(Received for publication, January 28, 1997, and in revised form, April 15, 1997)
From Second Department of Internal Medicine, Yokohama
City University School of Medicine, Yokohama 236, Japan and the
¶ Institute of Applied Biochemistry, University of Tsukuba,
Ibaraki 305, Japan
The protein product of the retinoblastoma
susceptibility gene, RB, is a nuclear phosphoprotein that modulates
transcription of genes involved in growth control via interactions with
transcription factors. Renin is a rate-limiting enzyme of the
renin-angiotensin system that regulates blood pressure and
water-electrolyte balance. Renin gene expression is regulated in a
tissue-specific and developmentally linked manner. Similarly, the
expression of RB is controlled in a differentiation-linked manner.
Thus, to investigate whether RB is involved in the regulation of renin
gene expression, we examined the effects of RB on transcriptional
activity of the mouse renin (Ren-1C) promoter. The
Ren-1C promoter contains two transcriptionally important
elements; the RU-1 (224 to
138) and RP-2 (
75 to
47) elements.
RB activated the Ren-1C promoter in human embryonic kidney
cells. The promoter element responsible for RB-mediated transcriptional
regulation was the RP-2 element. The results of DNA-protein binding
experiments showed that RB increased nuclear binding activity to the
RP-2 element, and site-directed mutation which disrupted binding of
nuclear factors to the RP-2 element markedly reduced RB-mediated
activation of Ren-1C promoter in human embryonic kidney
cells. These results indicate that the RP-2 element plays an important
role in RB-mediated transcriptional regulation of Ren-1C
promoter activity in human embryonic kidney cells, thereby suggesting
an interesting mechanism by which RB may modulate the renin-angiotensin
system.
Selective gene expression is mostly controlled at the level of
transcription (1). Regulation of transcriptional activity is achieved
through the binding of a series of transcriptional factors to
sequence-specific DNA elements (2-4). The identification of both
cis-acting elements and nuclear factors in specialized cells
facilitates understanding of the molecular mechanisms that underlie
tissue- and development-specific gene expression. Renin, an aspartyl
protease, plays an important role in the regulation of blood pressure
and water-electrolyte balance by catalyzing the rate-limiting step of
the renin-angiotensin system, and may be involved in the pathogenesis
of hypertension (5-7). The expression of renin gene is regulated in a
tissue-specific and development-linked manner (8, 9), and the main site
of production of circulating renin is the kidney. Transgenic studies
previously demonstrated that the 5-flanking region of the human renin
gene directs tissue-specific expression in the kidney (10, 11), and
that 5
-flanking sequences of the mouse renin gene (Ren-2)
directed tissue- and development-specific expression of the reporter
SV40 T antigen gene (12, 13). In in vitro studies, we
identified two transcriptionally important promoter elements (RU-1;
224 to
138, and RP-2;
75 to
47) in the mouse renin gene
(Ren-1C), and demonstrated that the combination of these
elements was responsible for cell type-specific transcriptional activity of Ren-1C gene in transfected human embryonic
kidney (HEK)1 cells (14, 15). Furthermore,
recent studies using pituitary and kidney cell lines indicated that
novel factors bound to the proximal promoter and regulated
transcription of human and mouse renin genes (16, 17). 5
-Flanking
sequences of the mouse, rat, and human renin genes show significant
homology, suggesting that the promoter regions may be involved in the
regulation of renin gene expression in the kidney (18, 19).
The protein product of the retinoblastoma susceptibility gene, RB, is a
nuclear phosphoprotein which plays a key role in the regulation of cell
growth and differentiation (20, 21). RB appears to control cell growth
in part by regulating gene expression (22, 23). Several cellular genes
encoding growth-regulatory factors such as c-fos (24),
c-myc (25), transforming growth factor-1 (26),
transforming growth factor-
2 (27), insulin-like growth factor-II
(28), interleukin-6 (29), and neu (30) have been identified
as targets of transcriptional control by RB. It is suggested that RB
regulates transcription by interacting with specific transcription
factors and modulating their activity. For example, RB is able to
interact with Sp-1 (28), E2F (31, 32), MyoD (33), Elf-1 (34), CREB/ATF
(27), N-Myc (35), c-Myc (35), PU.1 (36), and UBF (37), and with TFIID
directly through binding to a coactivator(s) (38). Although RB is
ubiquitously expressed, recently it has been reported that the amounts
of RB are varied in specific cell types of normal tissues, and maturing cells possess higher levels of RB than their progenitors, indicating that the expression of RB is regulated during the process of
differentiation in several cells and tissues (39). Renin is mainly
produced in the kidney, and renin gene expression is regulated in a
developmentally linked manner. Since RB mRNA is also expressed
abundantly in the kidney (40), it is possible that RB modulates
expression of the renin gene in the kidney.
