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Center for Research on Reproduction and Women’s Health, University of Pennsylvania (L.K.C., J.F.S.), Philadelphia, Pennsylvania 19104; Department of Molecular Biology and Biochemistry, University of California (T.F.O.), Irvine, California 92717; and Department of Cellular and Molecular Physiology, Pennsylvania State College of Medicine (J.M.M.), Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: Lane K. Christenson, Ph.D., 1354 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail: lchriste{at}mail.med.upenn.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cholesterol homeostasis in mammals is regulated by a unique family of transcription factors called sterol regulatory element-binding proteins (SREBP) (9, 10). These transcription factors are localized to the endoplasmic reticulum in an approximately 125-kDa precursor form under conditions where intracellular sterol/cholesterol stores are replete. When conditions of cellular sterol/cholesterol need arise, these membrane-bound proteins are cleaved by proteases, one of which is controlled by the sterol-sensitive SREBP-cleavage activating protein, thereby releasing an approximately 68-kDa transcription regulator (11). The mature SREBPs enter the nucleus where they activate genes involved in cholesterol biosynthesis, uptake, and metabolism [i.e. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, squalene synthase, low density lipoprotein (LDL) receptor, and cholesteryl ester transfer protein] (11, 12). In cell culture studies, the two major functional proteins, SREBP-1a and -2, which are derived from two different genes have similar activities (9, 10). However, SREBPs alone are weak transcriptional activators of sterol-responsive genes; they appear to require a partnership with other transcription factors (i.e. Sp1, CCAAT binding factor/nuclear factor Y, and cAMP response element-binding protein) to elicit transcriptional control over sterol metabolism (11, 13, 14). Recently, yin yang 1 (YY1), a DNA-binding zinc finger transcription factor that can act as both an activator and a repressor, was shown to disrupt SREBP-dependent gene activation (15, 16). In our effort to elucidate the mechanisms responsible for steroidogenic acute regulatory protein (StAR) gene expression, we carried out studies to determine whether the human StAR gene, another protein involved in cholesterol metabolism, is a target gene for SREBPs and YY1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The ß-galactosidase expression
vector [pCH110 (Pharmacia Biotech, Piscataway, NJ) or
pCMV-ß-gal] was used for normalization of luciferase data. Plasmids
for transfection were prepared using the QIAGEN Maxiprep
system (Valencia, CA).
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Enzymatic assays
Luciferase activity was determined in a LUMAT LB 9507
luminometer (E.G.&G Berthold, Nashua, NH) with the
Promega Corp. luciferin as substrate as previously
described (7). ß-Galactosidase activity was determined
by a standard colorimetric assay using
2-nitrophenyl-ß-D-galactopyranoside as
substrate. Luciferase activity for each well was determined by dividing
luciferase relative light units by the ß-galactosidase activity
(A420).
Western blot analysis
Nuclear extracts from human granulosa-lutein cells were
generated as previously described (20). Protein
concentrations of the extracts were determined by the Bio-Rad Laboratories, Inc. dye binding assay (Richmond, CA). Equal
amounts of protein (100 µg) were loaded onto SDS-PAGE gels for
electrophoresis. After electrophoresis, gels were transferred to
polyvinylidene difluoride membranes for probing with antibodies to
SREBP-1a (mouse monoclonal IgG-2A4, American Type Culture Collection, Manassas, VA) and YY1 (rabbit polyclonal sc-281,
Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Detection
of the antibodies used the ECL kit (Amersham Pharmacia Biotech, Arlington Heights, IL).
