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Experimental Studies |
Department of Pediatrics and the Metabolic Research Unit, University of California, San Francisco, California 94143-0978
Address all correspondence to and requests for reprints to: Dr. Walter L. Miller, Building MR IV, Room 209, University of California, San Francisco, California 94143-0978.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The transcriptional regulation of the genes for steroidogenic enzymes
is cell-specific and developmentally programmed (4, 5, 6). The activity of
the human promoter for the cholesterol side-chain cleavage enzyme,
P450scc (also termed CYP11A), has been examined in transfected Y1
murine adrenal (7, 8, 9, 10), MA-10 murine Leydig (11), and JEG-3 human
choriocarcinoma cells (12, 13, 14, 15). In Y1 or MA-10 cells, sequences between
-44 to -56 bp (10) and between -110 and -127 (9, 11, 15) function
as basal transcriptional elements, and sequences between -1541 and
-1676 bp (9, 10, 11) and possibly also between -100 and -118 (13, 14)
function as cAMP-responsive elements. However, when human P450scc
promoter/reporter constructions are transfected into JEG-3 cells,
sequences between -142 and -152 function as a positive basal element,
those between -152 and -177 function as a negative basal element, and
cAMP inducibility is conferred by sequences between -89 and -108 (12, 14, 15). Thus, the human P450scc promoter behaves similarly in mouse
adrenal Y1 and Leydig MA-10 cells, but behaves very differently in
human placental JEG-3 cells. The transcriptional regulation of the gene
for P450c17 (17
-hydroxylase/17,20-lyase) (also termed CYP17), has
been studied primarily with bovine (16, 17, 18) or rodent (19) promoters.
The single study of human P450c17 gene transcription found that the
promoter was highly active in mouse adrenal Y1 cells and minimally
active in mouse MA-10 cells, which is different from the patterns of
expression of the endogenous cellular genes or of the gene in normal
mouse tissues (20). Thus, there has been some concern about the
significance of results obtained when some human promoters are analyzed
in nonhuman cell systems.
The trans-acting nuclear factors needed for the specific activation of a particular promoter are not thought to vary much among mammalian species, so that mouse cells have been widely used for studies of the bovine and human genes for various steroidogenic enzymes (7, 8, 9, 10, 11, 13, 14, 16, 17, 18, 20, 21, 22). The substantial differences seen in P450scc promoter activity in mouse adrenal Y1 and Leydig MA-10 cells compared to human JEG-3 placental cells is generally thought to reflect a tissue-specific difference rather than a species-specific difference (12, 13, 14, 15), but the human P450scc and P450c17 promoters have not been studied in human Leydig or adrenal cells. Studies in primary cultures of human adrenal cells are compromised by poor availability, rapidly diminishing responsiveness to ACTH and cAMP (23), and irreversible senescence (24).
Therefore, to assess whether studies of the human P450scc and P450c17 promoters in mouse Y1 adrenal cells or other cells predict their activities in human adrenal cells, we examined the regulation of these promoters transfected into human adrenal NCI-H295 cells.
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Materials and Methods |
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NCI-H295 cells were grown as previously described (2). Adherent NCI-H295 cells (growing in monolayer in 10% FCS) were isolated by repeatedly aspirating off medium (with the suspended cells) and adding fresh medium over the course of several months; the resulting subline is termed NCI-H295A (adherent). Transfections of NCI-H295A cells were performed in DMEM with 3 g glucose/L (DMEM-H16) supplemented with 10% FBS and penicillin/streptomycin (20 U/mL; 20 µg/mL). Growth and transfections of Y1 (9), MA-10 (11), and JEG-3 cells (12) were previously described. For transfection, all cells were divided into 2-cm, six-well plates (Falcon 3046, Becton Dickinson Co., Lincoln Park, NJ) at approximately 50% confluence 24 h before transfection.