In the present study, we showed that RB activated transcription of the
Ren-1C gene in HEK cells. The proximal promoter region from
75 to +16 of the Ren-1C gene mediated the transcriptional activation by RB. We demonstrated that the RP-2 element (
75 to
47)
which overlapped the TATA-like region was the major contributor to the
regulation of Ren-1C promoter activity by RB in HEK
cells.
pUCSV3CAT contained the coding
sequence for chloramphenicol acetyltransferase (CAT) fused with the
simian virus 40 (SV40) enhancer/promoter sequence and polyadenylation
signal (41). pUCSV0CAT contained the SV40 polyadenylation signal 5 to
the CAT coding gene to efficiently terminate read-through transcription arising from prokaryotic sequences in the constructs (41). pUCSV3CAT was used as a positive control, and pUCSV0CAT was used as a background reference. Plasmid phuRb contained a KpnI (position
240)
to EcoRI (position +4602) human cDNA fragment cloned
into the SmaI and EcoRI sites of the control
vector pJ3
, which included the SV40 enhancer/promoter sequence and
poly(A) signal (kindly provided by Dr. Paul Robbins, Department of
Molecular Genetics and Biochemistry, University of Pittsburgh School of
Medicine, Pittsburg, PA) (24).
Ren-1C promoter-CAT hybrid genes, R365CAT (365 to +16),
R224CAT (
224 to +16), R183CAT (
183 to +16), R164CAT (
164 to +16), R114CAT (
114 to +16), R75CAT (
75 to +16), and R47CAT (
47 to +16)
were constructed as described previously (14). Construction of TK-CAT
was described previously (42). The RU-1 or RP-2 elements with or
without the other element were linked upstream of R47CAT or TK-CAT in
5
to 3
orientation. R365CAT was used as a template to construct
mutations in the RP-2 element (m(RP-2) element) by oligonucleotide-directed mutagenesis (43). The sequences of the
oligonucleotide used to create m(RP-2) element were
5
-CCCTGGGGTAccgAcTCAAAGCAGAGCCT-3
. Once the
mutations were obtained and confirmed by sequencing, the altered
381-base pair (
365 to +16) fragment was subcloned into the
BglII/HindIII sites of pUCSV0CAT (R365
m[RP-2]CAT). To construct plasmid pC/RP-2, six tandem copies of a
synthetic double-stranded RP-2 element
(5
-CCCTGGGGTAATAAATCAAAGCAGAGCCT-3
) were inserted into the
BglII site of pUC19 as described previously (44). To construct pC/m(RP-2), six tandem copies of the m[RP-2] element were
inserted into the BglII site of pUC19.
HEK cells were maintained in minimum essential medium containing 10% heat-denatured horse serum (14). HeLa and T98G cells were cultured in minimum essential medium supplemented with 10% fetal bovine serum. NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. These cell lines were kept in 5% CO2 and plated approximately 24 h before transfection at a density of 5 × 105 cells in 60-mm diameter plastic dishes.
DNA Transfection and CAT AssayRB expression vector phuRb
or pUC19 plasmid with Ren-1C promoter-CAT hybrid genes were
transiently cotransfected into cultured cells as described previously
(14, 15). Media were replaced with fresh media 24 h after
transfection, cells were harvested 48 h after transfection, and
aliquots of cell extracts containing equal amounts of total protein (40 µg) were used for CAT assay. CAT assay was performed as described
previously (14, 15), and results were normalized on the basis of
protein concentration or -galactosidase activity to correct for
differences in transfection efficiency. The conversion ratios of
[14C]chloramphenicol were measured with an image analysis
system (BAS2000; Fujix, Tokyo, Japan). All experiments were performed at least four times for each construct.
Nuclear extracts from HEK cells were prepared using a modification of the protocol of Dignam et al. (14, 45). The final protein concentration was 5-7 mg/ml. In some instances, cultured cells were transfected with the RB expression vector phuRb 36 h before the preparation of nuclear extracts.
