©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Regulation of the Vacuolar H-ATPase B2 Subunit Gene in Differentiating THP-1 Cells (*)

(Received for publication, December 8, 1994; and in revised form, January 30, 1995)

Beth S. Lee (§) David M. Underhill Monica K. Crane Stephen L. Gluck (¶)

From the Department of Medicine/Renal Division and the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(r) = 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.


INTRODUCTION

The mammalian vacuolar H-ATPase, or V-ATPase, (^1)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(0)-F(1) 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(r) = 70,000 or ``A'' subunit, the site of ATP hydrolysis, and a M(r) = 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(r) = 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(r) = 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(r) = 31,000) (19) and the C (M(r) = 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(r) = 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(r) = 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) . (^2)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.


EXPERIMENTAL PROCEDURES

Materials

Unless otherwise indicated, all reagents were obtained from Sigma and were reagent grade.

Isolation and Culture of Cells

Peripheral blood monocytes were isolated by countercurrent elutriation(33) . Monocyte-derived macrophages were obtained by culturing purified monocytes on plastic Petri dishes in RPMI 1640 (Sigma) with 12.5% human AB serum (North American Biologicals, Inc., Miami, FL), 2 mML-glutamine, 1 mM sodium pyruvate, and 20 µg/ml gentamicin at 37 °C in a 5% CO(2) incubator. THP-1 cells were obtained from the American Type Culture Collection, Rockville, MD, and were cultured in RPMI 1640 with 10% fetal bovine serum (HyClone, Logan, UT), 2 mML-glutamine, and 20 µg/ml gentamicin in a 5% CO(2) incubator. THP-1 cells were induced to differentiate by addition of 160 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). Upon addition of TPA, THP-1 cells, which are propagated in suspension, stop dividing, become adherent, and differentiate to a macrophage-like state. The human embryonic kidney cell line 293 was obtained from the American Type Culture Collection and cultured in modified Eagle's medium with Earle's salts, 10% fetal calf serum, and 20 µg/ml gentamicin.

Antibodies

Monoclonal antibody E11 was used for immunoprecipitation and immunoblot detection of the V-ATPase E (M(r) = 31,000) subunit(21) . Anti-peptide rabbit antiserum rB1-CT was utilized for detection of the B1 isoform subunit.^2 The B2 isoform was detected with rabbit antiserum rB2-NT, derived to a fusion protein construct encompassing the N-terminal 120 amino acid residues added to the carboxyl terminus of the Escherichia coli maltose binding protein.^2

Immunoprecipitation

For metabolic labeling of monocytes or macrophages, one 100-mm tissue culture plate of cells was incubated with 100 µCi of TranS-label (ICN) in 4 ml of methionine-free medium overnight. 1 times 10^6 human peripheral blood monocytes or macrophages were solubilized by 14 passes through a motorized Teflon-glass homogenizer (Wheaton, Millville, NJ) in solubilization buffer (20 mM Tris-Cl, pH 7.4, 5 mM sodium azide, 1 mM EDTA, 1 mM dithiothreitol, 1% Triton X-100, and 0.1% SDS). Insoluble material was pelleted at 150,000 times g for 1 h at 4 °C, and H-ATPase complexes were precipitated from the supernatant using monoclonal antibody E11 coupled to protein A-Sepharose as described previously(18) . After a 2-h incubation at 4 °C, the E11-coupled beads were pelleted briefly in a microcentrifuge and washed three times with ice-cold Net-gel buffer(34) . The supernatants from the first immunoprecipitations were removed to separate tubes, and a second precipitation was performed to verify quantitative recovery of V-ATPase complexes. Immunoprecipitated proteins were solubilized in SDS-PAGE gel-loading buffer, and one-third of each sample was loaded onto a 10% SDS-polyacrylamide gel. Following electrophoresis, the gel was fixed, treated in Fluorohance (RPI, Mount Prospect, IL), and juxtaposed to film.

