(Received for publication, December 8, 1994; and in revised form, January 30, 1995)
From the
Monocyte-macrophage differentiation was used as a model system
for studying gene regulation of the human vacuolar
H-ATPase (V-ATPase). We examined mRNA levels of
various V-ATPase subunits during differentiation of both native
monocytes and the cell line THP-1, and found that transcriptional and
post-transcriptional mechanisms could account for increases in cell
V-ATPase content. From nuclear runoff experiments, we found that one
subunit in particular, the B2 isoform (M
=
56,000), was amplified primarily by transcriptional means. We have
begun to examine the structure of the B2 subunit promoter region.
Isolation and sequencing of the first exon and 5`-flanking region of
this gene reveal a TATA-less promoter with a high G+C content.
Primer extension and ribonuclease protection analyses indicate a single
major transcriptional start site. We transfected promoter-luciferase
reporter plasmids into THP-1 cells to define sequences that mediate
transcriptional control during monocyte differentiation. We found that
sequences downstream from the transcriptional start site were
sufficient to confer increased expression during THP-1 differentiation.
DNase I footprinting and sequence analysis revealed the existence of
multiple AP2 and Sp1 binding sites in the 5`-untranslated and proximal
coding regions.
The mammalian vacuolar H-ATPase, or V-ATPase, (
)performs a diversity of functions in establishing and
maintaining organellar, cytoplasmic, and extracellular pH. This
multisubunit enzyme is responsible for acidification of endosomal (1, 2) and lysosomal (3) compartments,
contributing to cellular processes such as protein transport and
degradation. In specialized cell types such as the osteoclast (4) or renal intercalated cell(5) , the V-ATPase
secretes acid from the cell in a vectorial manner by polarization of
the enzyme to a specific membrane domain.
The overall structure of
the V-ATPase appears similar to that of the F-F
ATPases in that the enzyme has a ``studlike'' or
``peg'' shape, with the head forming a catalytic cytoplasmic
domain, while the stem attaches to a membrane-spanning domain forming a
proton channel. The catalytic domain contains a M
= 70,000 or ``A'' subunit, the site of ATP
hydrolysis, and a M
= 56,000 or
``B'' subunit whose precise function is unknown. Three copies
of each polypeptide assemble to form the head structure(6) .
Although two A subunit isoforms have been identified in
plants(7) , until recently, only one mammalian form of the A
subunit was thought to exist. This form is ubiquitously expressed in
all tissue types examined, and cDNA clones have been isolated by our
laboratory and others(8, 9, 10) . A cDNA
clone for a second A subunit isoform was recently isolated from a human
osteoclastoma(11) . RNA hybridization analysis of multiple
mammalian tissues revealed no expression of this form in tissues other
than the osteoclastoma.
The M = 56,000
or B subunit family includes at least two members whose cDNAs have been
isolated by our laboratory and
others(12, 13, 14) . One of these, the B2 or
``brain'' isoform, is expressed at moderate to high levels in
all tissues studied, while the B1 or ``kidney'' isoform is
amplified in kidney cortex and medulla, with lower levels of expression
in most other tissues. While different B isoforms predominate in
different tissues(14, 15) , it is not yet clear if
these isoforms vary in function.
Of the transmembrane polypeptides,
a cDNA clone has been obtained only for the M = 15,000 subunit, a proteolipid thought to be the major
structural component of the transmembrane proton
channel(16, 17) , although immunoprecipitation of
V-ATPase reveals the presence of multiple low molecular weight
polypeptides that may also reside in the membrane(18) . It is
believed that six proteolipid polypeptides assemble to form the
transmembrane channel (6) .
The function of other subunits
of the mammalian V-ATPase in catalysis has not been established. Those
subunits for which cDNA clones have been isolated include the E (M = 31,000) (19) and the C (M
= 44,000) (20) subunits.