Electrophoretic Mobility Shift AssayElectrophoretic
mobility shift assay (EMSA) was performed essentially as described
previously (14, 44-46). Briefly, nuclear extracts were preincubated
for 15 min on ice in 20-µl reaction mixtures containing 12 mM HEPES, pH 7.9, 60 mM KCl, 0.1 mM
EDTA, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 12% glycerol, and 500 ng of
double-stranded poly(dI-dC). Non-labeled competitor was included in
some of the binding reactions as indicated. The synthetic
double-stranded RP-2 element (5-CCCTGGGGTAATAAATCAAAGCAGAGCCT-3
;
75
to
47) and the double-stranded DNA fragment of RU-1 element (
224 to
138) were phosphorylated on its 5
-ends by T4 kinase and
[
-32P]ATP, and used as the probes. Aliquots of 0.1 to
0.4 ng (approximately 15,000 cpm) of the probe were added and
incubation was continued for 30 min at room temperature. The incubation
mixture was loaded on to a 4% polyacrylamide gel in a buffer
containing 50 mM Tris-HCl, pH 8.3, 192 mM
glycine, and 1 mM EDTA, and electrophoresed at 140 V for
3 h followed by autoradiography. Radioactivity of shifted bands on
the gel was quantified using a BAS2000 FUJIX BIO-Imaging Analyzer (Fuji
Photo Film). Double-stranded synthetic DNAs containing the consensus
binding sequences for Sp-1 were obtained from Stratagene (GELSHIFTM
KIT, La Jolla, CA). Oligonucleotides for the RP-2 element, m(RP-2)
element, Pit-1/GH-1 (47), and CREB/ATF (48) were synthesized on a
MilliGen/Bioresearch CycloneTM Plus oligonucleotide synthesizer, and
purified on OPC columns (Applied Biosystems, Foster City, CA) according
to the manufacturer's protocol.
DNase I footprinting was
performed essentially as described previously (44, 45). Briefly, the
Ren-1C promoter fragment from 114 to +16 relative to the
transcriptional start site was end-labeled with T4 polynucleotide
kinase and [
-32P]ATP, followed by digestion with
DdeI (
18) to generate a probe. After gel purification, the
probe (approximately 15,000 cpm) was incubated with nuclear extracts in
50-µl reaction mixtures containing 12 mM HEPES, pH 7.9, 60 mM KCl, 4 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol, and 1 µg
of double-stranded poly(dI-dC). The mixtures were incubated for 30 min
on ice, followed by 1 min at room temperature with addition of 50 µl
of a solution containing 12 mM HEPES, pH 7.9, 5 mM CaCl2, 5 mM MgCl2,
and 5-250 ng of DNase I. The reaction was stopped by addition of 100 µl of 12 mM HEPES, pH 7.9, 0.6 M sodium
acetate, pH 7, 0.5% SDS, 0.1 mM EDTA, and 20 µg of tRNA. The DNA was extracted with phenol/chloroform (1:1, v/v) and
precipitated with 2.5 volumes of ethanol prior to electrophoresis on a
6% polyacrylamide, 8 M urea sequencing gel. To define the
position of the protected region, G + A sequence ladders were prepared
(49).
Cotransfection of phuRb activated CAT expression
directed by the Ren-1C promoter from 365 to +16 (R365CAT)
in HEK cells (Fig. 1). In contrast, this promoter region
was not able to mediate the activation by RB in HeLa, T98G, and NIH 3T3
cells. This inactivity in nonrenal cells was not caused by a lower
transfection efficiency, since expression directed by pUCSV3CAT was
similar in HEK and these nonrenal cells.
Then, HEK cells were cotransfected with R365CAT and phuRb DNA or pJ3
plasmid (Fig. 2). The level of CAT activity was
proportional to the amount of cotransfected phuRb DNA in the range of 3 to 6 µg of DNA. In contrast, cotransfection of phuRb did not have any
significant effect on CAT expression directed by the TK promoter (TK-CAT).
Effects of RB on the Expression of Renin Promoter-CAT Fusion Genes in HEK Cells
Next, to define DNA sequences responsible for
transcriptional activation of the Ren-1C promoter by RB in
HEK cells, a series of 5-deletion mutants extending from
365 to
47
were constructed and tested for their ability to promote transcription.
HEK cells were cotransfected with a 5
-deletion construct and phuRb DNA or pJ3
plasmid (Fig. 3).
