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J. Biol. Chem., Vol. 277, Issue 11, 8827-8834, March 15, 2002

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Regulation of Enhanced Vacuolar H⁺-ATPase Expression in Macrophages^*

Shui-Ping Wang, Irina Krits, Shuting Bai, and Beth S. Lee^§¶

From the  Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110 and the ^§ Department of Physiology and Cell Biology, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43210

Received for publication, December 14, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The proton-translocating vacuolar ATPase (V-ATPase) acidifies the endocytic network of eukaryotic cells. Although all eukaryotic cell types require low to moderate levels of V-ATPase, some proton-secreting cells express amplified levels for use in specialized membrane domains. To characterize genetic elements required for this heightened expression, we studied transcription and stability of mRNA encoding the V-ATPase c subunit in a low expressing fibroblast cell line (NIH 3T3) and a high expressing macrophage cell line (RAW 264.7). Isolation of the promoter and mapping of the transcriptional start site indicated that the c subunit promoter is TATA-less and initiates transcription at a single site. Promoter activity was regulated through the same transcription factor binding sites in both cell types, which showed no discernible difference in rates of c subunit transcription. In contrast, c subunit transcripts showed markedly greater stability in RAW cells than in 3T3 cells, as did other constitutively expressed V-ATPase subunit transcripts. Only the B and `a' subunits, which are expressed in multiple isoforms, were not regulated solely by mRNA stability. These results suggest that overall expression levels of the V-ATPase are set primarily by regulation of mRNA stability and that transcriptional mechanisms determine subunit composition in varying cell types.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The proton-translocating vacuolar ATPase (V-ATPase)¹ utilizes energy of ATP hydrolysis to transport protons across cellular membranes. In eukaryotic cells, the action of V-ATPase is required for acidification of the endocytic network, and thus is crucial for protein processing, degradation, and trafficking. Two functionally and structurally distinct domains compose this enzyme (1). The V₁, or catalytic domain, contains the site of ATP hydrolysis and includes eight distinct polypeptide subunits. The V₀, or membrane-spanning domain, is composed of up to five distinct polypeptides and forms the pore through which protons traverse the membrane. The most abundant proteins of the V₀ domain are highly hydrophobic proteolipids that form hexameric rings within cell membranes (2). Although all eukaryotic cells require the V-ATPase for normal cellular function, some specialized, proton-secreting cells such as macrophages, osteoclasts, and renal tubule cells, express the enzyme at high levels on their plasma membranes and specialized intracellular compartments. Osteoclasts and renal intercalated cells shuttle V-ATPases between intracellular stores and the plasma membrane in response to extracellular stimuli (3, 4), whereas macrophages require high levels of V-ATPase for acidification of phagocytic vacuoles (5) and regulation of intracellular pH (6, 7). In addition, these cells and others that utilize V-ATPases for specialized purposes often express tissue-restricted isoforms of a few V-ATPase subunits. The B subunit, a component of the V₁ domain, is expressed as two isoforms, designated B1 and B2 (8, 9). Although B2 is widely expressed, B1 is restricted to a few tissues, including kidney-intercalated cells (10) and cells of the inner ear (11). In addition, the `a' subunit of the V₀ domain is expressed as three isoforms, designated a1-a3 (12, 13), and a more recently discovered human form, referred to here as a4 (14). Biochemical (15-17) and genetic data (11, 14, 18) indicate that both the B and a subunit isoforms are involved in intracellular targeting of V-ATPases and most likely are critical for transport to specialized membrane domains. Mutations in the B1, a3, and a4 isoforms cause human disease states, including malignant osteopetrosis (18-20) and renal tubular acidosis (11, 14).

Side-by-side comparisons of V-ATPase protein and mRNA for low and high expressing tissues show that subunit protein levels generally correlate with subunit mRNA levels, indicating that overall enzyme amounts are controlled by steady-state mRNA expression (10, 21). In addition, V-ATPase subunit mRNAs appear to be tandemly regulated. In previous studies, we found that the stoichiometry of V-ATPase mRNAs in bone marrow cultures reflected the stoichiometry of V-ATPase subunits in intact enzyme complexes (22). mRNA transcripts for subunits that are present in multiple copies in an intact V-ATPase were expressed at higher levels than mRNA transcripts for those subunits present in single copies. Furthermore, subunit levels appear to change in tandem during cellular processes that increase V-ATPase levels, such as cell differentiation (22, 23). These results suggest that expression of V-ATPase subunits is tightly controlled and that there may exist a universal mechanism that regulates expression levels of all subunits in a coordinated manner.

