Regulation of Niemann-Pick C1 Gene Expression by the 3'5'-Cyclic Adenosine Monophosphate Pathway in Steroidogenic Cells

Nicolas Y. Gévry, Enzo Lalli, Paolo Sassone-Corsi and Bruce D. Murphy

Centre de Recherche en Reproduction Animale (N.Y.G., B.D.M.), Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, Québec J2S 7C6, Canada; Institut de Génétique et Biologie Moléculaire et Cellulaire (E.L., P.S.-C.), Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale-Université Louis Pasteur, Illkirch, Communauté Urbaine de Strasbourg 67404, France

Address all correspondence and requests for reprints to: Dr. Bruce D. Murphy, Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, 3200 rue Sicotte, St-Hyacinthe, Québec J2S 7C6, Canada. E-mail: murphyb{at}medvet.umontreal.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Niemann Pick-C1 (NPC-1) protein is essential for intracellular transport of cholesterol derived from low-density lipoprotein import in mammalian cells. The role of the protein kinase A (PKA) pathway in regulation of expression of the NPC-1 gene was investigated. NPC-1 promoter activity was induced by treatment with dibutryl cAMP (dbcAMP), alone or in combination with the cAMP response element (CRE) binding protein (CREB) overexpressed in adrenal Y-1 cells. When the catalytic subunit of PKA was overexpressed in Y-1 cells, there were similar increases in NPC-1 promoter activity in the presence of CREB. Responses were attenuated by blockade of the PKA pathway, and in the Kin-8 cell line deficient in PKA. Promoter deletion analysis revealed that this response was present in promoter fragments of 186 bp and larger but not present in the 121-bp fragment. Two promoter regions, one at -430 and one at -120 upstream of the translation initiation site, contained CRE consensus sequences. These bound recombinant CREB in EMSA, confirming their authenticity as CREB response elements. Promoters bearing mutations of both CRE displayed no response to dbcAMP. The orphan nuclear receptor, steroidogenic factor-1 (SF-1), was implicated in NPC-1 transactivation by the presence of SF-1 target sequence that formed a complex with recombinant SF-1 in EMSA. Furthermore, transfection of a plasmid that overexpressed SF-1 into ovarian granulosa cells increased promoter activity in response to dbcAMP, an effect abrogated by mutation of the SF-1 target sequence. Chromatin immunoprecipitation assays demonstrated that the CRE region of the endogenous and transfected NPC-1 promoter associated with both acetylated and phosphorylated histone H-3 and that this association was increased by dbcAMP treatment. Treatment with dbcAMP also increased the association of the CRE region of the promoter with CREB binding protein, which has histone acetyltransferase activity. Together, these results demonstrate a mechanism of regulation of NPC-1 expression by the cAMP-PKA pathway that includes PKA phosphorylation of CREB, recruitment of the coactivator CREB binding protein and the phosphorylation and acetylation of histone H-3 to transactivate the NPC-1 promoter.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE NIEMANN-PICK C-1 (NPC-1) gene codes for a membrane glycoprotein that functions in processing of low-density lipoprotein (LDL) imported cholesterol and in intracellular cholesterol homeostasis (1). Its absence is characterized by pathological accumulation of cholesterol in endosomes and lysosomes, resulting in premature death, usually from neural manifestations of the excess cholesterol storage (2, 3). The mechanism of NPC-1 action in cholesterol transport has not been determined in its entirety but is the subject of intense current investigation. Recent evidence indicates that the protein resides primarily in the late endosomes (4). It displays domain homology with the resistance-nodulation-division protein pump family in prokaryotes (5) and is believed to be a bulk trans-organelle transporter of cholesterol.

Cholesterol is the parent molecule of the steroid hormones, and it is supplied principally by importation from extracellular sources (6). In nonrodent mammals, LDL cholesterol is the most significant contributor to steroidogenesis (6). Mice homozygous for mutations of NPC-1 have impaired basal and ligand-induced testosterone synthesis (7), and the females are infertile (Gévry, N. Y., and B. D. Murphy, unpublished). Furthermore, cats bearing inactivating mutations in NPC-1 display adrenal insufficiency (8). NPC-1 is highly expressed in steroidogenic theca and luteal cells in the pig ovary (9). Thus, it is expected that NPC-1 plays an important intracellular trafficking role in steroid synthetic cells.