Immunologic evidence exists for heterogeneity of the E
subunit(21) ; only a single form of the C subunit has been
reported(20) . Recent evidence indicates that while the C
subunit enhances activity of the V-ATPase, it is not required for
enzyme activity(22) , although gene deletion studies of the
homologous subunit in Saccharomyces cerevisiae indicate that
the subunit is essential(23) . An integral membrane protein of M
= 95,000-120,000 has been
copurified with the V-ATPase from some mammalian, plant, and yeast cell
extracts (reviewed in (24) ). Mutation of the gene for this
subunit in Saccharomyces results in loss of
bafilomycin-sensitive ATPase activity and proton transport. However, a
requirement for the M
=
95,000-120,000 subunit may not be universal among all species.
Active V-ATPase isolated from mammalian kidney (18) and
Golgi-enriched membranes lack this subunit(25, 26) .
The role of V-ATPase subunits in the amplification and targeting of
the enzyme is less clear. V-ATPase expression is greatly amplified in a
cell-specific manner in several types of renal tubular epithelial
cells, allowing them to transport hydrogen ion efficiently. We showed
recently that individual B subunit isoforms are highly amplified in
specific kidney cell types present in different segments of the nephron (14) . ()Such variations in V-ATPase composition and
abundance among different cell types suggest that the expression of
V-ATPase subunits may be under tissue-specific control mechanisms,
allowing each subunit to be amplified or suppressed as required in
different cells. We were therefore interested to identify a model that
would allow us to examine how such regulation of V-ATPase subunits
occurs and found that cells of the monocytic lineage increase their
expression of V-ATPase during their differentiation into macrophages.
Macrophages and other cells of the mononuclear phagocyte system perform several functions that require a high degree of V-ATPase expression. Mature macrophages develop a large reserve of lysosomes, required for intracellular digestive processes, that are acidified by a V-ATPase (reviewed in (27) ). Macrophages utilize a plasma membrane V-ATPase, in part, to regulate their intracellular pH in acidic environments(28, 29) . The osteoclast, a cell type closely related to the macrophage, exhibits amplified expression of the V-ATPase at its ruffled membrane upon attachment to bone(4) , creating an acidic resorptive space required for digestion of bone mineral and matrix proteins (reviewed in (30) ).
To investigate expression of the V-ATPase in differentiating macrophages, we have studied both human peripheral blood monocytes and a related cell line, THP-1. The human monocytic leukemia cell line, THP-1, has recently come into use as a model for monocyte-macrophage differentiation (reviewed in (31) ). Like their more widely used counterparts, HL-60 and U-937, THP-1 cells may be induced to differentiate toward a macrophage-like state by culture with phorbol esters(32) . The THP-1 line, however, is thought to mimic more closely monocyte-derived macrophages in expression of oncogenes and membrane proteins(31) .
We have examined V-ATPase subunit expression during monocytic differentiation and present evidence for both transcriptional and post-transcriptional control mechanisms. We show that increased expression of the B2 subunit isoform is mediated primarily at the level of transcription. We have isolated B2 gene fragments containing the first exon and 5`-flanking region and present an initial characterization of the promoter elements responsible for transcriptional control.
Densitometry of the autoradiograms was performed with a Hoefer Scientific (San Francisco, CA) model GS300 scanning densitometer in transmittance mode connected to a Spectra-Physics SP4270 integrator.
The B2 subunit cDNA probe used in
these studies for library screening and Southern hybridization was an
85-bp PCR-amplified cDNA spanning the region from -29 to +56
with respect to the translational start site. The oligonucleotides
designed to amplify this region were 18 mers of the sequences
5`-GCTGGGCCAGTCGGGACA-3` (sense direction) and 5`-ACGGGTAGCTCGGGTGCG-3`
(antisense direction). This probe was amplified from 50 ng of a B2
subunit cDNA clone ()derived from a human fetal brain
library (Clontech). PCR was performed under standard conditions using
the following cycling temperatures and times: 94 °C, 40 s; 58
°C, 1 min; 72 °C, 40 s. After 30 cycles, the product was run in
2.0% low melting-point agarose. The gel slice was excised and melted,
and PCR was performed from 5 µl of the gel slice as above, except 5
µCi of [
-
P]dCTP (3,000 Ci/mM)
were added to the reaction mixture. After 15 cycles, the product was
purified over a Sephadex G-50 spin column for use in hybridization.