Electrophorectic mobility shift assays (EMSAs)
Double stranded oligonucleotide probes, 25 bases in length with
4-base overhangs (CTAG), based on the wild-type human StAR promoter
were labeled with [
-32P]deoxy-CTP by fill-in
reaction with DNA polymerase I Klenow (large fragment; NEN Life Science Products, Boston, MA) or were end labeled as previously
described (21). Mutant probes (SREBP triple mutant,
SREBP/YY1 mutant, and YY1 mutant) differ from the wild-type probes at
the indicated (bold) bases in Fig. 1
. The human HMG-CoA
reductase and human LDL receptor oligonucleotides were previously
described (17, 21). The labeled probes were used in EMSAs
as described previously (7, 17, 21). Briefly, recombinant
SREBP-1a and YY1 and the truncated DNA-binding domains of SREBP-1a
(amino acids 321–491) and SREBP-2 (amino acids 331–481) were
incubated with labeled oligonucleotide probes for 20 min on ice before
loading onto prerun acrylamide gels. Recombinant proteins were used in
the gel shifts, because endogenous levels of the nuclear proteins do
not elicit an EMSA shift (22) even through under
experimental conditions SREBP-dependent promoter responsiveness can be
observed. Additionally, unlabeled oligonucleotides, wild-type and
mutant (1- to 20-fold excess), were added to selected samples to
demonstrate specificity of binding.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the
results from studies in COS-1 cells that were transiently transfected
with increasing doses of either the SREBP-1a or -2 expression plasmids
with either the 1.3-kb StAR-luciferase reporter or the LDL
receptor-luciferase reporter. StAR promoter activity increased in a
dose-dependent manner after transient expression of SREBP-1a, whereas
SREBP-2 was relatively ineffective in enhancing StAR promoter activity.
As expected, both SREBP-1a and -2 plasmids markedly stimulated the
activity of the transfected LDL receptor-reporter construct. When human
granulosa-lutein cells were transfected with the same reporters and the
SREBP-1a expression plasmid, we also observed a dose-dependent increase
in both LDL receptor and StAR promoter activities (Fig. 3A). As in the COS-1 cells, SREBP-2 was
relatively ineffective in stimulating StAR promoter activity compared
with SREBP-1a in human granulosa-lutein cells (Fig. 3B). However,
SREBP-1a and -2 stimulated LDL receptor promoter activity to a similar
extent in human granulosa-lutein cells (Fig. 3B).
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). The -1300, -235, -150, and -95
promoter constructs had similar strong responses to SREBP-1a. The -885
promoter construct gave the greatest response. The shorter StAR
promoters (-60 and -43) displayed significantly less
SREBP-1a-stimulated activity compared with the larger promoter
constructs.
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) in the -95 and
-235 StAR promoter constructs. Coexpression of SREBP-1a and the
wild-type -95 StAR reporter construct resulted in pronounced
enhancement (
20-fold greater) of StAR promoter activity over that in
cells transfected with the empty pCMV5 vector alone (Fig. 4, A and B).
A promoter construct in which all three putative SREBP-binding sites
were altered exhibited 20% the activity of the wild-type promoter in
response to SREBP-1a (Fig. 4B).
The putative SREBP-binding site could also act as a recognition site
for YY1. To discern whether YY1 was able to elicit changes in StAR
promoter activity, the StAR promoter was mutated in such a manner that
the putative YY1-binding site was disrupted, whereas the overlapping
SREBP-1a response element was retained (see Fig. 1). Mutation of the
YY1-binding site enhanced SREBP-1a-dependent StAR promoter activity
(mean relative luciferase units ± SEM, 1,056,399
± 41,627; n = 3) 2.5-fold compared with the -95 to +39 wild-type
StAR promoter (496,179 ± 104,401).
Absence of sterol regulation of StAR gene expression in
proliferating human granulosa-lutein cells
To determine whether modulation of endogenous SREBP levels affects
StAR promoter activity, we cultured COS-1 and human granulosa-lutein
cells under conditions that stimulate the production of the mature form
of SREBP-1a (i.e. lipoprotein-deficient medium) and then
added LDL and 27-hydoxycholesterol, which are known to suppress the
release of mature SREBPs (Fig. 5). These
studies were conducted with the -235 to +39 StAR reporter construct.