Immunoblots

For whole cell extracts, 0.5 times 10^6 human peripheral blood monocytes or macrophages were solubilized by heating for 10 min at 95 °C in SDS-PAGE gel sample buffer, and samples were applied directly to lanes of a SDS-polyacrylamide gel. After separation by SDS-PAGE, proteins were transferred to Immobilon-P membrane (Millipore, Bedford, MA), and the membrane was incubated overnight at 4 °C in blocking solution(34) . Primary antibody E11, as tissue culture supernatant, or rB1-CT or rB2-NT antiserum diluted 1:300 in blocking solution, was incubated with the membranes for 2 h at room temperature. Following washing of the membrane using standard conditions, horseradish peroxidase-conjugated secondary antibodies were incubated with the membrane(34) , and bound proteins were detected by chemiluminescence using the ECL reagents and protocol from Amersham Corp.

cDNA Probes and Antibodies

A 1.1-kb cDNA clone for the human V-ATPase M(r) = 15,000 (proteolipid) subunit was the kind gift from S. Reeders (Harvard University). A 0.27-kb ApaI-EcoRI fragment from the 3` end of this clone was used as a probe for RNA hybridization (Northern) analysis, while the intact cDNA in a pBluescript vector (Stratagene, La Jolla, CA) was used as a probe for the nuclear runoff assay. The cDNA for the human E subunit was isolated as reported previously(21) . The full-length 1.3-kb fragment in pBluescript was used for the nuclear runoff assay, while a 0.43-kb EcoRI-KpnI fragment from the 5` end was used for the RNA hybridization analysis. A cDNA clone for the human V-ATPase B1 (kidney) isoform cDNA was isolated by polymerase chain reaction from a human kidney cDNA library (a generous gift from Dr. Graham Bell, University of Chicago) using oligonucleotides derived from the published sequence(12) . A 400-base pair fragment from the 5` end was used directly for RNA hybridization analysis, and was subcloned into pBluescript for the nuclear runoff analysis. A cDNA clone for the human V-ATPase B2 (brain) isoform encompassing the start codon was isolated previously(14) . From this clone, a 0.56-kb EcoRI-ClaI fragment from the 5` end was used for both RNA hybridization and nuclear runoff analysis. Although the cDNA clones for the B1 and B2 subunit isoforms are highly homologous in some internal regions(14) , the 5` regions used here as isoform-specific probes exhibit an identity of only 65%, and therefore specific hybridization to a single isoform was easily attained under high stringency conditions. A 2.5-kb partial cDNA clone for the human V-ATPase A subunit was also isolated in this laboratory. (^3)This clone, which extends from nucleotide 840 in the coding region to the poly(A) tail, was used directly for RNA hybridization and was subcloned into pBluescript for the nuclear runoff assay. A cDNA probe for the human enzyme glyceraldehyde-3-phosphate dehydrogenase, obtained from Clontech (Palo Alto, CA), was used for normalization of mRNA levels in the RNA hybridization analysis.

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.

Isolation of RNA and Hybridization Analysis

Total cellular RNA was isolated from cells by treatment with guanidinium and acid-phenol(35) . Poly(A)-selected mRNA was prepared with a FastTrack commercial isolation kit (Invitrogen, San Diego, CA), using the manufacturer's protocol. RNA was electrophoretically separated in a formaldehyde-containing agarose gel under standard conditions, and was transferred to a Nytran membrane (Schleicher & Schuell, Keene, NH). Probes were radiolabeled with [alpha-P]dCTP using the Prime-a-Gene random priming labeling kit from Promega (Madison, WI), and were allowed to anneal bound RNA using a hybridization solution recommended by the manufacturers of Nytran. Final washes of the membrane prior to exposure to film were usually in 0.2 times SSPE, 0.1% SDS at 55 °C, unless specified otherwise in the text.

Nuclear Runoff Analysis

Preparation of THP-1 nuclei and radiolabeling of nascent transcripts were performed as described by Greenberg(36) . Radiolabeled RNA was isolated by the guanidinium thiocyanate acid-phenol method described above. Concentrations of radiolabeled RNA samples from different time points were adjusted to equal counts/min and were added to nylon membranes to which 10 µg of linearized denatured plasmid containing the appropriate cDNA probe had been applied in a slotted filtration manifold (Bio-Rad). Hybridization and washing of the membrane strips were performed as described by Greenberg(36) .