DNA sequencing was performed using the TaqTrack sequencing system
from Promega. Gene-specific oligonucleotides were used to prime
sequencing extension reactions, and the sequencing ladder was
visualized by incorporation of S-dATP into the extended
products.
293 cells were transfected by the method of calcium phosphate precipitation(39) . Each 100-mm plate (at about 80% confluence) was incubated overnight with a buffered calcium phosphate mixture containing 5 µg of the test plasmid and 2 µg of pRSVcat. The medium was changed the following morning, and after an additional 24 h, the cells were cultured in the presence or absence of 160 nM TPA for 5 h.
Luciferase activity was measured in an Optocomp II luminometer system (MGM, Hamden, CT). Cell extracts were injected with 100 µl of a luciferase assay reagent (Promega), and the resulting luminescence was measured for 20 s.
Binding sites for Sp1 and AP2 were detected using the Core DNase footprinting system (Promega), purified Sp1, and either purified AP2 or an extract from E. coli expressing recombinant AP2 (all from Promega). Binding reactions and DNase I digestion were performed following the manufacturer's instructions. The resulting products were separated by electrophoresis in a 6% denaturing polyacrylamide gel next to a sequencing ladder as a size marker.
Figure 1:
Immunoprecipitation of V-ATPase
complexes from monocytes and macrophages. V-ATPase complexes were
precipitated with monoclonal antibody E11 as described under
``Experimental Procedures'' and separated on 10 or 12.5%
SDS-polyacrylamide gels. For both native cells and THP-1 cultures,
precipitations were performed from 10 10
cells. The
A (M
= 70,000), B (M
=56,000), E (M
= 31,000), and proteolipid (M
= 15,000) subunits are indicated. Two additional
polypeptides at 45 and 40 kDa are present in the immunoprecipitates.
Immunoprecipitation in the presence of excess E11 peptide demonstrated
that the
40-kDa polypeptide is precipitated specifically with the
other V-ATPase subunits and probably represents the C subunit; the
45-kDa polypeptide is precipitated nonspecifically (data not
shown).
A pronounced increase in immunoprecipitable V-ATPase was observed in the macrophages in comparison with the monocytes. Densitometric measurements of these immunoprecipitates showed that in the macrophages, the A subunit increased 4.3-fold, and the B subunit increased 5.6-fold. Similar experiments in differentiating THP-1 cells showed increases of 4.0- and 3.6-fold for the A and B subunits, respectively (right panel).
Immunoblots were performed on
peripheral blood monocytes and monocyte-derived macrophages as a
second, independent method for assessing changes in V-ATPase content
during monocyte differentiation (Fig. 2). Gel-separated proteins
from 5 10
cells were transferred to a membrane and
probed with antibodies to the B1, B2, and E subunits. B1 subunit
protein (M
= 58,000) was not detectable
either in monocytes or in macrophages. In contrast, the B2 (M
= 56,000) and E subunit (M
= 31,000) were detectable on immunoblots
of both cell types, and levels of both subunits increased significantly
in the macrophages.
Figure 2:
Immunoblots of B1, B2, and E subunits
during monocyte-macrophage differentiation. Total cell homogenates were
prepared from 5 10
human monocytes (lane
2), monocyte-derived macrophages (lane 3), or 10 µg
of bovine kidney microsomes as a positive control (lane 1).
Proteins were fractionated and transferred to membranes as described
under ``Experimental Procedures,'' and probed with antibodies
to the V-ATPase B1 (M
= 58,000), B2 (M
= 56,000), or E (M
= 31,000) subunits as indicated. Size of immunoreactive
polypeptides is indicated as M
10
.
These results indicate that THP-1 cells may serve as a model system for the study of amplification of V-ATPase expression during monocyte to macrophage differentiation. We next examined whether the increase in V-ATPase content during differentiation was a result of increases in steady-state mRNA levels for any of the subunits.