Plasmids
The construction of the P450scc deletion/luciferase plasmids has
been previously described (12, 15). The 5'-flanking DNA of the human
P450c17 gene was obtained from the 5'-sequence from a 5.6-kilobase (kb)
pUC18 clone of the P450c17 gene (25). The 3'-overhangs were eliminated
from the pUC18 clone digested with DraIII. A 2.5-kb promoter
fragment was liberated with EcoRI and ligated into
pBluescript SK+ (Stratagene, La Jolla, CA) previously
digested with SmaI and EcoRI. This pBluescript
clone was then digested with PvuI (to cleave the vector),
HindIII, and BamHI, liberating a 2.5-kb promoter
fragment, which was then ligated into the pMG3 luciferase vector (12)
(prepared using BclI and HindIII digestion)
giving rise to the -2.5-kb luciferase clone. The pUC18 P450c17 genomic
clone was also digested with EcoRI and HindIII,
liberating a 5.4-kb EcoRI fragment. The -2.5-kb luciferase
clone was then digested with EcoRI and HindIII,
giving rise to the luciferase vector with 1.2 kb of promoter sequence
and two EcoRI sites. Ligation of these fragments produced a
3.7-kb P450c17 promoter luciferase reporter construct. Further
deletional constructions were performed by PCR amplification of
segments of the -2.5-kb luciferase clone as a template using the
(sense) oligonucleotides listed in Table 1
and the
antisense oligonucleotide 5'-CTAGAGGATAGAATGGCGCCG-3', which
corresponds to vector sequences downstream from the cloning site. All
constructions were verified with restriction enzyme digestion and
sequencing. Constructions consisting of 235 and 542 bp of the Z
promoter fused to the luciferase gene (-235Z luc and -542Z luc) (26)
were used as positive controls.
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Plasmids were purified on Qiagen columns (Qiagen, Chatsworth, CA). After the medium was aspirated off and the transfection medium was added, transient transfections were carried out for 24 h using calcium phosphate precipitates (27) with 5 µg promoter/reporter construct. Preliminary experiments showed no differences with incubations of 12, 24, and 48 h. Thus, the medium was changed again after the transfection period, and the cells were allowed to grow for an additional 48 h in the presence or absence of 100 µmol/L 8-bromo-cAMP (8Br-cAMP; Sigma Chemical Co., St. Louis, MO) before harvesting. Cells in suspension were harvested, and cellular extracts were prepared as previously described (9) for subsequent measurement of luminescence (12). Transfection efficiency was normalized by cotransfecting 1.0 µg/well of a ß-galactosidase (ß-gal) plasmid driven by the cytomegalovirus promoter, with subsequent measurement of ß-gal activity. Luciferase values were normalized by dividing all ß-gal values by the smallest values; the resultant value was then used to divide the luciferase values obtained from the corresponding cell extracts.
ß-Gal assay
To assess ß-gal activity, a reaction mixture was prepared using 450 µL/sample of Z buffer (100 mmol/L Na2HPO4, pH 7.0; 10 mmol/L KCl; and 1 mmol/L MgSO4), 100 µL/sample ONPG (4 mg/mL), and ß-mercaptoethanol (3.4 µL/1 mL Z buffer; both from Sigma Chemical Co.). Five hundred and fifty microliters of this reaction mixture were add to 50 µL of each cell extract and mixed at 20-s intervals. The reaction was allowed to proceed at room temperature until the solution was visibly yellow, and then 500 µL Na2HCO3 were added, again at 20-s intervals, to stop the reactions. Absorption was read at 420 nm, and the resultant value was used to divide the luciferase values obtained from the corresponding wells.
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Results |
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Initial experiments showed that NCI-H295 cells grown in suspension
could be transfected by several procedures, including lipofection,
diethylaminoethyl-dextran, and direct addition of calcium phosphate
precipitates. All three procedures yielded comparable, but low,
transfection efficiencies. Transfection with calcium phosphate
precipitates induced NCI-H295 cells in suspension to form monolayers,
and such transfections have been used successfully (26, 28). However,
we noted that with each passage of NCI-H295 cells, about 10–20% of
the cells remained adherent to the dishes, as noted previously (3, 29).
