in the JAR cell line (Fig. 7). Abrogation of CAMP Responsiveness of the CRE Motif

Regulation

of the hCRH CRE by the PKA Pathway

Treatment of transfected SK-N-MC cells with 25 PM forskolin produced a strong (29-fold) induction of the plasmid AMTV-LUC(wtCRE), (Fig. 9, bar 1). The specificity for stimulation by forskolin was underlined by replacement with the closely related analog l-deoxyforskolin, which did not substitute for forskolin (bar 2). In contrast, coexpression of the PKA catalytic subunit expression plasmid (MtC) successfully eliminated the requirement for forskolin (bars 3 and 4). In addition, cotransfection with 2.5 Fg or 5.0 fig CREB-A and -B cDNA expression vector caused an enhanced induction by forskolin (bars 6-l 0).

by Mutation

To provide a negative control experiment for regulation by CAMP we mutated the hCRH wtCRE oligonucleotide into a novel AP-1 site (TGAGTCA), which preferentially binds transcription factors of the fos/jun family. Single or multiple copies of mutated CRE (mtCRE) were in-

DISCUSSION CRH mRNA and immunoreactivity can be detected in many brain regions (2, 30, 31) which is consistent with

Fold Induction

in Luciferase

JAR constitutive

i-rso,

AMN-LUG

a-

AMTVLUC

(WCRE)

,

AMN-LUC

(WlCRE)

2

AMTV-LUC

(wtCRE)

3

AMTV-LUC

(wtCRE)

4

EfH+M

+I+

WM

+]+I+

wm

q;aJ

+[+I+I+

w

48

SK-N-MC

inductive

constttutive

LUC

1.o

LUC

2.1 f

LUC

3.1 f0.4

17.9io.2

1.5f

LUC

4.2 f 2.0

25.4

f 2.0

LUC

5.3 f 0.2

36.2

k 1.4

0.1

Activity

inductive

1.0 f 0.1

1.0

2.4f0.3

5.6 i0.3

1.4f0.3

6.6iO.2

0.4

27.9f

1.1

1.6 f0.4

37.4k

0.3

1.4*0.6

52.1

f 0.7

Fig. 7. Oligonucleotides Homologous with the hCRH CRE Motif Confer Inductive CAMP Responsiveness to the Heterologous MTV Promoter Single or multiple copies of the oligonucleotide wtCRE were cloned in forward orientation (arrows) into the modified heterologous AMTV promoter (hatched boxes). JAR or SK-N-MC cells were transfected with 5 pg of each construct, and basal expression was referred to the MTV-LUC promoter. Stimulation by 25 PM forskolin is compared with the basal level of expression for each construct. Results represent the mean & SEM from four independent experiments.

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hCRH

Promoter

Regulation

by CAMP

1937

wtCRE

5’- agctTAGGGCTCGTTGACGTCACCAAG

- 3’

mtCRE

5’- agctTAGGGCTCGTTGA

- 3’

GTCACCAAG

Fold Induction in Luciferase

Activity

SK-N-MC conslitutive

inductive

1.0

2.4 If: 0.3

LUC

0.9 f 0.2

2.6 k 0.1

LUC

2.6 f 0.4

2.5 + 0.1

LUC

7.3 f 2.3

1.9 + 0.4

1.3 * 0.3”

2.3 f 0.4’

AMTV-LUC C-9)

AMTV-LUC

(mtCRE),

AMTV-LUC

(mtCRE) 2

AMTV-LUC

(mtCRE)3

-

W-I -++I*

urn

wl-4

?x-)

1 ++N 1 -x-+ w

Fig. 8. Mutation

of the CRE Motif Abrogates Regulation by CAMP Sequences of the wtCRE and mtCRE are shown at the top of the figure. The diagram shows the inserted mtCRE oligonucleotides (open boxes) and their orientation (arrows) in the AMTV promoter (hatched boxes). Transfection of SK-N-MC cells was performed with 5 pg of each construct. Basal expression is referred to the AMTV-LUC promoter. Induction by 25 PM forskolin is compared with the basal level of each construct. The asterisk denotes basal and forskolin-dependent expression of AMTV-LUC(mtCRE)3 for low serum concentrations (1%). Results represent the mean f SEM from four independent experiments expressed in terms of induction.