Little is known about the transcriptional regulation of the NPC-1 gene. Portions of the sequence of the of the human (10), pig (9), and mouse (11) 5' flanking regions have been reported. These sequences have elements in common, including a CpG island and specificity protein-1 (SP-1) and activator protein-1 (AP-1) consensus sites. Watari et al. (10) demonstrated constitutive activity of the human promoter in steroidogenic and nonsteroidogenic cells and further demonstrated that the region found 220 bp upstream of the translation initiation codon directs constitutive transcription in human granulosa-lutein cells. They concluded that the NPC-1 promoter displays modest cell-specific responses and that the principal mechanism of regulation is posttranscriptional (10). In contrast, we demonstrated the presence of consensus elements for cAMP response element binding protein (CREB), GATA, and activator protein-1 (AP-1) in the first 400 bp upstream of the translation initiation site of the pig NPC-1 promoter (9). We further showed that cAMP analogs increased NPC-1 message abundance, and that dibutryl cAMP (dbcAMP) modulates the activity of the 1.8-kb pig promoter in three steroidogenic cell lines (9). In this report, we describe subsequent studies in which we identify promoter elements and mechanisms by which cAMP regulates NPC-1 transcription.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CREB and Protein Kinase A (PKA) in NPC-1 Transcription
Our previous results (9) demonstrated that the NPC-1 1.8-kb promoter-luciferase construct signal was significantly increased in three steroidogenic cell lines, Y-1, MA-10, and SVG40, by treatment with dbcAMP. This result was confirmed by transfection of the NPC-1 promoter fused to the luciferase reporter into Y-1 cells that were treated with 1 mM cAMP. The results (Fig. 1AGo) indicate that cAMP alone induced 4- and 5-fold increases in NPC-1 promoter activity at 12 and 24 h, respectively. To explore whether this effect was mediated through CREB, Y-1 cells were transfected with 80 ng of a CREB expression plasmid and concurrently treated with 1 mM cAMP. This resulted in time-dependent increases in promoter activity, with the first significant response observed as early as 3 h and 5-fold increases by 24 h (Fig. 1BGo). The role for endogenous CREB was then tested by transfection of mutant CREB (ACREB), a vector that serves as a dominant negative in competition with CREB for CRE (11). ACREB attenuated the cAMP response at 12 h and extinguished it at 24 h (Fig. 1AGo). We then undertook determination of whether PKA was in the cascade of events leading to NPC-1 transactivation, using three approaches. The first was cotransfection of a plasmid overexpressing the catalytic isoform of PKA. This resulted in a modest but significant increase in NPC-1 promoter activity (Fig. 2AGo, P < 0.05). The mutant form of PKA (mPKA) displayed no capacity to increase promoter activity. When plasmids expressing PKA and CREB were cotransfected, the NPC-1 reporter signal was increased 2- to 3-fold over PKA or CREB alone (Fig. 2AGo, P < 0.01). As further shown in Fig. 2AGo, the synergy between PKA and CREB did not occur in the presence of the dominant-negative form of CREB, ACREB. The second approach was to treat Y-1 cells with the PKA inhibitor H-89, resulting in significant reduction in basal (P < 0.05) and complete abrogation of cAMP induction of cAMP-induced promoter activity (Fig. 2BGo). No inhibition was observed when cells were treated with the MAPK inhibitor PD98059 (Fig. 2BGo). Further confirmation that cAMP was acting through PKA in activation of NPC-1 transcription was acquired by transfecting of the NPC-1 and Renilla constructs into two adrenal cell lines, Y-1 and Kin-8. When both lines were treated with doses of 50 µM-1 mM cAMP, there were dose-dependent increases in NPC-1 promoter activity (Fig. 2CGo). In the Kin-8 line, a Y-1 derivative deficient in but not devoid of PKA (12), NPC-1 promoter activity was significantly lower at 0.2–1.0 mM cAMP concentrations (Fig. 2CGo). Thus, there was a reduced NPC-1 promoter response to CREB in the presence of mPKA, when there was a pharmacological blockade of PKA and in a cell line deficient in PKA. Taken together, this is solid evidence that cAMP is stimulating NPC-1 transcription through the linear PKA pathway.



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Figure 1. NPC-1 Promoter Activity Is Increased by cAMP and CREB

A, Treatment of Y-1 cells transfected with a plasmid constitutively expressing CREB with 1 mM dbcAMP increases NPC-1 promoter activity. Cells were transfected overnight with a plasmid expressing the 1.8-kb porcine NPC-1 promoter construct fused to the luciferase reporter. Concurrently, cultures represented by black bars were transfected with the CREB plasmid or with the ACREB, a dominant-negative construct. All cultures were treated with dbcAMP and harvested at 0, 12, and 24 h after initiation of treatment. Bars represent mean ± SEM of three independent experiments. Differing superscripts represent means that are significantly different from one another at P < 0.05. B, The combination of dbcAMP and CREB increases NPC-1 promoter activity. Y-1 cells were transfected with a plasmids expressing CREB and the 1.8-kb NPC-1 promoter-luciferase construct, then treated with 1 mM dbcAMP. Cultures were terminated at 12 and 24 h and luciferase activity determined. Asterisks indicate differences from corresponding control values at P < 0.05.

 


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Figure 2. NPC-1 Promoter Is Induced by cAMP and Mediated through PKA

A, PKA and CREB interact to increase NPC-1 promoter activity. Y-1 cells were transfected with the promoter-luciferase construct and with a plasmid overexpressing the catalytic subunit of PKA, PKA and CREB, ACREB, mPKA and CREB, ACREB, and PKA or an empty plasmid (control). Where indicated, cells were treated with 1 mM dbcAMP and cultures were harvested at 24 h to determine luciferase signal abundance. Results represent means ± SEM, and the asterisks indicate means significantly in excess of control at P < 0.05. B, The PKA inhibitor H-89 attenuates NPC-1 promoter activity in response to dbcAMP. Y-1 cells were pretreated with 10 µM of the PKA inhibitor H89 or 50 µM of the MAPK inhibitor PD9809 for 1 h and than treated for 6 h with 1 mM dbcAMP. C, The response of the NPC-1 promoter is reduced in response to cAMP in the PKA deficient cell line Y-1 Kin 8. Y-1 and Kin-8 cells were transfected with the 1.8-kb NPC-1 promoter-reporter construct, then treated ascending doses of dbcAMP for 24 h. Mean ± SEM promoter activity, in terms of luciferase expression is presented, and means designated by asterisks are significantly different between the two cell types at P < 0.05.