Southern Analysis, Polymerase Chain Reaction (PCR), and DNA Sequencing

DNA hybridization (Southern) analysis was performed by standard procedures. Following electrophoretic separation of DNA, the DNA fragments were transferred to a Nytran membrane (Schleicher & Schuell) using a Vacu-Blot vacuum transfer apparatus (Hoefer). Hybridization of P-labeled probes to membrane-bound DNA was performed under standard conditions(37) . Final washes of the membrane prior to exposure to film were usually in 0.2 times SSPE, 0.1% SDS at 55 °C, unless specified otherwise in the text.

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 (^4)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 [alpha-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.

Isolation of Promoter Fragments

1 times 10^6 plaques from a WI38 human lung fibroblast genomic DNA phage library (courtesy of V. Sukhatme, Harvard University) were screened with an 85-bp PCR-amplified cDNA probe corresponding to a 5` region of the B2 subunit mRNA (described above). Hybridization and washing conditions were the same as those described for Southern analysis, above. Following two rounds of screening, two clones were isolated. From one of these, two overlapping subclones, pBSL217 and pBSL220 were isolated. Further details are described in the text.

Primer Extension and Ribonuclease Protection Assays

A commercial kit (Promega) was used for the primer extension analysis. Eight micrograms of total cellular RNA from resting or induced THP-1s were annealed with either of two P-end-labeled antisense primers: 5`-ATCTTGTCTCCTCTGTCC-3` (+2 to -16); or 5`-ACGGGTAGCTCGGGTGCG-3` (+56 to +39). The primer extension reactions were performed with avian myeloblastosis virus reverse transcriptase according to the manufacturer's instructions. Extension products were separated in a 6% polyacrylamide gel containing 1 times TBE (0.09 M Tris borate, 2 mM EDTA, pH 8.3) as a buffer, and using P-labeled X174-HinfI fragments as markers. The gel was fixed in 25% ethanol, dried, and exposed to film. Ribonuclease protection assays were performed using the RPA II kit (Ambion, Austin, TX). 0.5-1.0 µg of poly(A)-selected mRNA from TPA-induced THP-1s or 293 cells were hybridized overnight at 45 °C with a P-labeled RNA probe made using a Riboprobe (Promega) transcription system and T7 RNA polymerase. The probe contained 222 bp of 5`-untranslated and flanking regions (between a NarI site at -96 and an MluI site at -317) plus 33 bp of pBluescript polylinker. Following RNase digestion according to the manufacturer's protocol, protected fragments were separated in a 6% polyacrylamide gel with 1 times TBE buffer. The gel was fixed in 25% ethanol, dried, and exposed to film.

Promoter Fusion Constructs

For construction of promoter-luciferase fusion plasmids, the 5`-flanking regions from pBSL217 and pBSL220 were first spliced together in pBluescript at a common SpeI site. The resulting plasmid, pBSL222, contained approximately 4.5 kb of B2 subunit 5`-flanking region plus 30 bp of coding sequence. From this spliced promoter region, smaller fragments were isolated and cloned into pGL2-basic (Promega) upstream of the luciferase coding sequences.

Transfection of Cells

For optimal transfection efficiency, cells were cultured at a density of 2-5 times 10^5/ml, and were grown continuously for no more than 6 weeks. Transfections of THP-1 cells were performed essentially as described by Mackman et al.(38) . Each 100-mm plate of cells was transfected with 5 µg of the test plasmid and 2 µg of pRSVcat as an internal control. Forty-six h after transfection, cultures were divided in half, and each half was incubated an additional 5 h in the presence or absence of 160 nM TPA.

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 and Chloramphenicol Acetyltransferase Assays

Cell extracts for luciferase and chloramphenicol acetyltransferase activity measurements were prepared by standard procedures(40) . Chloramphenicol acetyltransferase activity was measured by a modification of the two-phase diffusion assay of Neumann et al.(41) , except the final reaction mixture contained 20 mM Tris-Cl (pH 8.0), 1 mM chloramphenicol, 30 µM acetyl-CoA, and 0.4 µCi of [^3H]acetyl-CoA (ICN, 5-15 Ci/mmol).

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.