Therefore, we isolated a subline of adherent (A) NCI-H295A cells to
determine whether they might be more easily transfected than standard
NCI-H295 cells in suspension. Calcium phosphate transfection of a Rous
sarcoma virus (RSV)-Luc vector showed that the transfection efficiency
with the monolayer NCI-H295A cells was 10-fold more efficient than
transfection of NCI-H295 cells in suspension (Fig. 1).
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We have recently characterized two powerful, adrenal-specific
promoters of the newly discovered human XB-S (28) and Z genes (26)
using transiently transfected NCI-H295 cells in suspension. Therefore,
we transfected NCI-H295A cells with the Z promoter (which is the more
powerful of these two), with the shortest constructions of P450scc
(-79) and P450c17 (-63) that yielded basal activity, and with our
longest available constructs of the human P450scc and P450c17
promoters. All of these promoters gave activities between 5–40% of
the maximal activity seen with the control RSV construct, RSV-Luc (Fig. 2). Thus, promoters previously shown to work in NCI-H295
cells in suspension had similar activity in NCI-H295A cells, and the
maximal activities of the human P450scc and P450c17 promoters were in
this readily detectable, informative range of luciferase activities.
Therefore, the NCI-H295A cells retain all of the features needed for an
informative host cell system.
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Regulation of P450scc promoter/luciferase reporter deletion constructs
The 5'-flanking DNA of the human P450scc gene regulates the
expression of reporter genes in a physiologically appropriate fashion
when transiently transfected into mouse adrenal Y1 (9, 10), mouse
testicular MA-10 (11), and human choriocarcinoma JEG-3 cells (12, 13, 14, 15).
To determine the transcriptional regulation conferred by this
5'-flanking DNA in NCI-H295A cells and to compare this regulation to
that found in other steroidogenic cells, we used our previously
described (12, 15) deletional constructions of the 5'-flanking regions
of the human P450scc gene linked to a firefly luciferase reporter gene.
Transient transfection into NCI-H295A cells revealed maximal
transcriptional activation within the first 79 bp upstream (-79) from
the transcriptional start site of the P450scc gene without an increase
by adding further sequences to -2327 (Fig. 3A). The
variation in the activities of the constructions from -79 to -2327
was not statistically significant by Student-Newman-Keuls analysis. To
locate cAMP-responsive sequences, we incubated the cells with 100
µmol/L 8Br-cAMP for 48 h, conditions that elicit the maximal
effect on P450scc mRNA (2). The responsiveness to 8Br-cAMP was also
conferred by sequences within the first 79 bp upstream from the
transcriptional start site (Fig. 3B). Again, the variations in the
activities of the longer constructions were not statistically
significant. Although the four longest constructions appeared to confer
minimal induction, they consistently conferred greater induction than
the 
luc control.
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Similar studies with P450c17 promoter constructions identified a
basal element within the first upstream 63 bp (Fig. 4A).
The variations in the activities of the constructions from -63 to
-184 were not statistically significant. One or more additional basal
elements in the -184 to -206 region elicited a more than 2-fold
increase in transcriptional activity that was statistically
significant. Addition of further upstream sequences to -3.7 kb had
little effect. Responsiveness to cAMP was conferred by sequences within
the first 63 bp, and addition of upstream DNA to 3.7 kb did not result
in a statistically significant change in cAMP induction (Fig. 4B).
Although the cAMP responsiveness of some of the longer constructs was
rather modest, all consistently conferred greater induction than
luc.