R365CAT activated CAT expression to a significant level (3.2-fold
increase) on cotransfection with phuRb, and only the 91-base pair
Ren-1C promoter sequences (75 to +16) were required to
elicit the RB-induced expression of the CAT reporter gene in HEK cells (Fig. 3, R75CAT). Although the induction ratios of all
mutants with deletion end points up to
75 (R75CAT) were relatively
constant, deletion of the sequences from
75 to
48 (RP-2 element)
abolished the induction of CAT activity by RB (R47CAT). These results
indicated that the induction of CAT expression by RB was dependent on
Ren-1C proximal promoter sequences, and suggested that the
RP-2 element from
75 to
47 was important for RB-induced
Ren-1C gene promoter activity.
To assess the functional
importance of the RU-1 and RP-2 elements in Ren-1C promoter
activation by RB, we first fused these elements with or without the
other element in 5 to 3
orientation to the Ren-1C promoter
(Fig. 4A). A construct with a combination of
the RU-1 and RP-2 elements ([RU-1/RP-2]R47CAT) efficiently activated
the Ren-1C promoter on cotransfection with phuRb (3.9-fold). The RP-2 element alone ([RP-2]R47CAT) also supported RB-inducible activation of Ren-1C promoter (4.3-fold), although the
relative CAT activity of [RP-2]R47CAT in the presence or absence of
RB was significantly lower than that of [RU-1/RP-2]R47CAT. In
contrast, [RU-1]R47CAT, which contained the RU-1 element alone, as
well as the control R47CAT, was not able to confer RB inducibility.
To further establish the functional roles of the RU-1 and RP-2 elements in directing RB-induced CAT expression, the RU-1 and/or RP-2 elements were linked upstream of a TK promoter-CAT hybrid gene. As shown in Fig. 4B, [RU-1/RP-2]TK-CAT elicited RB-induced expression of the CAT-reporter gene (3.2-fold activation). In addition, the RP-2 element alone mediated transcriptional activation by RB ([RP-2]TK-CAT, 2.5-fold activation). In contrast, the RU-1 element alone ([RU-1]TK-CAT) did not confer the RB inducibility although this element activated the TK promoter in an RB-independent manner. These results suggested that the RU-1 element functioned as a constitutive activator of TK promoter, and that the RP-2 element was necessary not only for basal Ren-1C promoter activity but also for RB-mediated activation of Ren-1C and TK promoters.
Effects of RB on Nuclear Factors Binding to RP-2 ElementThe
above results suggested that the RP-2 element was involved in
RB-mediated activation of CAT expression directed by the Ren-1C promoter. We previously demonstrated that HEK
cell-dominant nuclear factors bound to the RP-2 element by EMSA (14).
To examine the effects of RB on binding of nuclear factors to this
element, we first performed DNase I footprint analysis (Fig.
5). A labeled DNA fragment from 114 to
19 was
incubated with HEK cell nuclear extracts followed by DNase I digestion.
The results shown in Fig. 5A indicated that a sequence from
68 to
55 in the RP-2 element overlapping the TATA-like region
(TAATAAA;
67 to
61, Fig. 5B) was protected from
digestion by DNase I (lanes 2-5, denoted by the
hatched box, Fig. 5A). The pattern of DNase I
footprinting disclosed increased protection on cotransfection with
phuRb (lanes 8-11, Fig. 5A).
We next carried out EMSA using the RP-2 element as a probe (Fig.
6A). Incubation of HEK cell nuclear extracts
with this element produced a single shifted band, and cotransfection
with phuRb tended to increase the intensity of the shifted band derived
from nuclear factors binding to the RP-2 element. The shifted band was
specifically competed out by the unlabeled RP-2 element and by a DNA
fragment including the promoter region from 75 to +16, but not by a
promoter fragment from
47 to +16 (lanes 1-3, and lanes 9 and 10, Fig. 6A). Previous
studies showed that the human renin gene promoter basal and cAMP
stimulated activities in gonadocorticotrope and chorionic cells were
dependent upon the presence of the pituitary-specific trans-acting factor (Pit-1/GH-1)-binding site (
79 to
64)
bearing partial sequence similarity to the RP-2 element (50-52). In
addition, transcription factors Sp-1 and CREB/ATF have been reported to mediate transcriptional activation by RB (27, 28). Thus, it is possible
that a Sp-1, CREB/ATF, or Pit-1 family transcription factors bind to
the RP-2 element. However, double-stranded synthetic DNAs containing
the consensus binding sites for Sp-1, CREB/ATF, or Pit-1/GH-1 did not
compete efficiently with the RP-2 element binding activity (lanes
6-8, Fig. 6A).