Y

FOR

1 DEOXY FOR

’

+ Zn ++

,

,A

B

A

B ,

CREB

Fig. 9. Regulation

of the CRE Oligonucleotide by CAMP Proceeds by the PKA Pathway SK-N-MC cells were transfected with 5 Kg AMTVLUC(wtCRE)* (bars l-8). Treatment with 25 PM forskolin (bar 1) is compared with 25 FM 1-deoxy-forskolin (bar 2). Cotransfection of 5 pg MtC dispensed the need for stimulation by forskolin (bar 3) and increased further after stimulation of the metallothionein promoter with 100 PM ZnCI, (bar 4). Forskolinmediated activity was enhanced for cotransfection of 2.5 pg and 5 pg CREB-A or -B expression vector (bars 5-8). Results

represent the mean f SEM from four independent experiments expressed in terms of induction.

the important role CRH plays in linking the central nervous system and the adenohypophysial endocrine system (for review see Refs. 2 and 3). To study the molecular mechanism of CRH gene expression we investigated the regulation of the hCRH promoter in an in vitro transfection system. We recently observed stimulation of the intact hCRH gene promoter by CAMP in AtT 20 cells, an anterior pituitary cell line, and noted the existence of an 8-bp sequence (TGACGTCA) homologous with the concensus for a CAMP-responsive enhancer (8). Seasholtz et al. (7) reported similar results for stimulation of the transfected rat CRH promoter by forskolin in the pheochromocytoma-derived PC 12 cell line. Evidence that an intact PKA pathway is required for induction of the hCRH promoter by CAMP is provided in the present study by a strongly attenuated expression of the CRH(-666)CAT construct in the CAMP-unresponsive F9 cell line. In support of this finding, cotransfection of the catalytic subunit of PKA replaced forskolin-mediated stimulation in the SK-N-MC neuroblastoma cell line. Previous cotransfection studies using PKA expression plasmids demonstrated reduced (32) or comparable (33) levels of induction of CAMP-responsive reporter genes in comparison to stimulation by forskolin. However, in these studies the ratio of reporter to expression plasmid, the concentration of forskolin, and the employed cell lines were different from those used in the present investigation. Interestingly, we observed

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MOL 1938

ENDO.

1992

a 3-fold higher induction of the CRH promoter by the catalytic subunit when we expressed the cDNA from the strong cytomegalovirus promoter (data not shown). Thus we conclude that the metallothionein promoter provides only weak expression of the catalytic subunit of PKA in the cell lines used here. The results from cotransfection of a PKA plasmid and the hCRH promoter were strengthened by the observation that cotransfection of CREB-A or -B expression plasmids enhanced forskolin-mediated stimulation of CRH(-666)CAT, whereas cotransfection of the respective antisense constructs significantly inhibited forskolin induction. Transfection of a series of progressive 5’-end-deleted promoter constructs revealed an essential CAMP-responsive region between -226 and -143 bp, which exhibited a 7.2-fold and 2.5 fold difference in the forskolin induction ratio in the JAR and SK-N-MC cell lines, respectively. Our observation is consistent with previous findings by Montminy et al. (14) of a 4- to 5-fold decrease in the forskolin induction ratio for deletion of the CRE element of the somatostatin gene promoter. In the SK-N-MC cell line, additional binding sites for transcription factors modulated or induced by CAMP appear to reside between positions -143 and -55 of the hCRH promoter. However, no increase in forskolin induction of the plasmids CRH(-163)CAT and CRH(-143)CAT after cotransfection of 5 pg CREB-A or -B expression vector were observed. Consistent with this result, sequences downstream from -163 bp revealed no CRE motif homologies. This observation could indicate an additional cellspecific mechanism for CAMP-dependent regulation of the hCRH promoter, unrelated to the interaction between the CRE element and the CREB protein. Restriction fragments of various sizes of the hCRH gene promoter conferred CAMP responsiveness to the heterologous tk promoter and exhibited different levels of constitutive and inductive expression in the JAR and SK-N-MC cell lines. Therefore, our next step was to design an oligonucleotide homologous with the CRE motif and to test its functional properties in a heterologous promoter system. This wtCRE oligonucleotide conferred CAMP responsiveness to the tk promoter and promoted CAMP stimulation independent of its orientation, a property now considered a basic feature of transcriptional enhancer elements. Interestingly, wtCRE oligonucleotides inserted in front of the tk promoter closely mimicked the differential degree of constitutive and inductive expression of hCRH-tk constructs in the JAR cell line. However, single or multiple copies of the wtCRE oligonucleotide inserted into the unrelated AMTV promoter revealed a preferentially inductive expression in both the JAR and SK-N-MC cell lines. Consistent with previous reports (17, 29) our results suggest that the different levels of constitutive and inductive expression of the hCRH CRE element are predominantly determined by the intrinsic properties of the promoter employed. We therefore postulate that transcription factors binding to adjacent enhancer motifs in the tk promoter could interact with the CREB