Figure 3: V-ATPase subunit steady-state mRNA levels during monocyte-macrophage differentiation. Total cellular RNA was isolated from (A) THP-1 cells treated for indicated times with 160 nM phorbol ester, or from (B) human monocytes (day 0) or from monocyte-derived macrophages (day 5 in culture), separated on agarose gels, and transferred to membranes as described under ``Experimental Procedures.'' RNA hybridization analysis was performed with a cDNA fragment specific for the B2 subunit isoform, or with a probe for glyceraldehyde-3-phosphate dehydrogenase (GPDH) as a control for RNA loading (upper two panels). In the lower panel, poly(A)-enriched RNA was isolated from THP-1 cells treated for 0 or 24 h with 160 nM phorbol ester, separated, and transferred to membrane as above, and hybridization analysis was performed using cDNA probes for the other V-ATPase subunits indicated, or glyceraldehyde-3-phosphate dehydrogenase as a control.
Message levels for other V-ATPase subunits were examined by RNA hybridization analysis of poly(A)-enriched RNA from THP-1 cells before, and 24 h after, TPA induction, using subunit-specific cDNA probes (Fig. 3A, bottom). mRNA levels for neither the E nor proteolipid subunit showed any increase at the same time points used to analyzed B2 subunit message. The mRNA level for the A subunit, however, increased by about the same amount as the B2 mRNA. Because the A subunit message is detectable only in poly(A)-selected mRNA, we were unable to probe for its presence in native monocytes and monocyte-derived macrophages due to constraints on cell numbers available. RNA hybridization analysis of total cellular RNA from these cells, however, done with the other cDNA probes indicated in Fig. 3A, showed that mRNA transcripts for neither the E subunit nor the proteolipid subunit increased in monocyte-derived macrophages (data not shown). Although the B1 protein was not detectable in macrophages, mRNA for this subunit was detected in both monocytes and macrophages by RNA blot (data not shown) and nuclear transcription assays (see Fig. 4).
Figure 4:
Nuclear runoff analysis of V-ATPase
subunit transcripts in THP-1 cells before and after phorbol ester
induction. Nuclei were prepared from THP-1 cells before and 24 h after
induction with 160 nM phorbol ester as described under
``Experimental Procedures.'' Nascent transcripts were labeled
with [-
P]UTP and allowed to hybridize to
cDNA probes for the indicated V-ATPase subunits and controls bound to
nitrocellulose filters.
The B2 subunit showed the largest change in transcription rates of the several V-ATPase subunits tested. The B2 subunit transcript was barely detectable in control THP-1 cells and increased substantially by 24 h after TPA treatment. Densitometric quantitation in multiple experiments revealed an average increase of 3.5-fold (data not shown). This number compares favorably with the average 3.1-fold increase calculated from the RNA hybridization experiments and suggests that transcriptional regulation accounts for most or all of the increase in steady-state message levels. Nuclear runoff analysis performed at additional time points after treatment of THP-1 cells with TPA have indicated that enhanced transcription begins within 4 h (data not shown).
In contrast, there was little or no change in transcription of the proteolipid and E subunits. This result was not unexpected, as there was no change in steady-state mRNA levels for these subunits after treatment with TPA. There was a slight increase in transcription of the A and B1 subunits, but the changes were far less than that found in the B2 subunit isoform.
Because the B2 subunit appeared to be regulated primarily at the transcriptional level, we proceeded to examine the gene sequences that might control transcription by isolating and characterizing the 5` end of the B2 gene.
The same human genomic library was rescreened with a
PCR-amplified fragment corresponding to the region of the B2 subunit
from -29 to +56 (described under ``Experimental
Procedures''). This probe was chosen because it would not
hybridize to the genomic clones isolated initially, and the probe had
virtually no sequence identity with the B1 isoform. Following primary
and secondary screens, two clones, each of >20 kb, were isolated.