We performed EMSA using the RU-1 element as a probe to show binding of
phuRb () and phuRb (+) extracts to the RU-1 element as a control for
extract quality (Fig. 6B). The intensity of the shifted
bands derived from nuclear factors binding to the RU-1 element did not
show any significant change by cotransfection with phuRb (lanes
2 and 3, Fig. 6B). Therefore, with the
result of EMSA using the RP-2 element as the probe, the overexpression of RB is considered to enhance specifically the binding of nuclear factors to the RP-2 element.
From the above results, the RP-2 element seems to
exert a critical influence on RB-mediated promoter activity of the
Ren-1C gene in HEK cells. Thus, to evaluate the functional
significance of the RP-2 element in RB-mediated Ren-1C
promoter activity, we first assayed the effects of a mutation that
disrupted binding of nuclear factors to this element. Although the
DNA-protein complex formed by the RP-2 element binding activity could
be competed out by non-labeled RP-2 element, the m(RP-2) element, that
contained substitution mutations interrupting the TATA-like region
protected on DNase I footprinting analysis at positions 68 to
55,
did not compete out this binding (lanes 2-4, Fig.
6A). In transiently transfected HEK cells, Ren-1C
promoter-CAT hybrid gene with this mutated RP-2 element (R365
m[RP-2]CAT) showed a significant decrease in RB-mediated promoter
activity (Fig. 7). We next performed in vivo
competition experiments to confirm the functional role of the RP-2
element as an RB-mediated positive regulator. Six tandem copies of RP-2
or m(RP-2) elements were inserted into pUC19. These plasmids, named
pC/RP-2 or pC/m(RP-2), were cotransfected with R365CAT into HEK cells,
and the CAT activities were analyzed (Fig. 8). The
amount of transfected DNA was normalized by pUC19 to 18 µg. The
results obtained in this experiment showed that CAT activity decreased
with increasing amounts of pC/RP-2, but not with pC/m(RP-2). Therefore,
these functional assays further suggest that the RP-2 element
overlapping the TATA-like region from
67 to
61 is important for
RB-mediated activation of Ren-1C promoter in HEK cells.
The regulation of renin gene transcription is achieved via the
interplay of various signaling stimuli and trans-acting
nuclear factors (5-7). All of the mouse renin genes (Ren-1C,
Ren-1D, and Ren-2 genes) are expressed abundantly and
equivalently in the kidney, and 5-flanking regions of these genes are
highly homologous to around 79 nucleotides upstream from the
transcriptional start site (18, 19). We supposed that the highly
homologous 5
-flanking regions were involved in the basal and
stimulated transcriptional activity of these renin genes. Thus, in this
and previous studies we have focused on the promoter region from
365 to +16 of the Ren-1C gene (14, 15, 45). We previously
demonstrated that the Ren-1C promoter region from
365 to
+16 mediated HEK cell-dominant transcriptional activity and that
combination of the RU-1 (
224 to
138) and RP-2 (
75 to
47)
elements efficiently induced transcription from the Ren-1C
promoter (14). In this study, we showed that the promoter region from
365 to +16 (R365CAT) directed the activation of CAT expression by RB,
and indicated that the proximal promoter region from
75 to +16
(R75CAT) was sufficient to confer this inducibility by RB.
The functional role of the RU-1 element in RB-induced Ren-1C
promoter activity was similar to that in basal transcriptional activity. We previously showed that the RU-1 element efficiently directed transcription of Ren-1C promoter only in
combination with the RP-2 element (14, 45). Our results in this study showed that the RU-1 element alone did not confer RB responsiveness on
Ren-1C and TK promoters, and suggested that the RU-1 element is a constitutive activator-like region. On the other hand, the RP-2
element seemed to be essential for RB-mediated induction. We performed
DNase I footprint analysis to identify the nature of the DNA binding
activities which occurred at the RP-2 element. We demonstrated that
nuclear factors in HEK cell nuclear extracts bound to the region from
68 to
55 in the RP-2 element, overlapping the TATA-like region. The
activation of gene transcription in response to stimuli usually
involves the induction of nuclear binding factors. DNase I footprinting
in this study disclosed increased protection in the RP-2 element by RB,
and EMSA showed that the binding activities of nuclear factors to the
RP-2 element were increased by RB. The results of deletion analyses and
heterologous promoter assay indicated that the RP-2 element was able to
confer RB inducibility, and site-directed mutation that disrupted
nuclear binding activity to the RP-2 element greatly decreased this
RB-mediated activation of CAT expression. Furthermore, in
vivo competition of RB binding in transfected HEK cells markedly
decreased RB-mediated CAT expression directed by the Ren-1C
promoter. These results suggest that the RP-2 element is responsible
for RB-induced promoter activity of Ren-1C gene in HEK
cells.