Based on these data, we began to examine mRNA transcription and stability for one V-ATPase subunit in an effort to define the control mechanisms that mediate overall expression levels. We chose the proteolipid c subunit, because it is ubiquitously expressed, and the corresponding mRNA transcript is highly abundant (22, 24), facilitating analysis of its expression. We isolated the murine c subunit promoter and assayed transcription factor binding and promoter activity in two mouse cell lines, NIH 3T3 fibroblasts, and RAW 264.7 macrophages. Although macrophages express 6- to 8-fold more V-ATPase than fibroblasts, we found no difference in relative promoter activity or in the transcription factors used by each cell line. However, the c subunit mRNAs exhibited a greatly enhanced stability in RAW 264.7 cells, accounting for the differences in cellular expression levels. All other subunits tested showed similarly enhanced mRNA half-lives, with the exception of the B2 subunit. In addition, the a subunits appear to be regulated at least in part by transcriptional control. These data suggest that transcriptional control may determine the subunit content of the V-ATPase, whereas regulation of mRNA stability determines its overall expression levels.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of the c Subunit Promoter-- A DNA fragment of 116 bp, comprising a portion of the first exon of the human c subunit gene, was used to screen a pre-arrayed mouse genomic BAC library (Incyte Genomics, Palo Alto, CA). BAC clones that gave a positive signal in the initial screen were digested with multiple restriction enzymes and reprobed with the same 116-bp DNA fragment. Positive bands were cloned into the plasmid pBluescript KS⁺ (Stratagene, La Jolla, CA) for further experiments. Putative promoter regions were characterized by sequencing the first exon and surrounding regions, using the ABI Prism dRhodamine Terminator Cycle Sequencing kit (PE Biosystems, Foster City, CA). Sequencing reactions were analyzed by the Protein and Nucleic Acid Chemistry Laboratory at the Washington University School of Medicine.

Primer Extension Analysis-- An antisense oligonucleotide primer of the sequence 5'-GAAAAACGAAGAATATTCGG-3', corresponding to c subunit base pairs 23-42 (relative to the translational start), was synthesized and end-labeled with [-³²P]ATP using T4 polynucleotide kinase. Total RNA was purified from mouse kidney using the RNeasy Mini kit (Qiagen Inc., Valencia, CA). Primer extension reactions were performed as described previously (25), and the products were analyzed in a conventional 6% polyacrylamide gel containing 1× TBE (0.09 M Tris borate, 2 mM EDTA, pH 8.3) with a DNA sequencing ladder as the molecular weight standard.

Luciferase Reporter Constructs and Transfections-- Promoter fragments were subcloned into the firefly luciferase-encoding vector pGL3-basic (Promega, Madison, WI). All fragments of clone L5 contained the entire 5'-untranslated region (up to bp +158) and were truncated at upstream sequences. Deletions were performed by PCR and confirmed by DNA sequence analysis.

NIH 3T3, RAW 264.7, and LLC-PK₁ cells were obtained from the American Type Culture Collection (Manassas, VA) and were grown in Dulbecco's modified Eagle's medium (Invitrogen, Rockville, MD) supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37 °C in a 5% CO₂ atmosphere. IC-21 cells also were obtained from the American Types Culture Collection but were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 10 mM Hepes, and penicillin/streptomycin. Transfection was performed using the SuperFect reagent (Qiagen Inc.). Cells in 60-mm dishes at 50% confluence were transfected with ~1 µg of DNA. Promoter fragments in pGL3 were co-transfected at a ratio of 5:1 with the plasmid pRL-CMV (Promega, Madison, WI) as an internal standard for transfection efficiency. The total concentration of plasmid added to the transfection mixture was kept constant in all experiments. Luciferase activity was quantified using the Dual-Luciferase Reporter Assay system (Promega) and a Zylux FB12 tube luminometer, according to the manufacturers' recommendations.