From one of these clones, a 6.0-kb EcoRI fragment was found to
hybridize on Southern blots to the probe used for the initial library
screening. This fragment was subcloned into the EcoRI site of
pBluescript and designated pBSL217. Restriction mapping of this
fragment was performed by single and double digests of pBSL217 using
appropriate restriction enzymes. From these data, pBSL217 was found to
contain approximately 1.4 kb of 5`-flanking region upstream of the
translational start site. The original clone was digested with
several restriction enzymes and analyzed by DNA hybridization, using
the 5`
620 bp of pBSL217 as a probe. A hybridizing 4.0-kb SpeI fragment was found which overlapped pBSL217 by
approximately 0.8 kb. This fragment was subcloned into the SpeI site of pBluescript and designated pBSL220. A restriction
map of the 5`-flanking regions spanned by pBSL217 and pBSL220 is shown
in Fig. 5.
Figure 5: Map of the 5` regions of the B2 subunit gene. Two overlapping genomic clones, pBSL217 and pBSL220 are designated, along with their respective sizes and restriction enyzme sites. The first exon of the B2 gene is indicated.
Using gene-specific oligonucleotides as primers,
we sequenced 0.90 kb upstream, and >0.15 kb downstream of the
translational start site. This sequence included a region of 100%
identity to B2 subunit coding sequences, and is shown in Fig. 6.
This figure also indicates the major transcriptional start site, which
we have determined as discussed below. The promoter region exhibits a
number of characteristics common to recently studied genes. First, no
TATA or CAAT boxes are evident. TATA-less promoters generally may be
grouped in two categories, those exhibiting a pyrimidine rich initiator
(Inr) sequence (43) and those that are (G+C)-rich. While
the B2 subunit gene falls in the latter group, it is somewhat unusual
in using predominantly one initiation site (discussed below). Most
genes of this type exhibit multiple initiation points that are used at
similar frequencies. The (G+C)-rich character of the first exon
extends into the coding region and stops at +100. Over this
region, the exon sequences are 78% G+C. Notably, the region
between the transcriptional start site and +100 corresponds
precisely to the sequences of the B2 subunit cDNA that exhibit no
significant homology to the B1 isoform (14) . Downstream of
+100, the percent G+C of the first exon drops to 60%; in this
region the two isoforms show >70% identity at the nucleotide level.
This suggests that the (G+C)-rich character of the B2 subunit
first exon may play a specific role in regulation of this gene. A
second notable feature of the B2 subunit gene is the presence of two
GA/CT stretches, from -653 to -626, and from -313 to
-220. These regions are similar to previously described
``GAGA boxes'': purine- or pyrimidine-rich sequences present
in the promoters of many genes (44) . The protein that binds to
these stretches, ``GAGA factor'' is a transcriptional
activator that probably acts by rearranging chromatin
structure(45) . Finally, the (G+C)-rich region of the
5`-untranslated region and coding sequences contain multiple sites for
Sp1 and AP2 binding (discussed below), although the 5`-flanking region
does not.
Figure 6:
Sequence of the B2 subunit gene first exon
and 5`-flanking region. The first exon is designated in boldface type;
the 5`-flanking sequences and first intron are in normal type
face. The start of transcription is indicated as the first base in bold type(-207), and the ATG translation start codon is
marked with an asterisk. GA/CT stretches are indicated by a
single underline. Sp1 and AP2 binding sites are noted. These sequence
data are available from EMBL/GenBank/DDBJ under accession
number Z37165.
Figure 7:
Primer extension mapping of the 5` end of
the B2 subunit transcript. Eight micrograms of total cellular RNA from
TPA-treated THP-1 cells were allowed to hybridize with each of two
end-labeled primers spanning from -16 to +2 (lane
1) or from +39 to +56 (lane 2). P-Labeled
X174-HinfI fragments are shown as
size markers. The single major bands in each lane differ in size by 54
bases, as expected, and indicate a 5`-untranslated region of 207
bp.
Figure 8:
Ribonuclease protection mapping of the 5`
end of the B2 subunit transcript. A P-labeled RNA probe
spanning from -317 to -96 was allowed to hybridize to
0.5-1.0 µg of poly(A)-enriched RNA from either THP-1 (lane 3) or 293 cells (lane 4), and digested with
RNase. Lane 2 shows the probe incubated with no RNA to control
for probe self-hybridization. Lane 1 is an overnight exposure
of undigested probe; lanes 2-4 are 5-day exposures of
the hybridizations indicated. The major protected fragment of 111 bp
and minor protected fragment of 36 bp are
indicated.