 
Regions of the NPC-1 Promoter-Mediating cAMP Responses
We then undertook deletion analysis to determine the promoter regions involved in cAMP-CREB responses (Fig. 3Go). Two potential CRE sites had been previously identified by MATInspector (Abteling Genetik, Braunschweig, Germany) analysis of the 5' flanking region of the NPC-1 promoter, at -430 and -120 bp upstream of the translation initiation codon (ATG). Among constructs tested, the minimal promoter fragment required for both basal and CREB-cAMP responses consisted of nucleotides found -186 bp upstream of the transcription initiation codon and including one of the potential CRE (Fig. 3Go). A second fragment, of -296 bp, which also contains the most proximal of the two CRE sites, displayed a similar response. Promoter fragments from -944 and -636 bp upstream of the ATG, and containing the two CRE sites had increased CREB-cAMP response relative to the two shorter constructs (P < 0.05). The two longest fragments, -1811 and -1266, displayed a third class of response that was greater than the all more abbreviated promoter constructs (P < 0.05).



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Figure 3. Deletion Constructs of the NPC-1 Promoter and the CREB-cAMP Response

Promoter fragments fused to the luciferase reporter were transfected into Y-1 cells with or without the plasmid expressing CREB. All cultures were treated with 1 mM cAMP for 24 h. Approximate location of the potential CRE sites is indicated by the black oval. Means ± SEM of triplicate transfection experiments are presented. Means bearing the same superscript (a, b, c) are in homogenous subgroups (P < 0.05).

 
To test the ability of each of the potential CRE sites to interact with CREB, double-stranded oligonucleotides were synthesized representing the core CRE sequence and flanking nucleotides (Table 1Go). Recombinant CREB was then employed in EMSA to establish whether these were authentic CREB binding sites. The results (Fig. 4AGo) revealed formation of a well-defined low-mobility complex for recombinant CREB with a consensus CRE sequence and with each of the oligonucleotides representing the putative CRE of the NPC-1 promoter. Addition of unlabeled oligonucleotides representing each of the sequences resulted in dose-dependent inhibition of formation of the low mobility complex (Fig. 4AGo). Oligonucleotides bearing mutations in the core CRE consensus sequence (Table 1Go) were incapable of binding recombinant CREB and likewise incapable of interfering with the formation of complex between recombinant CREB and the NPC-1 CRE sites (Fig. 4BGo).


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Table 1. Sequences of Oligonucleotide Primers Employed in EMSAs

 


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Figure 4. CREB Binds CRE Consensus Sequences in the NPC-1 Promoter

A, EMSAs demonstrate the authenticity of CRE sites in the NPC-1 promoter. Recombinant CREB was incubated with double-stranded oligonucleotides with consensus CRE sequence (csCRE, Table 1Go) or the proximal (pCRE) or distal (dCRE) sequence. A, Increasing amounts of unlabeled oligonucleotide were added to demonstrate specificity. B, Unlabeled or mutated (mt, Table 1Go) oligonucleotides were added to the reaction tube. The complex formed with recombinant CREB is indicated by the arrow.

 
To determine the biological authenticity of the endogenous CREB binding to the proximal and distal CRE sites of pig NPC-1 promoter, nuclear extract from dbcAMP-stimulated Y-1 cells was subjected to EMSA with cognate labeled oligonucleotides in the presence or absence of antibody specific to the ATF family [CREB, CRE modulator, and activating transcription factor-1 (ATF-1)]. A complex formed between the nuclear extract and each of the probes that migrated similarly to the complex formed with the recombinant CREB. The incubation with the Y-1 nuclear extract with the ATF antibody was found to interfere with the complex formation and to result in a super shift of the band (Fig. 5Go). Together, these observations confirm that the elements in the NPC-1 promoter with consensus to known CRE binding sequences are authentic CRE sites.



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Figure 5. Supershift EMSA Showing Specificity of CREB Complex Formation with Nuclear Extract from Y-1 Cells

Aliquots of 10 µg of recombinant CREB (rCREB) or nuclear extract from Y-1 cells stimulated with 1 mM dbcAMP (Y-1 NE) were incubated in presence of oligonucleotide sequences representing the distal (dCRE) and proximal (pCRE) cAMP response elements from the NPC-1 promoter (Table 1Go). The formation of the CREB-DNA complex was decreased when ATF-1 antibody was added to incubation mixture, and unshifted (CREB) and shifted (SS) bands are indicated by arrows.

 
To define the importance of the two CRE sites in transactivation of the NPC-1 promoter, three new promoter-reporter constructs (1.8 kb) were developed, so that one or both CRE were mutated. These were then cotransfected into Y-1 cells with the plasmid expressing CREB and stimulated with dbcAMP, as in previous experiments. The results (Fig. 6Go) demonstrate that either of the CRE sites can direct the response to cAMP and CREB and either CRE site can induce promoter activity equivalent to the wild-type construct of the same length. Mutation of both sites attenuated the cAMP-CREB response (Fig. 6Go), indicating that CREB transactivates transcription through one or both cAMP sites.



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Figure 6. Mutation of Both CRE Eliminates the Response to CREB and cAMP

The proximal (mpCRE) and distal (mdCRE) CRE sites in the 1.8-kb porcine NPC-1 promoter were mutated. Constructs were transfected into Y-1 cells along with the plasmid constitutively expressing CREB. Cultures were treated with 1 mM dbcAMP overnight and harvested for luciferase reporter quantification. When both sites were altered, the promoter activity response to CREB and cAMP was eliminated (P < 0.05).