Gel Mobility Shifts and Supershifts-- Nuclear extracts from NIH 3T3 and RAW 264.7 cells were isolated as described (26). Double-stranded oligonucleotide probes were ³²P-labeled using T4 polynucleotide kinase and purified over a G-50 NICK Spin column (Amersham Biosciences, Inc., Piscataway, NJ). Six micrograms of nuclear extract either alone or premixed for 10 min with 100× competitor oligonucleotide were added to 50,000 cpm of probe, and incubated 20 min at room temperature prior to gel electrophoresis. For supershifts, extracts were mixed with 50,000 cpm of probe for 30 min at 4 °C, then 4 µg of purified antibody was added and incubated for an additional 30 min at 4 °C. Antibodies against the Sp1 and AP-2 families of transcription factors were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Reactions were run in precast 1× TBE minigels containing 5% acrylamide (Bio-Rad, Hercules, CA).

Nuclear Run-off Analysis-- Preparation of NIH 3T3 and RAW 264.7 nuclei and radiolabeling of nascent transcripts were performed as described (27). Radiolabeled RNA was isolated using RNAzol (Tel-Test, Inc., Friendswood, TX) followed by ethanol precipitation in the presence of ammonium acetate to remove unincorporated nucleotides. Radiolabeled RNA samples from 3T3 and RAW cell nuclei were adjusted to equal counts/min and were added to nitrocellulose membranes to which 5 µg of linearized, denatured plasmid had been applied using a slotted filtration manifold (Bio-Rad, Hercules, CA). Hybridization and washing of the membrane strips were performed as previously described (27).

Actinomycin D Treatment and Northern Blotting-- NIH 3T3 or RAW 264.7 cells were treated over time with 5 µg/ml actinomycin D (Sigma Chemical Co., St. Louis, MO). Total RNA was prepared using RNAzol (Tel-Test Inc.), following the manufacturer's instructions. Five micrograms of each RNA sample was separated in a formaldehyde gel system as previously described (23), and the RNA was transferred to GeneScreen Plus membrane (PerkinElmer Life Sciences, Boston, MA). The blots were probed with ³²P-labeled cDNA fragments of various murine V-ATPase subunits generated by reverse transcription-PCR from murine marrow cultures, as previously described (22), as well as ³²P-labeled probes for murine -actin and murine 18 S rRNA (both from Ambion, Austin, TX). Final washing conditions for all probes were 0.2× SSC, 1% SDS, at 65 °C. Hybridized probe was quantified using phosphorimaging and ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA).

Immunoblots and Immunocytochemistry-- Polyclonal antiserum generated against a C-terminal peptide of the a1 subunit was a kind gift of Dr. Xiao-Song Xie (University of Texas-Southwestern Medical Center, Dallas, TX). Antiserum derived against the V-ATPase a3 subunit was generously provided by Dr. Jan Mattsson (AstraZeneca Corp., Sweden). Polyclonal antiserum against the B2 subunit was described previously (28). For immunoblots, equivalent amounts of whole cell lysates from NIH 3T3 and RAW 264.7 cells were run in 10% SDS-PAGE gels (Bio-Rad, Hercules, CA) under standard conditions. Proteins were transferred to Hybond-P membranes (Amersham Biosciences, Inc.) and probed with antisera at the following dilutions: a1, 1:5000; a3, 1:1250; B2, 1:1000. Primary antibodies were detected using horseradish peroxidase-coupled secondary antibodies (Southern Biotechnology, Birmingham, AL) and chemiluminescence detection reagents (Pierce, Rockford, IL). Results were visualized using a Chemi-Doc image analysis system (Bio-Rad).