Figure 9: Promoter activity studies of B2 gene fragments in THP-1 and 293 cells. B2 gene promoter constructs with a luciferase reporter were transfected into either THP-1 (solid bars) or 293 cells (open bars) and tested for inducibility of luciferase activity with TPA. The region of the B2 gene promoter included in each construct is indicated. The B1 isoform construct contains 4.0 kb from the 5`-flanking region of the B1 subunit gene. Induction is expressed as percent of control luciferase activity following TPA treatment. Cells were cotransfected with pRSVcat, and all activity measurements were normalized against CAT activity. Assays were performed at least three times for each construct/cell combination. Data bars indicate mean inducibility ± S.E.
Because the B2 subunit is
ubiquitously expressed in all cell types that have been examined in
this laboratory, it was not possible to create a negative control for
basal expression levels. It was found, however, that 293 cells (another
human cell line) transfected with the same reporter constructs showed
no induction of luciferase activity after treatment with TPA (Fig. 9, open bars). A luciferase construct containing
a 4.0-kb fragment of the human B1 subunit isoform promoter region ()was also tested in THP-1 cells and was unable to mediate
induction of luciferase activity by TPA (Fig. 9, solid
bars).
Figure 10: DNase I footprinting of transcription factors Sp1 and AP2 in the first exon of the B2 gene. A DNA probe spanning from -338 to +84 was tested for its ability to bind purified Sp1 (left panels), and either purified AP2 or an AP2-enriched E. coli extract (right panels). Amounts of protein added are indicated above each lane. Schematics of the relative positions of factor binding sites are shown.
The V-ATPase is a multisubunit complex, with a variable composition in different tissues and subcellular membrane fractions (14, 49) . A multiplicity of genetic controls may be requisite to accomplish this type of cell-specific amplification. However, no studies of the mechanisms behind this cell specificity have been reported to date. In this report, we have identified a model system for examination of V-ATPase gene control mechanisms.
We found
that regulation of the B2 isoform occurs primarily by changes in
transcription, in contrast to other V-ATPase subunits examined here.
This observation suggests that cells increase the content of proton
pumps with a specific structure by amplifying expression of a desired B
subunit isoform, while regulating the remaining subunits through other
means. Although we have not addressed B1 regulation in this study, we
found that kidney cells, which express a high level of B1 isoform mRNA,
may also increase the content of V-ATPases containing the B1 subunit by
enhancing its transcription. We have isolated 5`-flanking
regions from the human B1 gene, and are currently in the process of
analyzing the control elements responsible for enhanced transcription
through the use of promoter-reporter constructions in transfected cell
systems.
From our immunoblot analysis, we showed that
monocytes and macrophages express only the B2 isoform of the B subunit.
Although B1 protein was undetectable, the message for this isoform was
found in both monocytes and macrophages. Translation of the B1 protein
may be repressed in these cells. On hybridization analysis of
poly(A)-enriched RNA, B1 isoform levels were comparable to those of the
A subunit, yet the A subunit protein is easily detected in both
monocytes and macrophages.
Our initial search for the B2 subunit gene resulted in isolation of a clone containing an exon with 86% identity to the B2 cDNAs already described. We were unable to detect a corresponding mRNA transcript in a number of tissues tested. Although we cannot rule out the possibility that this gene is expressed in a tissue or cell type we did not test, our findings suggest that this clone represents a pseudogene. V-ATPases are ancient enzymes, and their coding sequences may have been prone to rearrangement or inactivation during the course of evolution. Our laboratory also has identified a number of pseudogenes of the E subunit during screens of genomic libraries(21) .