 
Interactions with Steroidogenic Factor-1 (SF-1)
The magnitude of response of the NPC-1 promoter in steroidogenic cells varies with the steroidogenic cell type, with Y-1 cells providing the greatest signal (9). We hypothesized that these differences were due to variation in the abundance of the orphan nuclear receptor, SF-1, which is robustly expressed in Y-1 cells (13) and has been shown to synergize with CRE in induction of cAMP responses (14). To test this hypothesis, a plasmid constitutively overexpressing SF-1 was cotransfected along with the 1.8-kb NPC-1 promoter construct, into the cell line that provided the most modest dbcAMP response, the SVG40 human granulosa cells. Transfection of SF-1 alone increased promoter activity by approximately 2-fold (P < 0.05). The response was greater when cells thus treated were incubated with 1 mM cAMP (P < 0.01, Fig. 7AGo). The combination of SF-1 and CREB provided a response similar to SF-1 alone or SF-1 along with dbcAMP. A startling increase was achieved when cells expressing both SF-1 and CREB were treated with dbcAMP, to more than 5-fold over control (P < 0.001). Finally, the specificity of the SF-1 response was tested by cotransfection of a plasmid expressing SF-1 in reversed orientation, resulting in a greatly reduced response to CREB and dbcAMP, and suggesting the authenticity of the SF-1 response (Fig. 7AGo).



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Figure 7. The Orphan Nuclear Receptor SF-1 Interacts with CREB in Induction of NPC-1 Promoter Activity

A, SVG40 cells were transfected with a plasmid overexpressing SF-1 alone or in combination with the CREB plasmid, or a plasmid bearing a mutant SF-1 (SF-1r). Cultures marked cAMP were treated with 1 mM dbcAMP for 24 h before luciferase assay. Bars represent means ± SEM of triplicate experiments. Means greater than the control mean at *, P < 0.05; **, P < 0.01. B, EMSA demonstrating complex formation by oligonucleotides representing the consensus SF-1 recognition site (csSF-1, Table 1Go) and the porcine promoter SF-1 (pSF-1, Table 1Go) with rSF-1. All concentrations of the unlabeled csSF-1 competitor prevented protein-oligonucleotide complex formation. C, Mutation of the SF-1 consensus site in the NPC-1 promoter prevents its interaction with recombinant SF-1. The pSF-1 probe and the mutated form (Table 1Go) were incubated in presence of rSF-1 and/or in the presence of mutated or wild-type unlabeled oligonucleotide competitors.

 
Inspection of the NPC-1 promoter suggested one potential SF-1 binding site was present, between the two CRE sites, at 245 bp upstream of the ATG. Oligonucleotides containing this sequence and the consensus SF-1 sequence were synthesized and employed in EMSA. The results (Fig. 7BGo) indicate that the consensus SF-1 oligonucleotide bound to recombinant SF-1, resulting in a single complex. The putative SF-1 sequence of the NPC-1 promoter migrated identically in the presence of recombinant SF-1. Furthermore, unlabeled oligonucleotides from the NPC-1 SF-1 sequence completely displaced labeled SF-1 oligonucleotides from the recombinant protein, indicating the specificity of the response (Fig. 7BGo). We then mutated the SF-1 binding sequence in the NPC-1 promoter (Table 1Go), which had little effect on the binding of wild-type SF-1 oligonucleotide to recombinant SF-1 (Fig. 7CGo). Furthermore, the mutated form did not bind to recombinant SF-1 (Fig. 7CGo). Taken together, these experiments identify an SF-1 binding site that appears to interact with the cAMP pathway in synergistic activation of NPC-1 promoter. To further confirm this, the SF-1 site was mutated in the 1.8-kb promoter fragment, and the ability of the constitutive expression of SF-1 to enhance promoter activity was tested. The results (Fig. 8AGo) demonstrate that this mutation greatly attenuated the transactivation of the promoter in response to SF-1. In addition, mutation of either or both the proximal and distal CRE site eliminated the response to constitutive SF-1, indicating that these elements are functionally linked in regulation of the NPC-1 promoter. We then undertook the determination of whether the SF-1 site was significant to the dbcAMP induction of promoter activity using the 0.6-kb promoter fragment mutated at the putative SF-1 site, the proximal CRE site, the distal CRE site or both CRE sites. The results indicate, as expected, mutation of the CRE sites reduces the promoter activity in the presence of cAMP (Fig. 8BGo). Similar attenuation was observed when the SF-1 site was mutated, further demonstrating a role for this promoter sequence in regulation of NPC-1 transcription.



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Figure 8. SF-1 Site Mutation Reduces the Response of the NPC-1 Promoter to SF-1 and cAMP

A, Mutation of CRE or SF-1 sites in the 1.8-kb NPC-1 promoter abrogates SF-1 + dbcAMP induction of transcription. The human granulosa cell line SVG-40 was transfected with promoter constructs mutated at the proximal (mpCRE) or distal (mdCRE) or both (mpCRE/mdCRE) of the CRE sites, or at the SF-1 site (mpSF-1). Cells were then treated with 1 mM dbcAMP for 24 h and promoter activity assessed by luciferase assay. Bars represent the mean ± SEM of three experiments and means bearing an asterisk have significantly lower activity compared with the wild-type, dbcAMP-treated promoter at P < 0.05. B, Mutation of the CRE or SF-1 sites attenuates the NPC-1 promoter activity in response to cAMP in Y-1 cells. *, Mean response differs from the wild-type promoter (pGL3–636) at P < 0.05.