DNase I Footprinting

A genomic EcoRV-SacI fragment spanning from -338 to +84 with respect to the start of translation was subcloned into pGEM-3Z (Promega). The fragment was radiolabeled at one end with [-P]ATP and T4 polynucleotide kinase (Promega) and was purified from pGEM-3Z following restriction enzyme digestion and agarose gel electrophoresis(38) . The probe was further purified with an Elutip-D column (Schleicher & Schuell), following the manufacturer's instructions, except the DNA was eluted in 0.4 ml of a high salt buffer containing 2.0 M NaCl, 20 mM Tris-Cl (pH 7.4), and 1 mM EDTA.

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.


RESULTS

Characterization of V-ATPase Protein Levels in Monocytic Cells

Since macrophages develop an extensive lysosomal system as part of their capacity to digest pathogens and present antigen, we examined whether differentiating monocytes could serve as a model system for studies of V-ATPase gene regulation. Fig. 1shows an immunoprecipitation of assembled V-ATPase complexes from fresh human peripheral blood monocytes and from macrophages obtained by culturing monocytes in vitro for 5 days (left panel). The immunoprecipitates contained several subunits known to be present in the mammalian vacuolar H-ATPases, including the A (70 kDa), B (56 kDa), C (40 kDa), E (31 kDa), and proteolipid (15 kDa) subunits. (^5)


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 times 10^6 cells. The A (M(r) = 70,000), B (M(r)=56,000), E (M(r) = 31,000), and proteolipid (M(r) = 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 times 10^5 cells were transferred to a membrane and probed with antibodies to the B1, B2, and E subunits. B1 subunit protein (M(r) = 58,000) was not detectable either in monocytes or in macrophages. In contrast, the B2 (M(r) = 56,000) and E subunit (M(r) = 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 times 10^5 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(r) = 58,000), B2 (M(r) = 56,000), or E (M(r) = 31,000) subunits as indicated. Size of immunoreactive polypeptides is indicated as M(r) times 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.

Steady-state mRNA Levels of Only the A and B2 V-ATPase Subunits Increase during Monocytic Differentiation

In order to examine the mechanisms regulating changes in V-ATPase subunit expression during monocyte differentiation, we performed an RNA hybridization analysis on total cellular RNA isolated from THP-1 cells at different time points after TPA treatment. Fig. 3A (top) shows an RNA blot probed with a partial cDNA fragment specific for the B2 brain isoform. The experiment demonstrates that steady-state levels of the B2 subunit message increase after TPA treatment, reaching maximal levels at 24 h (further time points not shown). To determine whether this regulation occurs in native monocytes as well as the THP-1 cell line, we analyzed mRNA levels for the B2 subunit in total RNA from human peripheral blood monocytes and from monocyte-derived macrophages after 5 days in culture (Fig. 3B). As shown, mRNA levels for the B2 subunit were similarly increased following differentiation of native monocytes. In both experiments, the membranes were also probed with a cDNA to glyceraldehyde-3-phosphate dehydrogenase (GPDH) as a control for applied mRNA. In several experiments, densitometry measurements yielded an average 3.1-fold increase (3.1 ± 0.4, n = 4) in the B2 subunit message in THP-1 cells when normalized for glyceraldehyde-3-phosphate dehydrogenase. Similarly, the mRNA level for the B2 subunit increased 3.1-fold in human monocyte-derived macrophages as compared to peripheral blood monocytes (Fig. 3B).


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 [alpha-P]UTP and allowed to hybridize to cDNA probes for the indicated V-ATPase subunits and controls bound to nitrocellulose filters.



The B2 Brain Isoform Is Regulated Primarily at the Transcriptional Level

To determine whether the increases in steady-state mRNA levels of the A and B2 subunits were due to transcriptional or post-transcription mechanisms, we performed a nuclear runoff analysis on nuclei from THP-1 cells before, and 24 h after, TPA treatment. Radiolabeled transcripts were allowed to hybridize to cDNA probes for several V-ATPase subunits, and to the cloning vector pBluescript and plasmids containing beta-actin and c-fos cDNAs as controls. Although c-fos is known to be activated transcriptionally immediately upon initiation of monocytic differentiation, transcription returns to baseline levels by a few hours after induction in THP-1 cells and can therefore be used as a standard for transcript levels (42) .