In these experiments we were unable to detect sterol regulation of StAR
promoter function in either COS-1 or human granulosa-lutein cells, even
though LDL receptor promoter activity was suppressed in the
sterol-replete medium.
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depicts the results of Western analysis
for these transcription factors. Granulosa-lutein cells cultured under
control conditions (without serum) contained mature SREBP-1a (66 kDa)
within their nuclei (Fig. 6A). Addition of 27-hydroxycholesterol caused
a marked decline in mature SREBP-1a levels within granulosa-lutein
nuclei, as would be anticipated.
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). Treatment of
granulosa-lutein cells with LDL and 27-hydroxycholesterol did not
elicit significant changes in nuclear YY1 levels.
Transcription factors SREBP-1a, SREBP-2, and YY1 interact with the
-82 to -70 region of the StAR promoter
The DNA-binding domains of SREBP-1a and -2 were both able to cause
a gel shift of the -90 to -65 StAR promoter fragment (Fig. 7A), although the complexes formed with
SREBP-1a (amino acids -321 to -490) were more prominent than those
formed with SREBP-2 (amino acids -331 to -481) at equivalent amounts
of protein. The gel shift bands observed for the StAR oligonucleotide
with the recombinant SREBPs were identical to those observed for the
control HMG-CoA reductase oligonucleotide. Both SREBP-1a and -2
DNA-binding domains exhibited a dose-dependent interaction with the
wild-type StAR oligonucleotide. However, at equivalent recombinant
SREBP protein input, the interaction between the StAR promoter
oligonucleotide and the recombinant SREBP DNA-binding domains was much
weaker than that for the HMG-CoA reductase oligonucleotide, which
contains a well characterized SREBP-binding site (Fig. 7A).
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). Again, the
gel shift bands for the StAR oligonucleotide and the HMG-CoA reductase
exhibited identical mobility, while the amount of interaction was
significantly less for the StAR promoter at equivalent amounts of input
protein. To demonstrate specificity, a mutant oligonucleotide (triple
mutant) in which all three putative SREBP-binding sites were mutated
formed only a weak complex with the highest dose of recombinant
SREBP-1a and was about 50-fold less than that with the wild-type StAR
oligonucleotide.
Comparison of the LDL receptor and StAR oligonucleotide binding to the
full-length SREBP-1a was also completed (Fig. 8). Similar to the HMG-CoA reductase, the
LDL receptor oligonucleotide bound SREBP-1a with greater affinity than
the StAR oligonucleotide. We observed dose-dependent binding of
SREBP-1a to both the LDL and StAR oligonucleotides in gel shift assays.
However, the affinity of the LDL receptor oligonucleotide for SREBP-la
was approximately 8-fold greater than that for the StAR
oligonucleotide. This relative difference in binding affinity
correlates well with the trans-activation studies carried
out in transiently transfected COS-1 cells with the LDL receptor and
StAR reporter constructs shown in Fig. 2. Additionally, we observed
that mutation of the YY1 site had no effect on SREBP-1a binding.
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and 9) that could be competed away with
the addition of unlabeled StAR oligonucleotide. The StAR
oligonucleotide with a single base pair change in the proximal SREBP
site (YY1 mutant) that should disrupt only YY1 binding and have no
effect on SREBP’s ability to bind caused a significant reduction in
recombinant YY1’s ability to elicit a shift of the labeled probe (Fig. 9). Disruption of the proximal
SREBP/YY1-binding site by a single base pair substitution designed to
prevent binding of both SREBP and YY1 also caused a loss in recombinant
YY1’s ability to elicit a gel shift. The positive control HMG-CoA
reductase oligonucleotide that also contains a SREBP/YY1 motif elicited
a gel shift at the same location and of a similar magnitude as that of
the StAR promoter. Additionally, we demonstrated that these mutations
were specific, as incubation with a 20-fold excess of the SREBP/YY1 and
YY1 mutant (cold competitor) failed to cause a loss in YY1’s ability
to elicit a gel shift with the wild-type StAR oligonucleotide.