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The results for the human P450scc promoter shown in Fig. 3
are
substantially different from what we (9) and others (7, 8, 10, 13, 14)
reported previously for the activity of this promoter transfected into
mouse adrenal Y1 cells. Similarly, the results for the human P450c17
promoter in Fig. 4 are substantially different from what we (20) and
others (30) found when that promoter was analyzed in mouse adrenal Y1
cells. To ensure that there were no subtle but important changes in our
materials or procedures that might have made our present results in
NCI-H295 cells differ from our previous results in Y1, MA-10, and JEG-3
cells, we transfected selected P450scc and P450c17 constructions into
Y1, MA-10, JEG-3, and NCI-H295A on the same day using the same DNA
preparation in each cell type (Fig. 5). For P450scc, the
-79 construction showed maximal activity in NCI-H295 cells, with no
change in the longer constructions, as in Fig. 3. In JEG-3 cells, the
-152 construction showed tremendous activity, but the -79 and -2327
constructions did not, as reported previously (12, 15). The P450scc
results in Y1 and MA-10 cells were equivalent, with most basal activity
seen in the -79 and -152 constructions, consistent with our finding
of basal elements between 0 and -79 and between -110 and -127, as
described previously (9, 11). For P450c17, basal activity was again
seen at -63 and -206 in NCI-H295A cells, as shown in Fig. 4; the -63
activity was a bit lower in this single experiment, but was within the
range of the experiments constituting Fig. 4. In JEG-3 cells, a very
modest level of activity (5 times Luc) was seen with the -63
construction and was less with longer constructions, consistent with
the nearly absent activity of this promoter in JEG-3 cells reported
previously (20). The activities in Y1 and MA-10 cells were
qualitatively similar, but quantitatively much greater in MA-10 cells,
as described previously (20), and the most basal activity was found in
the -206 construction, consistent with our previous report of a basal
element between -184 and -235 (20). Thus, the data presented in Fig. 5 reconfirm results that we and others reported previously for the
activities of the P450scc (7, 8, 9, 10, 11, 12, 13, 14, 15) and P450c17 (20) promoters.
Therefore, the differences between the data in Figs. 3 and 4, and the
previously reported data are due to host cell-specific differences and
not to experimental variation or artifact.
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Discussion |
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Human adrenal NCI-H295 cells should be an ideal model in which to study the regulation of steroidogenic enzyme genes, because these cells express all of the genes for the steroidogenic enzymes in a hormonally regulated fashion that appears to reflect the regulation of primary cultures of human fetal adrenal cells (2). NCI-H295 cells were originally selected as floating aggregates to eliminate fibroblast contamination (1), but subpopulations of these cells can be selected that adhere to plastic dishes (29, 32). We developed a subline of adherent NCI-H295A cells that was 10-fold more susceptible to calcium phosphate transfection than those grown in suspension, thus facilitating the examination of the activities of the human P450scc and P450c17 promoters. We also used these cells to analyze the activity of the human TN-X gene in the 21-hydroxylase locus (33). By transiently transfecting deletional promoter/reporter constructions for the human P450scc and P450c17 genes, we identified the regions conferring basal and cAMP-induced transcription of these two genes in human adrenal cells. Our data demonstrate that the transcriptional regulation of these genes in human adrenal cells differs substantially from the widely used Y1 model.
In NCI-H295A cells, the P450scc gene demonstrates both basal and
cAMP-induced transcriptional activity within the first 79 bp upstream
from the transcriptional start site. The cAMP-responsive region
identified in the human adrenal NCI-H295A cells is different from those
found in the other cell types studied. Table 2 lists the
regulatory regions identified in previous studies of the human P450scc
promoter in other cells. Examination of the sequence of the first 79 bp
of the human P450scc promoter reveals the possible CAAT-box CATT at
-66 to -63, a putative binding site for cAMP response element-binding
protein at -50 to -57, an SF-1 site at -38 to -46, and an unusual
TATA box sequence at -29 to -24. cAMP inducibility of both the human
aromatase gene (34) and the rat P450c17 gene (35) is conferred by an
SF-1 site. Thus, the cAMP inducibility of the -79 construct may be
conferred by the SF-1 site at -38 to -46.