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.

Isolation of the B2 Subunit First Exon and 5`-Flanking Sequences

1 times 10^6 plaques of a human genomic library were initially screened with a restriction fragment of the B2 subunit cDNA corresponding to nucleotides -19 to +530 with respect to the translational start(14) . Because this probe displayed only 65% identity with the corresponding region of the B1 isoform, the isoforms could be distinguished using high stringency hybridization and washing conditions. After three rounds of screening, two overlapping clones of >20 kb were isolated. From one of these isolates, a 5.0-kb XbaI fragment that hybridized to the original probe on a Southern blot was subcloned. Sequencing of this fragment using primers corresponding to the B2 subunit coding region revealed a region of homology with the B2 subunit coding region that extended from +67 to +259. However, this sequence showed only 86% identity with the B2 subunit cDNA sequence. This sequence may represent a third B subunit isoform or a pseudogene. This fragment was used as a probe for RNA hybridization analysis on poly(A) mRNA from multiple human tissues (Clontech), but no hybridization was detected.

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.



Determination of the Transcriptional Start Site

Because the sequence data from the regions 5` to the B2 subunit coding region showed no apparent TATA box or transcription initiator (Inr) sequences, both primer extension and ribonuclease protection assays were performed to determine the transcriptional start site(s) of the B2 subunit message. Primer extension analysis was performed on total cellular RNA from both resting (data not shown) and induced THP-1s (Fig. 7). Two primers were used for each RNA sample. Primer 1 (lane 1) corresponds to the region from +2 to -16, and primer 2 (lane 2) corresponds to +56 to +39. Fig. 7shows that extension from each of these primers results in one major product, differing in size by 54 bp, as expected from differences in the annealing sites of the two primers. Primer 1 yielded a product of 209 bp; primer 2 gave a product of 263 bp. From the length of the extension products, the 5`-untranslated region of the B2 mRNA is calculated to be 207 bp. The same results were obtained when using RNA from resting THP-1 cells (data not shown). To confirm that the cloned genomic sequences contain the transcriptional start site, we performed a ribonuclease protection assay using an RNA probe containing sequences from -96 to -317 (NarI to MluI), plus 33 bases of pBluescript polylinker, for a total of 255 bases. We allowed the probe to hybridize with poly(A)-selected mRNA from either induced THP-1 cells, 293 cells, or a negative control. Fig. 8shows that a major fragment was protected for both THP-1 and 293 cells. When these same reactions were run on a sequencing gel with a sequencing ladder marker, the size of the protected fragment was determined to be 111 bases (data not shown), confirming the location of the transcriptional initiation site obtained from the primer extension assays. However, in addition to the major band, a minor protected fragment of 36 bases was also seen in the ribonuclease protection assay (Fig. 8). This result suggests a possible minor, alternative transcriptional start site at -131. However, this result was not confirmed by the primer extension analysis.


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.



Transfection of Promoter-Reporter Fusion Constructs in THP-1 Cells

To determine whether the THP-1 model of monocyte differentiation may be used for dissecting control elements of the B2 subunit, we prepared several constructs with different promoter fragments ligated to the 5` end of a luciferase reporter gene, and transfected them into THP-1 cells. The initial promoter fragments terminated at -96 (NarI site) and therefore contained the start of transcription, and about one-half of the 5`-untranslated region. Upstream boundaries of the promoter fragments ranged from -274 to -2.4 kb. THP-1 cells showed similar levels of basal luciferase activity when transfected with each of the constructs (data not shown). In transfected THP-1 cells incubated for 5 h in the presence of TPA, luciferase activity increased between 3- and 3.5-fold for each of the constructs tested (Fig. 9, solid bars). These values correlate well with the increase in transcription rates found in the nuclear runoff analysis for the B2 subunit (3.5-fold; see Fig. 4). This indicated that the response element(s) for TPA inducibility lie proximal to the first exon, within the range of -274 to -96. Because the sequences in this region upstream of the transcriptional start site contained only GAGA sequences and a short (12 bp) (G+C)-rich stretch, it was surmised that sequences downstream of the start site may contain important regulatory elements. A promoter-luciferase construct from -274 to -199 was therefore prepared, which lacked all but 8 bp of the 5`-untranslated region. In contrast to all other B2 promoter fragments tested, this construct did not exhibit any induction of luciferase activity by phorbol ester treatment. These results demonstrate the importance of the sequences downstream of the transcriptional start site in phorbol ester induction of promoter activity.