Conversely, incubation with as little as 2-fold greater amounts of the
wild-type StAR oligonucleotide caused a pronounced loss in YY1 binding
to the labeled StAR oligonucleotide (20-fold; results shown in Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SREBPs are basic helix-loop-helix leucine zipper transcription factors that regulate a variety of genes involved in cholesterol metabolism (LDL receptor, HMG-CoA reductase and synthetase, squalene synthase, and cholesterol ester transferase protein) (11, 12) and fatty acid synthesis (fatty acid synthase and acetyl-coenzyme A carboxylase) (11, 23, 24). In cell culture systems, both SREBP-1a and -2 increase promoter activity through sterol response elements with equal effectiveness (9, 10). In preliminary studies we reported that SREBP-1a could significantly enhance StAR promoter activity after transient expression in CV-1 and COS-1 cells (25). The present studies confirm and extend these observations to show that human granulosa-lutein cells also exhibit a significant increase in StAR promoter activity after exogenous expression of SREBP-1a. These studies demonstrate a differential response between SREBP-1a and SREBP-2 in a natural promoter context. Mutational analysis of the human squalene synthase gene indicates that this gene may also respond to SREBP-1a and SREBP-2 differentially (26). Interestingly, the squalene synthase promoter is more sensitive to SREBP-2, contrasting with our observations that the StAR promoter has greater affinity for and is more responsive to SREBP-1a. In addition to demonstrating that mutation of the two of the three SREs could completely abolish SREBP-1a-dependent promoter activity while not affecting SREBP-2 responsiveness, these investigators demonstrated that mutation of two GC elements known to interact with Sp1 affected SREBP-2-dependent promoter activity while not affecting SREBP-1a-dependent promoter activity. Recently, Gauthier et al. (12) observed that the SREBP-binding site within the cholesterol ester transfer protein promoter was more responsive to SREBP-1a than to SREBP-2. These experiments give precedent for a differential response between SREBP-1a and -2 that can depend upon either the response element or other transcriptional activators. Although there was a selectively greater response to SREBP-1a, we observed that the isolated recombinant DNA-binding domains of both SREBP-1a (amino acids 321–490) and SREBP-2 (amino acids 331–481) bound the -90 to -65 StAR promoter oligonucleotide, further confirming that this is a specific interaction. As both SREBP-1a and -2 bound to the StAR promoter in the gel shift assays, although the interaction was greatest for SREBP-1a, it is possible that chromatin structure and or other proteins (transcription factors/coactivators) account for the selective SREBP-1a response of the StAR promoter. Alternatively, the differential response of the StAR promoter to SREBP-1a and SREBP-2 may reflect the apparent differences in binding affinities revealed in the gel shift assays.
SREBPs were originally identified as transcription factors that recognize a 10-bp sterol regulatory element within the HMG-CoA synthase and LDL receptor promoters (9, 10). We have identified a series of three potential SRE half-sites within the first 115 bases from the transcription start site of the StAR promoter. Mutational analysis indicated that these three sites are essential for maximal SREBP-1a-induced trans-activation. These studies, however, do not rule out the possibility that an additional site(s) located within this region of the promoter responds to SREBP-1a. Although we have concentrated on the narrow region between -90 to -65 of the StAR promoter, other SREBP-binding elements outside of this region may be present in the StAR gene. Our data indicate that, at least in COS-1 cells, the region between -95 and -60 is where SREBP-1a exerts its greatest effects. Additionally, mutation of the binding sites within the StAR promoter caused a pronounced loss of the ability of SREBPs to bind to this promoter, as indicated by gel shift analysis.