The structure of the first exon and surrounding regions of the confirmed B2 isoform gene is typical of a number of recently described ``TATA-less'' genes. (G+C)-rich promoter regions of this type are found commonly in ``housekeeping'' genes, as well as those encoding cell growth-related products, such as growth factor receptors and oncogenes(50, 51, 52) . Less typical of TATA-less genes, however, is the presence of a single, major transcriptional start site in the B2 gene. (G+C)-rich TATA-less promoters routinely initiate transcription at multiple start sites, although initiation at a single site has been documented in a minority of cases(53, 54) . Like many others in this class of promoter, the B2 gene exhibits multiple potential Sp1 and AP2 binding sites. The consensus binding sites for these transcription factors are themselves rich in G+C content. More interesting from an evolutionary standpoint, however, is the location of these consensus sequences in the B2 gene. All of the potential Sp1 and AP2 binding sequences reside within the first exon, rather than within upstream sequences. Two AP2 binding sites reside just downstream from the translational start site. The (G+C)-rich character of the 5`-untranslated region extends into the coding region, and its 3` boundary corresponds precisely to the point at which the B1 and B2 isoforms begin to show a high degree of identity. Thus, the sequence differences between these two isoforms at their amino termini may be less important for the function of the proteins than for regulation of their expression.
Genomic sequence of only one other mammalian
V-ATPase subunit has been described in the literature(17) . The
230-bp region immediately 5` to the human proteolipid subunit coding
sequences similarly shows a high G+C content, with putative Sp1
elements, but no TATA or CAAT boxes. However, the transcription
initiation site was not mapped, so important elements may lie further
upstream from the region sequenced. Our laboratory has isolated the
promoter region of the human B1 isoform, and while it also is
TATA-less, the overall structure of this region shows little similarity
to the B2 isoform.
We have begun to examine promoter elements responsible for induction of B2 expression during monocytic differentiation and have found that elements downstream of the transcriptional start site are critical for B2 regulation. We found that a 179-bp fragment surrounding the transcriptional start site (-274 to -96) could mediate TPA induction as efficiently as a fragment containing an additional 2.3 kb of 5`-flanking region. Deleting all but 8 bp of the 5`-untranslated region abolished the phorbol ester sensitivity. Because the B2 gene is expressed in all cell types tested, we were unable to test these promoter fragments for cell-specific basal expression levels. We were, however, able to show that another human cell line, 293, was unable to mediate the phorbol ester-induced expression by B2 promoter construct.
Induction of B2 promoter activity may be mediated by a unique protein-binding sequence; alternatively, the numerous potential AP2 binding sites present in the 5`-untranslated sequence may mediate this effect. No other known sequences which mediate phorbol ester responses were found in this region. AP2 is a well characterized transcription factor that mediates responses to phorbol ester and cAMP(48) . It is also responsive to retinoic acid stimulation of teratocarcinoma cells and may be required for retinoic acid-induced differentiation in developing vertebrates(55) . AP2 expression is amplified in a few tissues during embryogenesis, including developing brain and kidney, two tissues that express very high levels of B2 isoform mRNA. Expression of AP2 in brain and kidney continues into adulthood(56) . More experimentation will be required to determine whether AP2, or some other factor is responsible for the effects of phorbol ester on B2 subunit transcription in THP-1 cells.
In addition to AP2 sites, we found two Sp1 binding sites in the 5`-untranslated region. Sp1 is a ubiquitously expressed protein with binding sites in many promoters. It is often found in clusters and is thought to be involved in linking distant control elements(57, 58) . Unlike AP2, it is not thought to regulate phorbol ester responsiveness of promoters.
These studies represent the first attempts to examine mechanisms of genetic regulation of the mammalian vacuolar ATPase. We have shown that amplification of certain subunits occurs in differentiating monocytes, and that transcriptional and post-transcriptional mechanisms are likely to be involved. We have also isolated B2 subunit gene fragments that mediate this amplification. Experiments like these in our monocyte system and in other cell systems should provide us with an understanding of mechanisms controlling tissue-specific expression of the V-ATPase. These kinds of studies will be most illustrative for tissue and cell types that express very high levels of this enzyme, such as kidney, brain, and the osteoclast, a cell type related to the macrophage.