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 (^6)was also tested in THP-1 cells and was unable to mediate induction of luciferase activity by TPA (Fig. 9, solid bars).

Transcription Factor Binding Sites in the 5`-Untranslated and Coding Regions

Sequence analysis of the 5`-untranslated region of the B2 promoter revealed several potential Sp1 and AP2 binding sites. Both of these factors bind to consensus sequences with a high G+C content. To determine whether these sequences could function as actual binding sites, we performed DNase I footprinting analysis on a promoter fragment extending from -338 to +84 (EcoRV-SacI fragment) incubated in vitro with AP2 and Sp1 proteins. Results are shown in Fig. 10. In the left panel, the promoter fragment was incubated with purified Sp1 protein obtained from a commercial source. Two clear Sp1 binding sites (GC boxes) were found, centered around -110 and -77. Both sites conform well to the weight matrix analysis prediction of Bucher (46) for GC boxes. In the right panel, the same fragment was tested for the presence of AP2 binding sites, using both purified AP2 and recombinant E. coli AP2 extracts. Five AP2 sites were found, including two in the coding region of the B2 mRNA, centered around -123, -63, -24, +7, and +16 (see Fig. 6for details). With the exception of the site at -24, all conform to the consensus GSSGNNGSS. This is in good agreement with the palindromic consensus sequence proposed by Williams and Tjian (47) for the core AP2 binding site, GCCNNNGGC. The remaining site at -24 is a palindromic sequence, CTGGGCCAG, with similarity to the high affinity AP2-binding site in the human growth hormone gene(48) .


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.




DISCUSSION

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.^6 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.^6 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.^6

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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants AR32087, DK09976, and DK38848 and George M. O'Brien Center for Kidney and Urological Diseases Grant DK45181 (to S. L. G.) and by a research grant from the Arthritis Foundation (to B. S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medicine, Renal Division, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2728; Fax: 314-362-8237; glucklab{at}imgate.wustl.edu.

Sandoz Pharmaceutical Corporation Established Investigator of the American Heart Association.

(^1)
The abbreviations used are: V-ATPase, vaculolar H-ATPase; TPA, 12-O-tetradecanoylphorbol-13-acetate; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); kb, kilobase(s).

(^2)
R. Nelson, D. Underhill, P. Hmiel, and S. Gluck, submitted for publication.

(^3)
M. Marushack, B. S. Lee, and S. L. Gluck, unpublished data.

(^4)
B. S. Lee and S. L. Gluck, unpublished data.

(^5)
The labeling of the proteolipid (15 kDa) subunit relative to the A and B subunits is somewhat weaker than might be expected, given the reported stoichiometry of two proteolipid subunits per A and B subunit(6) . It is possible that some of the immunoprecipitated H-ATPase represents the V(1) complex that has dissociated from the membranes.

(^6)
R. Nelson, S. Bae, and S. L. Gluck, unpublished results.


ACKNOWLEDGEMENTS

We are indebted to Dr. Howard Welgus, Washington University, for suggesting the use of the THP-1 cell line and for many valuable suggestions. We thank Suzanne Pontow and Dr. Philip Stahl for providing the human monocytes used in these studies, Dr. Raoul Nelson and Xiaoli Guo for helpful discussions, Dr. Kenneth Murphy for use of the luminometer, Bisola Ojikutu for aid with the transfections, and Irina Krits for technical assistance.