Vol6No.

11

protein in its unphosphorylated state and provide an enhanced basal expression of the reporter plasmid. However, this putative interplay appears to interfere with transcriptional activation by PKA, indicating a dual role for CRE-binding proteins in the regulation of genes containing a CRE motif in their promoter region. Similarly, the phosphoenolpyruvate carboxykinase CRE (34) and CRE elements within several viral enhancers (35, 36) may be required for basal expression. In line with these observations, studies on the adenovirus E4 gene suggest that proteins binding to the CRE increase the binding of TATA factors to the promoter (37). In contrast to the findings on the CRE of the CG-ol glycoprotein hormone promoter (17) we observed no inhibitory influences on basal or stimulated activity for multiple copies in front of the tk or within the AMTV promoter. This could reflect differences either in the promoter systems employed or in the CREs tested. A clear consensus has not yet emerged for the minimum size of a CRE sufficient for enhancer activity. Previous reports (14, 17) demonstrated that the mere presence of a CRE core motif is not sufficient for full CAMP-responsive enhancer activity in front of a heterologous promoter. Thus, contextual sequences adjacent to the CRE are permissive for transcription (17). However, we observed no impairment of CAMP induction of the construct CRH(-226)CAT, although 11 bp within a stretch of 14 bp upstream of the truncation point at -226 bp were replaced by Hindlll linker and pBLCAT3 vector sequences. This observation indicates either a small impact of permissive sequences at the native 5’-end of the hCRH gene on transcriptional activity of the CRE or differences in the requirements of CAMP responsiveness when tested in heterologous and intact promoter systems. Whereas CREs within CAMP-responsive genes appear to contain a variation of the TGACGTCA element, the CGTCA sequence is the most highly conserved aspect (17). The activity of the 5’-end-truncated construct CRH(-226)CAT was consistently maintained despite a mutation of the first nucleotide of the CRE motif (5’-GGACGTCA-3’). In contrast, responsiveness to CAMP was completely abolished when we mutated the hCRH wtCRE sequence into a novel AP-1 site (TGAGTCA). The slightly elevated basal levels of mtCRE constructs probably resulted from the high serum concentration (10%) routinely employed in our assay conditions, which could support stimulation of the fos/jun complex. Thus trans-activation of the MTV promoter via the inserted mtCRE sites was abolished under low serum conditions (1%). Oligonucleotides homologous with the CRE motif and adjacent contextual bases from genes that are not clearly CAMP-responsive are much less active or even nonfunctional in front of a heterologous promoter (17). The forskolin-dependent induction of a 27-bp segment of the hCRH gene coupled to a heterologous promoter suggested that this oligonucleotide served a role which was functionally equivalent to that of the native CRE, where the stimulation of transcription in response to

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hCRH Promoter Regulation by CAMP