Previous studies showed that RB stimulated promoter activity through CREB/ATF and Sp-1 binding sites (27, 28), and increased transcription of the phosphoenolpyruvate carboxykinase gene through an as yet undefined site (53). The RP-2 element has no apparent homology with CREB/ATF or Sp-1-binding sites. The CREB/ATF-binding site is known to confer cAMP-responsive transcriptional activity. We and others previously demonstrated that the RP-2 element and upstream CREB/ATF sites were involved in cAMP-mediated transcription of the mouse and human renin genes (45, 54-56). However, the synthetic DNA containing the consensus binding site for CREB/ATF did not compete for RP-2 element binding, indicating that CREB/ATF family transcription factors did not participate in RB-mediated activation by the RP-2 element.
Studies have demonstrated that RB can either positively or negatively
regulate expression of several genes through cis-acting elements in a cell type-dependent manner (57). At present
the exact mechanism by which RB activates transcription of the renin gene through the RP-2 element is unclear. RB may be directly involved in the binding activity of RP-2 element-binding factor through repression of transcription of a repressor protein or a protein that
inhibits DNA binding, or through a direct and positive interaction with
transcription factors (58). For example, RB stimulates Sp-1-mediated
transcription of the c-jun gene by liberating Sp-1 from a
Sp-1-negative regulator that specifically inhibits Sp-1 binding to a
c-jun Sp-1 site (59). Although the negative regulatory element has not been identified in the RP-2 element, CREB/ATF and
negative regulatory element-binding protein bind to the far upstream
5-flanking region of the mouse renin genes (Ren-1D,
619
to
597; Ren-2,
670 to
648) (54) and an interaction
between these nuclear proteins is suggested to play a role in the
tissue-specific regulation of renin gene transcription (55).
Furthermore, RB may also be indirectly involved in the binding activity
of RP-2 element-binding factor. The arrest of cell cycle progression in G1 in response to the overexpression of RB may result in a
post-transcriptional modification increasing transcription factor
binding to the RP-2 element.
Previous studies indicated binding of nuclear proteins from the pituitary cell line gonadocorticotrope to a region on the human renin promoter that is analogous to the RP-2 element, and suggested that transcription factors related to Pit-1/GH-1 could be responsible for binding to the RP-2-analogous element in the human renin gene (50-52, 60). Although originally identified as a Pit-1/GH-1-binding site, this sequence in the RP-2 element has only partial similarity to the Pit-1/GH-1 recognition sequence. In addition, Germain et al. (16) examined the cis-acting elements of the proximal promoter involved in cAMP-induced human renin gene transcription using renin-producing chorionic cell and kidney cortex cell nuclear extracts. They showed that tissue-specific factors distinct from Pit-1/GH-1 specifically bound the RP-2-analogous element in the human renin gene. Furthermore, Petrovic et al. (17) showed that the human Pit-1/GH-1 sequence did not form complexes with nuclear proteins from renal renin-expressing As4.1 cells nor was it able to compete with the nuclear binding activity in As4.1 cells to the RP-2 element. In this study, double-stranded synthetic DNA containing the consensus binding sites for Pit-1/GH-1 did not compete with the RP-2 element binding activity in HEK cells, supporting the suggestion that the RP-2 element-binding factor is distinct from the Pit-1/GH-1 family transcription factors. Although this factor may still belong to the POU homeodomain class family, its precise identity of the factor is unknown at present.
The data presented here demonstrated that RB activates the
Ren-1C gene proximal promoter in HEK cells, and that this
effect is mediated mainly via a proximal promoter element from 75 to
47 (RP-2 element) overlapping the TATA-like region. Transcription factors distinct from the CREB/ATF or Pit-1/GH-1 family appear to
mediate this RB response, and nuclear factors binding to the RP-2
element seem to be regulated by RB in the activation process. RB-mediated modulation of promoter activity may have a role in the
tissue-specific and developmentally linked regulation of renin gene
expression. Further studies are obviously warranted to investigate the
molecular relationship between RB and regulation of renin gene
transcription in response to various stimuli.