YY1 is ubiquitously expressed throughout the body (27). As expected, Western analysis clearly identified full-length YY1 (68-kDa) protein within nuclear extracts obtained from human granulosa cells. The role of YY1 in StAR gene expression is complicated by the fact that this protein has diverse roles; YY1 is known to act as both a transcriptional enhancer and repressor as well as a possible initiator binding protein depending upon the cell host and promoter context (28). Moreover, YY1 can activate and repress the same promoter if the intracellular milieu is altered, the binding element within the promoter is inverted, or the surrounding DNA sequence context is altered (29). We found a YY1 site within the StAR promoter that overlaps with the proximal SREBP-1a-binding site. We demonstrated that mutation of the YY1-binding site (i.e. maintaining the ability of SREBP-1a to trans-activate) enhanced the SREBP-1a response severalfold. This observation is consistent with studies of the HMG-CoA synthase promoter, which has a YY1-binding site (16). In several well characterized promoters in which YY1 has overlapping binding sites with transcriptional activators, YY1 has been shown to displace the activators and thus repress gene expression (16, 29). Additionally, YY1 could act as an active repressor once it is bound to the promoter. Our data indicating that SREBP-1a has a greater stimulatory effect in the YY1 mutant construct support such a mechanism. Our EMSA results clearly demonstrate that YY1 can specifically bind to the wild-type StAR promoter, and mutation of a single base that eliminates the YY1 site prevents a 20-fold excess of this oligonucleotide from competing away the specifically bound StAR oligonucleotide. Conversely, a 2-fold excess of the cold wild-type StAR oligonucleotide reduced the amount of labeled StAR oligonucleotide bound to YY1. The HMG-CoA synthase and reductase promoters also contain both SREBP- and YY1-binding elements (15), and recent studies examining these promoters indicate that YY1 is able to inhibit SREBP-dependent induction of HMG-CoA synthase elements (15, 16), but not the HMG-CoA reductase gene promoter (15). Mutation of the YY1 site within the StAR oligonucleotide failed to increase SREBP-1a binding in gel shift analyses. This suggests that endogenous YY1 most likely inhibits SREBP-1a-dependent StAR trans-activation when it is able to bind to the site adjacent to the SREBP-binding site in the StAR promoter.
The physiological significance of SREBP-1a action on StAR transcription remains to be determined. It is evident that changes in levels of SREBP-1a in response to alterations in cholesterol availability do not affect StAR gene expression. This might be predicted, as it would be counterproductive for a steroidogenic cell to reduce StAR gene expression, and thus steroid synthesis, while simultaneously trying to accumulate substrate for hormone production. However, a role for SREBP-1a in the tropic hormone up-regulation of StAR gene expression can be envisioned in certain circumstances. As granulosa cells luteinize during the periovulatory period, they dramatically increase their capacity to synthesize cholesterol de novo, take up LDL, and produce progesterone (30). This is a situation in which messenger RNAs for HMG-CoA reductase and low/high density lipoprotein receptors and StAR all rapidly accumulate to high levels (31, 32, 33). Lopez and McLean (34) recently observed that hCG-induced granulosa cell luteinization is associated with an 11-fold increase in the mature form of SREBP-1a in the rat ovary. These high levels of mature SREBP-1a would be expected to result in coordinated up-regulation of genes involved in cholesterol acquisition (LDL receptor and HMG-CoA reductase) and metabolism to steroid hormones (StAR). Thus, the StAR promoter may be conditionally responsive to SREBP-1a. This conditional response, observed only in the presence of elevated SREBP-1a levels, may be explained by the relatively lower affinity of the SREBP binding motif in the StAR promoter for SREBPs compared with the motifs in the LDL receptor, HMG-CoA reductase, and other SREBP-regulated genes.
In conclusion, we have shown that the human StAR promoter displays a selective response to SREBP-1a, that a SREBP-binding site is located in a transcription factor-binding site-rich region of the promoter adjacent to a YY1-binding site, and that changes in endogenous SREBP-1a levels in response to cholesterol availability that impact LDL receptor promoter activity do not affect StAR promoter function, suggesting that the human StAR promoter is conditionally responsive to high levels of SREBP-1a.
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Acknowledgments |
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Footnotes |
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Received June 19, 2000.
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