CAMP proceeded by the CAMP-dependent PKA pathway. In support of this hypothesis, overexpression of the catalytic subunit of the CAMP-dependent PKA (16, 38) dispensed the need for forskolin in stimulating AMTV-LUC(wtCRE), activity. Recently, Yamamoto et al. (38) reported a lo-fold difference in the transcriptional potential of the two homolog rat CREB proteins. In line with the study by Berkowitz and Gilman (39), we observed no significant differences between the two isoforms of human CREB proteins. One possible explanation is that the additional 1Camino acid peptides found in CREB-B present a target for cell-specific cellular protein kinases and thus reflect the differences in the assay systems used. In the nervous system a broad variety of neuropeptides and neurotransmitters regulate cellular activity and transcriptional responses of target genes through receptors coupled to adenylate cyclase. Recent studies by Herman et al. (9, 10) demonstrated that neuronal inputs to CRH neurons of the parvocellular region of the nucleus paraventricularis provide tonic inhibition of CRH mRNA production. Thus maintenance of the basal tone of CRH mRNA synthesis has a strong neuronal component, and further studies are required to define the afferent neuronal pathways and their coupling to the adenylate cyclase complex. In addition, the study of CRH gene regulation by CAMP in primary cultures of hypothalamic neurons will help to verify the results of the present study and might provide further information on the regulation of the CRH gene in vivo. However, the hypothalamic expression of the related CAMP-binding proteins CREBPl (40) CAMP-responsive element modulator (41), and members of the fos/ jun family (42) provide a complex framework for the regulation of the CRH gene by the PKA pathway. Heterodimerization between CREBPl and junD (43, 44), transcriptional activation of junD by PKA (45) and expression of various splice variants of the CAMPresponsive element modulator gene with transcriptionally inhibitory and stimulatory properties (41) will impart a fine-tuning of CAMP-dependent gene expression of the CRH gene. Further studies on the molecular interplay of these transcription factors may increase our understanding of the mechanism(s) underlying excessive production of CRH as believed to be the case in clinical disorders such as depression or anorexia nervosa (3). The molecular basis for such a development includes the present results, which demonstrate the presence of a functional CRE in the promoter region of the gene, thus constituting CRH gene regulation by various transmitter systems linked to modulation of intracellular levels of CAMP.

MATERIALS Reporter

AND METHODS

Plasmids

The reporter plasmid pBLCRHCAT3 containing a fstl fragment (-666 to +122 bp) of the hCRH 5’-flanking region cloned in

1939

front of the coding region for the CAT gene was constructed as described previously (8) and is now designated CRH(-666)CAT. The plasmid CRH(-917)CAT was created by /-/indIll and partial Pstl digestion of the parent promoter (8) and the desired fragment was inserted at the Hindlll/Pstl site of the reporter plasmid pBLCAT3. A series of sequential BAL-31 nuclease deletions of the plasmid CRH(-666)CAT was created by linearization at the 5’-flanking Hindlll site and subsequent digestion with BAL-31 nuclease for varying lengths of time. The digested ends were repaired by T,-DNA-polymerase and fused to HindIll linkers (5’-CCAAGCTTGG-3’) before religation. Fragments of the desired size were subcloned into the /-/indlll/BamHI site of pBLCAT3 (46). These constructs were sequenced to ensure the integrity and the truncation point of the hCRH promoter fragments. Heterologous constructs contain restriction fragments of the hCRH promoter in front of the tk promoter of pBLCAT2 (46). The plasmid CRH(-917/-36)CAT comprises an 881-bp Hindlll/Sall fragment isolated from the parent hCRH promoter (8) and inserted into the corresponding restriction sites of the pBLCAT2 polylinker region. The construct CRH(-666/ -36)CAT con&ts of a 630-bp fragment released from the plasmid CRH(-666)CAT by double digestion with HindIll and .%/I and ligated into pBLCAT2. For deletions upstream of position -36 bp the fstl fragment of CRH(-666)CAT was inserted in 3’5’ orientation into the vector oTl9 (GIBCO-BRL, Gaithersburg, MD). CRH(-666/-127)CAT’ was obtained by linearization of the pT19CRH plasmid at the Apal restriction site within the hCRH promoter. Protruding ends were blunted with T,-DNA-polymerase and fused to Sal1 linkers (5’GGTCGACC-3’). A 539-bp hCRH promoter fragment was released by Hindlll/Sa/l restriction and cloned into the polylinker of pBLCAT2. CRH(-332/-129)CAT was created by restriction’ of the plasmid CRH(l666)CAT by Haelll. The desired fraament was fused to Xbal linkers (5’-GCTCTAGAGC3’) befo& insertion into the corresponding restriction site of pBLCAT2. Oligonucleotides homologous with the CRE motif of the hCRH gene (wtCRE) and oligonucleotides containing a mutated CRE motif (mtCRE) correspond to position -238 to -216 bp of the hCRH promoter. They were synthesized as complementary pairs of single-stranded deoxynucleotides with cohesive Hindlll sites at the 5’-end and cloned upstream of the heterologous tk promoter of pBLCAT2. In addition, these oligonucleotides were inserted into the HindIll site of the reporter plasmid AMTV-LUC. AMTV-LUC was constructed as described for AMTV-CAT (47). The following oligonucleotides were used in the present study: wtCRE: 5’-agctTAGGGCTCGTTGACGTCACCAAG-3’ 3’-ATCCCGAGCAACTGCAGTGGTTCtcga-5’ mtCRE (AP-1): 5’-agctTAGGGCTCGTTGAGTCACCAAG-3’ 3’-ATCCCGAGCAACTCAGTGGTTCtcga-5’ All constructs were sequenced to confirm the orientation and integrity of the oligonucleotides. Expression