 
Histone Modification and NPC-1 Transcription
To test the possibility that modification of histones is related to the cAMP-induced induction of NPC transcription, we first treated Y-1 cells with one of two inhibitors of histone deacetylase (HDAC), Trichostatin A (TSA), or butyrate. Both reagents have the net effect of hyperacetylation of histones (15). Northern analysis (Fig. 9AGo) revealed that both HDAC inhibitors increased the abundance of NPC-1 mRNA in this cell model. This effect appeared to be additive or synergistic with cAMP, as the combination induced further increases in NPC-1 transcript abundance. Chromatin immunoprecipitation assays (ChIPs) were employed to examine this phenomenon further. Two trials were undertaken, in the first was Y-1 cells were transfected with the 1.8-kb NPC-1 promoter and incubated with dbcAMP for 6 h. In the second trial, primary porcine granulosa cells in culture were treated with dbcAMP for 6 h. After incubation in both cell types, antibodies directed against histone H-3 acetylated on lys-14 were used to precipitate total chromatin which was reversibly cross-linked to DNA to which it bound. The DNA associated with acetylated histone was amplified by PCR using primers specific to the NPC-1 promoter region of 500 bp that included both CRE. There was no PCR amplification of immunoprecipitates using IgG alone (Fig. 9BGo), and primers directed to the open reading frame of the NPC-1 were used as control (data not shown). As can be seen in Fig. 9BGo, treatment of Y-1 cells with 1 mM dbcAMP caused a 2- to 3-fold increase in this NPC-1 promoter fragment binding associated with acetylated histone H-3. The results were nearly identical for the endogenous promoter in primary pig granulosa cells (Fig. 9BGo). Similar studies were then performed using an antibody directed against histone H-3 phosphorylated at ser-10. The results (Fig. 9BGo) indicate a greater response, with 4- to 5-fold more of the transfected promoter sequence precipitating with phosphorylated H-3 in the presence of 1 mM dbcAMP. Association of phosphorylated H-3 with the endogenous promoter was increased 2- to 3-fold in pig granulosa cells treated with dbcAMP. Finally, we immunoprecipitated a coactivator protein known to have histone acetyl transferase (HAT) activity, CREB binding protein (CBP). This coactivator was found not to be associated with the CRE region of the NPC-1 promoter in Y-1 cells in the absence of dbcAMP but provided a strong band in the presence of this intracellular messenger. It associated with the endogenous promoter in pig granulosa cells, and this association was increased 3- to 5-fold in the cells treated with dbcAMP. Together, these results indicate that dbcAMP increases the acetylation and phosphorylation of histones associated with the NPC-1 promoter and induces the recruitment of CBP to the promoter region that includes both CRE elements.



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Figure 9. Chromatin Modification Is Associated with cAMP Induction of NPC-1 Expression

A, Northern blot showing the 5.0-kb Y-1 transcript that hybridizes with the NPC-1 probe. Cells were untreated or treated with 1 mM dbcAMP, the HDAC inhibitors TSA (100 ng/ml) or 15 mM Na butyrate (But), or the HDAC inhibitors in the presence of 1 mM dbcAMP. B, Immunoprecipitation assays employing antiserum against acetylated histone H-3 (H-3 Ac), histone H-3 phosphorylated at ser-10 (H-3 P) or CBP. Cultures of the Y-1 cell line were transfected with the NPC-1 promoter. Native promoter association with the proteins of interest was tested in primary porcine granulosa cells (PGC). Control cultures are in the columns designated C. Those in the marked cAMP were treated with 1 mM dbcAMP for 6 h, after which chromatin was immunoprecipitated and a 500-bp promoter sequence containing both CRE elements was amplified by PCR. DNA control (designated Input) for the procedure was established by amplification of an equivalent amount of DNA that had not been subject to immunoprecipitation. Control for antibody specificity was established by precipitation with rabbit Igs, designated IgG.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The NPC-1 protein mediates an essential step in the pathway by which LDL-borne cholesterol is assimilated into intracellular cholesterol pools (16). The LDL pathway serves as the most important primary source of cholesterol for steroidogenesis in many species (6). Steroidogenesis is provoked by extracellular hormones via intracellular pathway that includes cAMP, PKA, and the phosphorylation of CREB (17). As many of the elements that modulate LDL importation (e.g. the LDL receptor) are likewise driven by the downstream effectors of cAMP, it was expected that NPC-1 would be similarly regulated. Nonetheless, the prevailing view (10, 18, 19) is that NPC-1 functions as a housekeeping gene, and whatever control exists is posttranscriptional. Our findings provide strong evidence in contrast to this view. Overexpression of the catalytic subunit of PKA, or the PKA phosphorylation target, CREB, increased the signal from NPC-1 promoter-luciferase constructs. The response to overexpression of CREB was dramatically increased in cells treated with dbcAMP, or in cells where the PKA catalytic subunit was expressed. The failure of mutant constructs of both CREB and PKA to produce these effects, and their abrogation by blockade of PKA activity, indicate the specificity of this response. Furthermore, NPC-1 promoter activity was reduced in the Kin-8 cell line, known to be deficient in PKA (12). It is clear that CREB is not the sole transcription factor activated by cAMP (17), nor is CREB activated solely by cAMP through PKA (20). Nonetheless, the ensemble of the data confirms our preliminary observations of cAMP regulation of NPC-1 expression (9), and make a strong case for regulation of the porcine NPC-1 gene by the cAMP pathway (21). We suspect this to be the case for the mouse NPC-1 gene as well, as MATInspector analysis of the 5'flanking region in this species revealed five potential CRE sites in the first 500 bp upstream of the ATG (11). Furthermore, the 2.0-kb mouse promoter-luciferase construct transfected into Y-1 cells responded to cAMP and CREB with the same magnitude of increase as the pig promoter (Gévry, N. Y., and B. D. Murphy, unpublished observations).

The deletion analysis of the porcine NPC-1 promoter conforms to the results for the human sequence for constitutive expression reported by Watari et al. (22), if the promoter constructs in that study are numbered from the translation initiation codon. In the human sequence, elements essential to the constitutive response are found between -232 and -158 from the ATG (22). The corresponding sequence in the pig is -186 to -121. In the pig, the constitutive response was constant throughout the larger fragments, whereas in the human sequence, constructs larger than 232 bp upstream of the ATG displayed more robust activity than did the -177 fragment.