REFERENCES

  1. Galloway, C. J., Dean, G. E., Marsh, M., Rudnick, G., and Mellman, I. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3334-3338 [Abstract]
  2. Yamashiro, D. J., Fluss, S. R., and Maxfield, F. R. (1983) J. Cell Biol. 97, 929-934 [Abstract]
  3. Moriyama, Y., Takano, T., and Ohkuma, S. (1986) Biochim. Biophys. Acta 854, 102-108 [Medline] [Order article via Infotrieve]
  4. Blair, H., Teitelbaum, S., Ghiselli, R., and Gluck, S. (1989) Science 245, 855-857 [Medline] [Order article via Infotrieve]
  5. Gluck, S., Kelly, S., and Al-Awqati, Q. (1982) J. Biol. Chem. 257, 9230-9233 [Abstract/Free Full Text]
  6. Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802 [Abstract/Free Full Text]
  7. Starke, T., Linkkela, T. P., and Gogarten, J. P. (1991) Z. Naturforsch. 46c, 613-620
  8. Pan, Y.-X., Jin, S., Strasser, J. E., Howell, M., and Dean, G. E. (1991) FEBS Lett. 247, 147-153 [CrossRef]
  9. Marushack, M. M., Lee, B. S., Masood, K., and Gluck, S. (1992) Am. J. Physiol. 263, F171-F174
  10. Puopolo, K., Kumamoto, C., Adachi, I., and Forgac, M. (1991) J. Biol. Chem. 266, 24564-24572 [Abstract/Free Full Text]
  11. van Hille, B., Richener, H., Evans, D. B., Green, J. R., and Bilbe, G. (1993) J. Biol. Chem. 268, 7075-7080 [Abstract/Free Full Text]
  12. Südhof, T. C., Fried, V. A., Stone, D. K., Johnston, P. A., and Xie, X.-S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6067-6071 [Abstract]
  13. Bernasconi, P., Rausch, T., Struve, I., Morgan, L., and Taiz, L. (1990) J. Biol. Chem. 265, 17428-17431 [Abstract/Free Full Text]
  14. Nelson, R. D., Guo, X.-L., Masood, K., Brown, D., Kalkbrenner, M., and Gluck, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3541-3545 [Abstract]
  15. Puopolo, K., Kumamoto, C., Adachi, I., Magner, R., and Forgac, M. (1992) J. Biol. Chem. 267, 3696-3706 [Abstract/Free Full Text]
  16. Nelson, H., and Nelson, N. (1989) FEBS Lett. 247, 147-153 [CrossRef][Medline] [Order article via Infotrieve]
  17. Gillespie, G. A. J., Somlo, S., Germino, G. G., Weinstat-Saslow, D., and Reeders, S. T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4289-4293 [Abstract]
  18. Gluck, S., and Caldwell, J. (1987) J. Biol. Chem. 262, 15780-15789 [Abstract/Free Full Text]
  19. Hirsch, S., Strauss, A., Masood, K., Lee, S., Sukhatme, V., and Gluck, S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3004-3008 [Abstract]
  20. Nelson, H., Mandiyan, S., Noumi, T., Moriyama, Y., Miedel, M., and Nelson, N. (1990) J. Biol. Chem. 265, 20390-20393 [Abstract/Free Full Text]
  21. Hemken, P., Guo, X.-L., Wang, Z.-Q., Zhang, K., and Gluck, S. (1992) J. Biol. Chem. 267, 9948-9957 [Abstract/Free Full Text]
  22. Puopolo, K., Sczekan, M., Magner, R., and Forgac, M. (1992) J. Biol. Chem. 267, 5171-5176 [Abstract/Free Full Text]
  23. Ho, M. N., Hill, K. J., Lindorfer, M. A., and Stevens, T. H. (1993) J. Biol. Chem. 268, 221-227 [Abstract/Free Full Text]
  24. Manolson, M. F., Proteau, D., and Jones, E. W. (1992) J. Exp. Biol. 172, 105-112 [Abstract/Free Full Text]
  25. Young, G. P., Qiao, J. Z., and Al-Awqati, Q. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9590-9594 [Abstract]
  26. Moriyama, Y., and Nelson, N. (1989) J. Biol. Chem. 264, 18445-18450 [Abstract/Free Full Text]
  27. Gabig, T. G., and Babior, B. M. (1981) Annu. Rev. Med. 32, 313-326 [Medline] [Order article via Infotrieve]
  28. Swallow, C. J., Rotstein, O. D., and Grinstein, S. (1989) J. Surg. Res. 46, 588-592 [CrossRef][Medline] [Order article via Infotrieve]