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). Unfortunately, none of the cell models
previously available exhibit endogenous expression of P450c17, even
though regulation of human P450c17 promoter/reporter constructs has
been possible using the Y1 mouse adrenocortical cells. P450c17 promoter
analysis in the NCI-H295A cells identified both basal and
cAMP-responsive elements within the first 63 bp and a second basal
element between -184 and -206 bp. The first 63 bp of the human
P450c17 promoter contains the unusual TATA box TTTAAA at -19 to -24,
which is also found in rat, mouse, pig, and bovine P450c17 promoters
(19), an ASP/Sp1 site at -8 to -19, and an SF-1 site at -58 to -50.
As discussed above, such SF-1 sites may confer both basal and
cAMP-inducible expression. The -184 to -206 region that confers
additional basal activity corresponds well with the observation that
substantial basal activity lies between -184 and -235 when the
promoter is transfected into mouse adrenal Y1 cells (20). The site
within this sequence that confers this activity is not known, although
it does contain a sequence resembling an SF-1 site at -195 to
-200. Both the amount of transcription and the locations of the identifiable regulatory regions in the P450scc and P450c17 5'-flanking regions differed substantially in NCI-H295 cells compared to their activities in other cell systems used to date. The levels of activity seen with both the P450scc and P450c17 promoters is consistent with the levels of expression seen with other promoter/reporter constructions (26, 28) and with the RSV-Luc construction, all transfected into the subline of adherent NCI-H295A cells. Tissue-specific differences in the activities of transcriptional regulatory elements probably account for the differences between the behaviors of these promoters in NCI-H295A adrenal cells and their behaviors in human placental JEG-3 cells. However, species specificity probably accounts for the qualitative differences in the cis-acting regions identified in the human NCI-H295A cells compared to the mouse Y1 adrenal and MA-10 Leydig cells. The 5'-flanking region of the human P450c17 gene promotes transcription of reporter genes in Y1 cells (20), even though neither the mouse adrenal nor Y1 cells express their endogenous P450c17 gene.
As we have only examined 2327 and 3700 bp of 5'-flanking DNA of the P450scc and P450c17 genes, respectively; it is possible that transcriptional regulation of these genes in the human adrenal may require additional 5'- or 3'-flanking sequences for endogenous promoter activity. Many other genes, such as those for ß-globin (36, 37) or human chorionic somatomammotropin (38, 39), require the activity of distant locus control regions. Similarly, only 230 bp of 5'-flanking DNA is needed to elicit physiologically significant basal and cAMP-responsive expression of the mouse P450c21 promoter in mouse adrenal Y1 cells (40, 41), but two additional regions located 5.3 and 5.8 kb upstream are required for this promoter to function in the mouse adrenal in vivo (42). Thus, additional distant elements may be needed for complete expression of the human P450scc and P450c17 genes in the highly differentiated human adrenal NCI-H295 cells, although 2337 bp of the human P450c17 promoter suffices for adrenal-specific expression in transgenic mice (43). Similarly, as NCI-H295 cells resemble zonally undifferentiated fetal adrenal cells (2), there may be additional differences due to developmental staging in the behaviors of these promoters in NCI-H295 cells compared to their expression in human adrenals in vivo. However, despite these potential difficulties, NCI-H295 cells remain the only cellular model of the human adrenal cortex developed to date, and the present studies clearly show that mouse adrenal Y1 and Leydig MA-10 cells are not appropriate models for studying the activities of these human promoters.
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Acknowledgments |
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Footnotes |
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Received September 3, 1996.
Revised October 22, 1996.
Accepted October 22, 1996.