Our previous study indicated the presence of two sites with sequence homology to CRE elements, one at -120 and the second at -430. EMSA employing recombinant CREB demonstrated that oligonucleotides derived from these sites formed a complex that migrated identically to the consensus CRE sequence, indicating their authenticity as sites of CREB binding in the porcine promoter. Deletion analysis revealed that the 1.8-kb promoter, in the presence of two intact CREs, provided the greatest response. More abbreviated fragments containing both sites displayed a significantly higher promoter-driven luciferase signal than did those containing a single site. Mutational analysis indicated that the full-length promoter required only one of the two sites for maximal promoter activity in the presence of CREB and cAMP. These results suggest the presence of other sites on the full-length promoter that respond to phosphorylated CREB. These may be sequences with lower homology to the consensus CRE (23).

Promoter analysis indicated that orphan nuclear receptor SF-1 also plays a role in cAMP induction of NPC-1 transcription, as its overexpression increased the full-length signal. Furthermore, there were additive effects with SF-1 and dbcAMP. A DNA sequence representing an SF-1 consensus recognition site is present at 245 bp upstream of the ATG (9). In the present study, EMSA, employing recombinant SF-1, confirmed its authenticity, and mutation of the site eliminated its capacity to respond to the constitutive SF-1 and dbcAMP. We propose that SF-1 binds to its cognate promoter element and synergizes with CREB to increase transactivation of the NPC-1 gene. This interaction was first suggested by Parker and Schimmer (24) for the cytochrome P450 side chain cleavage enzyme and has been observed with other genes associated with steroidogenesis (25). A spectrum of interaction between SF-1 and cAMP in modulation of promoter activity has been described, ranging from additivity to synergy (26). Current data indicate that the NPC-1 promoter best fits the latter description. The SF-1-cAMP interaction explains the consistently higher NPC-1 promoter activity in response to cAMP in the Y-1 line, as these cells strongly express SF-1 (13). SF-1 shows a pattern of expression generally specific to endocrine and neural tissues (27). Although NPC-1 is ubiquitously expressed (10), we showed higher NPC-1 transcript levels in steroidogenic tissues (9), consistent with interaction between SF-1 and cAMP in its regulation. Several mechanisms of SF-1-cAMP interaction have been proposed, including direct interactions between CREB and SF-1 and recruitment of coactivators by the orphan nuclear receptor (26, 28). Indeed, in the present investigation, mutation of the proximal or distal CRE site eliminated the promoter response to constitutive SF-1 and dbcAMP. Further investigation will be necessary to determine the mechanisms of SF-1 and CREB in transactivation of the NPC-1 gene.

Among the important roles played by coactivators in transcription is the covalent modification of histones, which may be obligatory for the transactivation by CREB (29). Acetylation of histone H-3 in response to 8-Br-cAMP regulates the promoter of another cholesterol transport gene, steroidogenic acute regulatory protein (30). We therefore employed two HDAC inhibitors, TSA and butyrate, that have the net effect of hyperacetylating histones and consequent activation of transcription (31). Increases in dbcAMP-induced accumulation of NPC-1 transcripts in Y-1 cells occurred in the presence of HDAC inhibitors, indicating that the NPC-1 transcriptional response was coupled to acetylation of histone(s). This concept was further tested by ChIP analysis employing an antiserum against acetylated histone H-3 and primers that bracket the CREs in the promoter. Clear increases in promoter abundance associated with acetylated chromatin ensued after dbcAMP treatment both in Y-1 cell expressing a transfected promoter, and in primary porcine granulosa cells. These results occurred over the same time frame observed with another cholesterol transfer protein, the steroidogenic acute regulatory protein (30). These results are further in harmony with observations of histone H-3 acetylation associated the transactivation of a further two genes in the cholesterol homeostasis group, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and the LDL receptor (32). It is possible that acetylation of other histones, particularly H4, may be associated with the NPC-1 promoter in response to cAMP. This concept merits further study.

CBP is a coactivator that associates with phosphorylated CREB (33) and SF-1 (34) and appears to bridge between CREB and the transcriptional complex (35). It also has inherent HAT activity and may recruit other proteins with HAT activity (31). It has been shown to be necessary for transactivation of genes in the cholesterol homeostasis pathway (36). In the present investigation, the association of CBP with the CRE region of both the endogenous and transfected NPC-1 promoters in response to dbcAMP provides circumstantial evidence that CBP contributes to acetylation of histones associated with transactivation of the NPC-1 gene.

A second chromatin modification of importance to gene transcription is phosphorylation (37). Histone H-3 can be phosphorylated in vitro by the catalytic subunit of PKA (38) and FSH-induced histone H-3 phosphorylation in rat granulosa cells is blocked by PKA inhibitors (39). Phosphorylation and acetylation of histone H-3 are linked, and phosphorylation may precede acetylation (31, 37). Furthermore, global phosphorylation of cells has pervasive effects on acetylation of histones (40). Our ChIP analysis permits us to conclude that both the endogenous and transfected NPC-1 promoter sequences associate with phosphorylated as well as acetylated histone H-3. Furthermore, there is a substantial increase in NPC-1-association with phosphorylated histone H-3 after dbcAMP stimulation. Although histone H-3 phosphorylation has been traditionally linked to cell proliferation (37), there is precedent for an association with differentiation (38). NPC-1 expression increases in pig granulosa cells during differentiation (9), and the present study supports the view that histone H-3 phosphorylation plays a role in this process.