  29. Swallow, C. J., Grinstein, S., and Rotstein, O. D. (1990) J. Biol. Chem. 265, 7645-7654 [Abstract/Free Full Text]
  30. Baron, R. (1989) Connect. Tissue Res. 20, 109-120 [Medline] [Order article via Infotrieve]
  31. Auwerx, J. (1991) Experientia 47, 22-31 [Medline] [Order article via Infotrieve]
  32. Tsuchiya, S., Kobayashi, Y., Goto, Y., Okimura, H., Nakae, S., Konno, T., and Tada, K. (1982) Cancer Res. 42, 1531-1536
  33. Bauer, J., and Hannig, K. (1988) J. Immunol. Methods 112, 213-218 [Medline] [Order article via Infotrieve]
  34. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , pp. 18.26-18.75, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  35. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  36. Greenberg, M. E. (1987) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds) pp. 4.10.1-4.10.8, Wiley, New York
  37. Brown, T. (1987) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds) pp. 2.10.1-2.10.16, Wiley, New York
  38. Mackman, N., Brand, K., and Edgington, T. S. (1991) J. Exp. Med. 174, 1517-1526 [Abstract]
  39. Graham, F. L., and van der Eb, A. J. (1973) Virology 54, 536-539 [Medline] [Order article via Infotrieve]
  40. Kingston, R. E. (1987) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds) pp. 9.6.1-9.6.9, Wiley, New York
  41. Neumann, J. R., Morency, C. A., and Russian, K. O. (1987) BioTechniques 5, 444-447
  42. Auwerx, J., Staels, B., and Sassone-Corsi, P. (1990) Nucleic Acids Res. 18, 221-228 [Abstract]
  43. Smale, S. T., and Baltimore, D. (1989) Cell 57, 103-113 [Medline] [Order article via Infotrieve]
  44. Gilmour, D. S., Thomas, G. H., and Elgin, S. C. R. (1989) Science 245, 1487-1490 [Medline] [Order article via Infotrieve]
  45. Tsukiyama, T., Becker, P. B., and Wu, C. (1994) Nature 367, 525-532 [CrossRef][Medline] [Order article via Infotrieve]
  46. Bucher, P. (1990) J. Mol. Biol. 212, 563-578 [Medline] [Order article via Infotrieve]
  47. Williams, T., and Tjian, R. (1991) Genes & Dev. 5, 670-682
  48. Imagawa, M., Chiu, R., and Karin, M. (1987) Cell 51, 251-260 [Medline] [Order article via Infotrieve]
  49. Wang, Z.-Q., and Gluck, S. (1990) J. Biol. Chem. 265, 21957-21965 [Abstract/Free Full Text]
  50. Ishii, S., Xu, Y.-H., Stratton, R. H., Roe, B. A., Merlino, G. T., and Pastan, I. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4920-4924 [Abstract]
  51. Mavrothalassitis, G. J., Watson, D. K., and Papas, T. S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1047-1051 [Abstract]
  52. Ye, K., Dinarello, C. A., and Clark, B. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2295-2299 [Abstract]
  53. Araki, E., Shimada, F., Uzawa, H., Mori, M., and Ebina, Y. (1987) J. Biol. Chem. 262, 16186-16191 [Abstract/Free Full Text]
  54. Jakobovits, E. B., Schlokat, U., Vannice, J. L., Derynck, R., and Levinson, A. D. (1988) Mol. Cell. Biol. 8, 5549-5554 [Medline] [Order article via Infotrieve]
  55. Williams, T. Admon, A., Lüscher, B., and Tjian, R. (1988) Genes & Dev. 2, 1557-1569
  56. Mitchell, P. J., Timmons, P. M., Hébert, J. M., Rigby, P. W. J., and Tjian, R. (1991) Genes & Dev. 5, 105-119
  57. Briggs, M. R., Kadonaga, J. T., Bell, S. P., and Tjian, R. (1986) Science 234, 47-52 [Medline] [Order article via Infotrieve]
  58. Su, W., Jackson, S., Tjian, R., and Echols, H. (1991) Genes & Dev. 5, 820-826

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.