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References |
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) gene. J Biol Chem. 265:3304–3312.-hydroxylase/17,20 lyase) in
cultured human fetal adrenal cells. J Clin Endocrinol Metab. 65:170–175.-hydroxylase/17,20 lyase): similarity to the gene for P450c21. DNA. 6:439–448.[Medline]
-hydroxylase/17,20-lyase) in cultured human granulosa
cells. J Clin Endocrinol Metab. 63:202–207.-hydroxylase/c17,20 lyase). Mol Endocrinol. 10:147–158.This article has been cited by other articles:
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M. Y. H. Zhang, X. Wang, J. T. Wang, N. A. Compagnone, S. H. Mellon, J. L. Olson, H. S. Tenenhouse, W. L. Miller, and A. A. Portale Dietary Phosphorus Transcriptionally Regulates 25-Hydroxyvitamin D-1{alpha}-Hydroxylase Gene Expression in the Proximal Renal Tubule Endocrinology, February 1, 2002; 143(2): 587 - 595. [Abstract] [Full Text] [PDF]
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N. A. Hanley, W. E. Rainey, D. I. Wilson, S. G. Ball, and K. L. Parker Expression Profiles of SF-1, DAX1, and CYP17 in the Human Fetal Adrenal Gland: Potential Interactions in Gene Regulation Mol. Endocrinol., January 1, 2001; 15(1): 57 - 68. [Abstract] [Full Text]
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J. W. M. Martens, D. H. Geller, W. Arlt, R. J. Auchus, V. S. Ossovskaya, H. Rodriguez, A. Dunaif, and W. L. Miller Enzymatic Activities of P450c17 Stably Expressed in Fibroblasts from Patients with the Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 4338 - 4346. [Abstract] [Full Text]
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J. K. Wickenheisser, P. G. Quinn, V. L. Nelson, R. S. Legro, J. F. Strauss III, and J. M. McAllister. Differential Activity of the Cytochrome P450 17{alpha}-Hydroxylase and Steroidogenic Acute Regulatory Protein Gene Promoters in Normal and Polycystic Ovary Syndrome Theca Cells J. Clin. Endocrinol. Metab., June 1, 2000; 85(6): 2304 - 2311. [Abstract] [Full Text]
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N. Huang and W. L. Miller Cloning of Factors Related to HIV-inducible LBP Proteins That Regulate Steroidogenic Factor-1-independent Human Placental Transcription of the Cholesterol Side-chain Cleavage Enzyme, P450scc J. Biol. Chem., January 28, 2000; 275(4): 2852 - 2858. [Abstract] [Full Text] [PDF]
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Y. Chuman, Z. Zhan, and T. Fojo Construction of Gene Therapy Vectors Targeting Adrenocortical Cells: Enhancement of Activity and Specificity with Agents Modulating the Cyclic Adenosine 3',5'-Monophosphate Pathway J. Clin. Endocrinol. Metab., January 1, 2000; 85(1): 253 - 262. [Abstract] [Full Text]
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S. D. Wijesuriya, G. Zhang, A. Dardis, and W. L. Miller Transcriptional Regulatory Elements of the Human Gene for Cytochrome P450c21 (Steroid 21-Hydroxylase) Lie within Intron 35 of the Linked C4B Gene J. Biol. Chem., December 31, 1999; 274(53): 38097 - 38106. [Abstract] [Full Text] [PDF]
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M.-C. Hu, S.-J. Chou, Y.-Y. Huang, N.-C. Hsu, H. Li, and B.-c. Chung Tissue-Specific, Hormonal, and Developmental Regulation of SCC-LacZ Expression in Transgenic Mice Leads to Adrenocortical Zone Characterization Endocrinology, December 1, 1999; 140(12): 5609 - 5618. [Abstract] [Full Text]
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D. Monte, F. DeWitte, and D. W. Hum Regulation of the Human P450scc Gene by Steroidogenic Factor 1 Is Mediated by CBP/p300 J. Biol. Chem., February 20, 1998; 273(8): 4585 - 4591. [Abstract] [Full Text] [PDF]
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P. Zhang and S. H. Mellon Multiple Orphan Nuclear Receptors Converge to Regulate Rat P450c17 Gene Transcription: Novel Mechanisms for Orphan Nuclear Receptor Action Mol. Endocrinol., June 1, 1997; 11(7): 891 - 904. [Abstract] [Full Text]
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F. Gizard, B. Lavallee, F. DeWitte, and D. W. Hum A Novel Zinc Finger Protein TReP-132 Interacts with CBP/p300 to Regulate Human CYP11A1 Gene Expression J. Biol. Chem., August 31, 2001; 276(36): 33881 - 33892. [Abstract] [Full Text] [PDF]
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