In summary, we present data demonstrating that NPC-1 transcription is regulated by the cAMP pathway. We propose that ligands that activate this signaling cascade direct NPC-1 expression via CREB, which associates as a dimer with the palindromic CRE sites (21) present in the porcine promoter. Phosphorylation of CREB is sufficient to activate expression of its target genes, but additional cofactors are necessary for maximal response (21). An important cofactor is CBP, which is recruited to the transcriptional complex by phosphorylation of CREB. CBP has histone acetylase activity, resulting in acetylation of histone H-3 associated with the NPC-1 promoter, thereby increasing CREB-induced transactivation. There is evidence that SF-1-induced transactivation is enhanced in steroidogenic enzyme genes by SF-1-CBP interactions (34). Concurrent histone H-3 phosphorylation by PKA or by another kinase downstream from PKA further enhances the transcriptional responses, by mechanisms that are not well understood. It is certain that there are other pathways that modulate the expression of this gene, in particular, those associated with intracellular sterol regulation. These are under active investigation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructions
The 1.8 kb of NPC-1 gene promoter was cloned into pGL3 vector (Promega Corp., Nepean, Ontario, Canada) and various deletion constructs were prepared by PCR with EcoRI and MluI insertions for directed cloning. Potential CRE and the SF-1 sites were mutated with the QuikChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA). The wild-type and mutant expression vectors pRC/Rous sarcoma virus-PKA were a gift from Dr. R. Maurer (Oregon Health Sciences University, Portland, OR); pRc/Rous sarcoma virus-CREB from Dr. R. Goodman (Oregon Health Sciences University, Portland, OR); pRc/cytomegalovirus (CMV)-ACREB from Dr. C. Vinson (National Cancer Institute, National Institutes of Health, Bethesda, MD); and expression vectors pRC/CMV-cSF-1, pRC/CMV-recombinant SF-1 (rSF-1) and pGEX-1{lambda}T-SF-1 for the production of fusion proteins were kindly provided by Dr. K. L. Parker (University of Texas Southwestern, Dallas, TX). All of plasmids for transfection were prepared using the Maxi Prep kit (QIAGEN, Mississauga, Ontario, Canada).

Cell Culture, Transient Transfections, and Promoter Activity Assays
Porcine granulosa cells were aspirated from 3- to 5-mm follicles from ovaries recovered from pigs at slaughter and cultured in MEM (Life Technologies, Burlington, Ontario, Canada) containing 1 mg/liter insulin (Sigma, Oakville, Ontario, Canada), 0.1 mM nonessential amino acids (Life Technologies), 5 x 104 IU/liter penicillin (Life Technologies), 50 µg/liter streptomycin (Life Technologies), 0.5 mg/liter fungizone (Life Technologies), and 10% fetal calf serum (Life Technologies). Y-1 mouse adrenal tumor cells (CCL-79, ATCC, Manassas, VA) and Y-1 Kin-8 (gift of Dr. B. Schimmer, University of Toronto, Toronto, Ontario, Canada) were maintained in DMEM/F12 (Life Technologies) supplemented with 10% horse serum, 2.5% fetal bovine serum, and antibiotics. SVG40 human granulosa cells (gift of Dr. P. Leung, University of British Columbia, Vancouver, British Columbia, Canada) were cultured in Opti-MEM (Life Technologies), supplemented with 5% fetal bovine serum and antibiotics. The cells were transfected with 100 nM/well with deletion constructs of the pig NPC-1 promoter in the vector pGL3 using Effectene reagent (QIAGEN) according to the manufacturer’s protocol. Cells were cotransfected with the simian virus 40 (SV40) Renilla luciferase control vector pRL.SV40 (Promega Corp.) at a ratio of 10:1 of pNPC-LUC:pRL.SV40 to normalize results for transfection efficiency. The cotransfection experiments were performed with the transfection of 80 ng of CREB expression vectors and/or 80 ng PKA and/or 20 ng for the SF-1 expression vectors or an empty expression vector for correction of total DNA. Some cultures were treated with 1 mM dbcAMP (Sigma) for 12 h or 24 h and/or pretreated with the PKA inhibitor H89 or the MAPK inhibitor PD98059 (10 µM and 50 µM, respectively; Sigma) for 1 h. Luciferase activity was detected by the Promega Corp. Dual Luciferase Assay system and chemiluminescence measured in a Berthold 9501 luminometer. Control transfections included the inclusion of an equal amount of the promoter-less pGL3 basic plasmid (Promega Corp.).

EMSA
Nuclear extract were prepared from Y-1 cells treated with 1 mM cAMP for 24 h according to the method of and Andrews and Faller (41). The EMSAs were performed as described (42) with some modifications. Double-stranded oligonucleotides corresponding to the NPC-1 promoter region (Table 1Go) were labeled with {alpha}-32P-deoxy-CTP by polynucleotide kinase. In general, Y-1 nuclear extract, or recombinant CREB or SF-1 were incubated with 1 ng of labeled probe in binding buffer [20% glycerol; 5 mM MgCl2; 2.5 mM EDTA; 2.5 mM dithiothreitol; 250 mM NaCl; 50 mM Tris-HCl, pH 7.5; 0.25 mg/ml poly(deoxyinosine-deoxycytidine)-(deoxyinosine-deoxycytidine)] for 20 min at room temperature. For the supershift assay, ATF-1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used and the binding reactions were incubated for a further 45 min on ice. The binding reaction products were loaded on a 6% nondenaturing polyacrylamide gel in 0.25x Tris-buffered EDTA. Unlabeled wild-type and mutant oligonucleotides were added to demonstrate specificity of binding.