Plasmids

The expression vector plasmids containing the mouse metallothionein promoter (Mt-1) cloned in front of the cDNA for the a-form of the mouse CAMP-dependent protein kinase catalytic subunit (MtC) or a mouse-human hybrid fl-globin gene (MtGlobin) were generous gifts of Dr. D. Stanley McKnight (University of Washington, Seattle, WA) (48). The cDNAs of human CREB-A and CREB-B subcloned in ~BsM13+ were kindlv provided by Dr. L. Berkowitz (39). The cDNAs were released by EcoRl restriction and inserted into the EcoRl site of the SV40 promoter-driven expression vector pSG5 (Stratagene, La Jolla, CA). Sense and antisense orientations were determined by restriction site analysis.

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MOL 1940

ENDO.

Cell Culture, Galactosidase

1992

Transfection, Activity

Vo16No.11

and Assay

of CAT,

LUC,

and 4.

Monolayer culture of SK-N-MC (neuroblastoma, human; HTB lo), F9 (teratocarcinoma, mouse; CRL 1720) and JAR (choriocarcinoma, human; HTB 144) were grown in Dulbecco’s modified Eagle’s medium or RPM1 supplemented with 10% fetal calf serum. SK-N-MC, JAR, and F9 cells were transfected by electroporation (BTX 600, San Diego, CA) after determination of the optimal electric field strength (48). Cells were replated and forskolin (Siama. St. Louis. MO), 8-Br-CAMP (Siama), and the phosphodiegterase inhibitor IMX (Sigma) wereadded to the media 3 h later at final concentrations of 25 FM, I mM, and 0.5 mM, respectively. 1-Deoxy-forskolin (Sigma) was used at a final concentration of 25 PM. For cotransfection experiments with MtC, 100 PM zinc chloride (Sigma, tissue grade) were added to the cultures 12 h before harvest to induce expression from the Mt-1 promoter. Typically, 5 pg reporter plasmid were used along with 7.5 pg carrier DNA (pGEM4, Promega, Madison, WI) and 2.5 pg of the reporter pCHll0 (an SV40 promoter-driven @-galactosidase expression vector) as an internal control for transfection efficiency. Cells were harvested 24 h after transfection, and CAT activity (50) was measured as described previously (8) with the following modifications: the amount of cell extract was adjusted to P-galactosidase values, and incubations were performed for l-2 h with 0.125 &i [‘4C]chloramphenicol at 60 mCi/nmol. Acetylated and nonacetylated forms of [“‘Clchloramphenicol were separated by TLC. After autoradiographic visualization spots were cut from the plate and counted in a liquid scintillation counter. Assays for P-galactosidase and LUC activity were performed as described elsewhere (51, 52). Results represent the average of four to six independent experiments as indicated in the figure legends.

Acknowledgments The technical assistance of R. Beck, the help of B. Burkart with the graphs, and the technical support of E. Boll are gratefully acknowledged. The SK-N-MC and F9 cell lines were kindly provided by H. Lorke (Tumorbank Deutsches Krebsforschungs Zentrum, Heidelberg, Germany).

5.

6.

7.

8.

9.

10.

11.

12.

13.

14. Received April 24, 1992. Revision received September 3, 1992. Accepted September 3, 1992. Address requests for reprints to: Dr. Dietmar Spengler, Max Planck Institute of Psychiatry, Clinical Institute, Department of Neuroendocrinology, Kraepelinstrasse 10, 8000 Munich 40, Germany. * Present address: Institute for Small Animal Research (FAL), Molecular Genetics Research Unit, Dornbergstrasse 25-27, 3100 Celle, Germany.

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