RNA Analysis
RNA was isolated from untreated cells or from cells treated with 1 mM dbcAMP (Sigma), TSA (100 ng/ml; BIOMOL Research Laboratories, Plymouth Meeting, PA), or 15 mM sodium butyrate for 4 h. Northern blot analysis of total RNA was performed as previously described (9). In brief, cultured cells were homogenized in 4 M guanidine isothiocyanate (Life Technologies), 26.5 mM sodium acetate (Sigma), and 0.12 M ß-mercaptoethanol (Sigma) and stored at -70 C until analysis. Total RNA was purified with QIAGEN Easy Spin columns (QIAGEN). Aliquots of 15 µg total RNA were subjected to electrophoresis on 1% agarose-formaldehyde gels using a 20 mM morpholinopropanesulfonic acid buffer (pH 7.0), transferred overnight to nylon membranes, and cross-linked for 30 sec at 150 mJ in a UV chamber (GS Gene Linker, Bio-Rad Laboratories, Inc., Richmond, CA). Blots were hybridized with a 1-kb probe from the 5'region of the porcine NPC-1 open reading frame. All probes were labeled by random priming (Roche Molecular Biochemicals, Laval, Québec, Canada). Hybridized blots were subjected to phosphorimaging for visualization and quantitative estimate of the most prominent NPC-1 transcript.

ChIP
ChIPs were performed as described by Kuo and Allis (43) with minor modifications. The endogenous promoter was tested in primary porcine granulosa cells cultured for 24 h after plating. The 1.8-kb porcine NPC-1 promoter was transfected into Y-1 cells according to the procedures described above. At 24 h after transfection, Y-1 cells and primary porcine granulosa cells were treated with 1 mM dbcAMP (Sigma) for 6 h and cross-linked by addition of formaldehyde to the medium at a final concentration of 1% for 10 min at 37 C. Cells were washed in PBS, resuspended in 200 µl of ChIP lysis buffer [1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 50 mM Tris-HCl (pH 8.0), and protease inhibitors] and sonicated with a Branson (Danbury, CT) Sonifier 450 at power setting 2 with 10-sec pulses at duty cycle 90. The chromatin solution was diluted 10-fold in ChIP dilution buffer (0.01% SDS; 1.1% Triton X-100; 1.2 mM EDTA; 16.7 mM Tris, pH 8.1; 16.7 mM NaCl; and protease inhibitors). One tenth of the lysate was used for purification of total DNA. Each sample was precleared by incubating with 80 µl salmon sperm DNA/protein A-agarose 50% gel slurry (Upstate Biotechnology, Inc., Lake Placid, NY) for 30 min at 4 C. An aliquot of 5 µg of anti-acetyl histone H-3, antiphosphorylated H-3, or CBP antibodies (Upstate Biotechnology, Inc.) were added and immunoprecipitated at 4 C overnight. The immunoprecipitate was collected using salmon sperm DNA/protein A-agarose and washed once with the following buffers in sequence: low-salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tri-HCl, pH 8.1; 150 mM NaCl); high-salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.1; 500 mM NaCl); LiCl wash buffer (0.25 M LiCl; 1% Nonidet P-40; 1% sodium deoxycholate; 1 mM EDTA; 10 mM Tris-HCl, pH 8.1); TE (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). DNA-histone or DNA-CBP protein cross-links were reversed by incubation at 65 C for 4 h followed by proteinase K treatment. DNA was recovered by purification with the Qiaquik PCR purification column (QIAGEN). PCR was performed using total DNA as control, and immunoprecipitated DNA in presence of 2 µCi {alpha}-32P-deoxy-CTP with a temperature cycle of 45 sec at 94 C, 45 sec at 52 C, and 30 sec at 72 C. The primers used for the PCR were CHA (5'-AAGGGG AGAAATGAGTTGAAGC-3') and CH1 (5'-GAATTCCAGCAGGAGGAGGCCGAA-3'). Primers for the open reading frame of the gene were employed as a control to demonstrate specificity of amplification of DNA associated with immunoprecipitated chromatin or CBP. As a second control, rabbit IgG was used in place of the histone or CBP antibodies to precipitate cell lysates. PCR products were separated on 6% nondenaturing polyacrylamide gels and subjected to phosphorimaging for visualization and quantification.

Statistical Analysis
Each experiment was performed in triplicate. Luciferase data are expressed as the mean ± SEM. Analysis of variance was performed, and in the presence of a significant F value, followed by the Tukey-Kramer test to establish differences between treatment means. The minimum level of significance accepted was P < 0.05.


    ACKNOWLEDGMENTS
 
The technical assistance of Mira Dobias and Sandra Ledoux is gratefully acknowledged.


    FOOTNOTES
 
This work was supported by Grant MT-11018 from the Canadian Institutes of Health Research (to B.D.M.) and by grants (to P.S.-C.) from Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire Régionale, Fondation de la Recherche Médicale, Rh\|[ocirc ]\|ne-Poulenc Rorer Inc. (Bioavenir, France), and Association pour la Recherche Contre le Cancer.

Abbreviations: ACREB, Mutant form of CREB; ATF-1, activating transcription factor-1; CBP, CREB binding protein; ChIP, chromatin immunoprecipitation assay; CMV, cytomegalovirus; CRE, cAMP response element; CREB, CRE binding protein; dbcAMP, dibutyrl cAMP; HAT, histone acetyl transferase; HDAC, histone deacetylase; LDL, low-density lipoprotein; mPKA, mutant form of PKA; NPC-1, Niemann Pick-C1; PKA, protein kinase A; rSF-1, recombinant SF-1; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor-1; SV40, simian virus 40; TSA, trichostatin A.

Received for publication March 7, 2002. Accepted for publication January 10, 2003.


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