For immunostaining, IC-21 cells were plated on glass coverslips and, after an overnight incubation, were fixed and permeabilized in a solution containing 2% formaldehyde, 0.2% Triton X-100, and 0.5% deoxycholate in a physiological salt solution. Free aldehydes were quenched using 10 mM sodium borohydride, and the cells were preblocked in a solution of 0.8% bovine serum albumin, 0.1% gelatin, and 10 µg/ml goat IgG in phosphate-buffered saline. Primary antibodies were then added in a solution of 0.08% bovine serum albumin, 0.01% gelatin, and 1 µg/ml goat IgG in phosphate-buffered saline. Following a 2-h incubation, cells were washed and incubated with Alexa 568-conjugated goat anti-rabbit secondary antibodies (Molecular Probes, Eugene, OR) for 1 h. After further washing, the coverslips were mounted on slides in a mixture of 9 parts glycerol/1 part phosphate-buffered saline plus an anti-fading reagent. Cells were visualized using a Nikon Eclipse E800 microscope and SPOT image analysis software (Diagnostic Instruments, Sterling Heights, MI).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation and Sequencing of the c Subunit Exon I and 5'-Flanking Regions-- Five positive clones from a murine BAC library were identified using a 116-bp DNA fragment from the first exon of the human c subunit gene. Following digestion of these clones with multiple restriction enzymes and Southern blotting, subclones were generated for further study. Three were identified that contained the putative first exon and promoter region of the c subunit gene. These subclones included pL5, an 8-kb BamHI fragment; pD6, a 9-kb BamHI fragment; and pP10R, a 9-kb EcoRI fragment. DNA sequencing analysis revealed that pL5 contained the first exon of the c gene, with a consensus intron boundary at the same position as that found in the human gene (29). In contrast, pD6 encoded the entire c subunit coding region without introns, and its upstream sequence contained a long stretch of sequence corresponding to retroviral long terminal repeats. These results indicate that clone pD6 represents a pseudogene resulting from an event of retroviral reverse transcription and integration. Clone pP10R also encoded the c subunit without introns; however, the putative promoter region of pP10R was unlike that of pD6, indicating that it may represent a second pseudogene.

To examine this possibility that pL5 might represent a true c subunit gene fragment, we isolated regions of the three clones upstream of their coding sequences, fused them to a firefly luciferase reporter, and assayed their relative activities as promoters. As shown in Table I, the upstream fragment of pP10R showed no activity higher than the vector alone (pGL3-basic). Although pD6 elicited some activity, pL5 was by far the strongest promoter, with activity comparable to the CMV promoter. These data, coupled with the sequence information above, strongly suggested that clone pL5 contained the true c subunit promoter elements.

Fig. 1A illustrates a restriction map of the 8-kb L5 clone, whereas Fig. 1B shows the nucleotide sequence of ~1.1 kb beginning in the 5'-flanking region and ending at the base immediately upstream of the coding region.² Nucleotides are numbered relative to the site of transcription initiation, which were determined as discussed below.

View larger version (46K): [in this window] [in a new window]   Fig. 1.   Restriction map and sequence of the c subunit promoter. A, a restriction map of the L5 subclone is shown with the position of the first exon. B, the sequence of the c subunit 5'-untranslated region (underlined) and 1.1 kb of the 5'-flanking region are shown. The GC box centered at 34 (see below) is boxed.

Transcription of the c Subunit mRNA Initiates at a Single Site via an Initiator (Inr) Element-- To pinpoint the transcriptional start site(s) of the c subunit, the 5'-end of the native c subunit transcript was mapped by primer extension analysis. A strong band was produced (Fig. 2, arrow) that indicated transcriptional initiation 158 bases upstream of the translation start site, in the sequence 5'-CCATCTT-3', where the adenosine residue represents the start of transcription. A second experiment with a primer ~100 bp upstream of the one used in Fig. 2 gave the same results (data not shown). This start sequence corresponds well, although not perfectly, to the loose consensus sequence for an initiator (Inr) box, 5'-Py₂AN(T/A)Py₂-3', where Py = pyrimidine (30). No corresponding TATA box was evident 25-30 bp upstream of the transcription initiation site (see Fig. 1B). Although most abundantly expressed genes contain TATA boxes, Inr elements can serve as functional analogs in directing transcription initiation by RNA polymerase II (31).

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Mapping the 5'-end of the c subunit mRNA. Total cellular mouse kidney RNA was allowed to anneal to an oligonucleotide primer corresponding to a sequence within the first exon of the c subunit, and the primer was extended with reverse transcriptase. A single strong band of 200 bp was produced. Band sizes were determined by running an unrelated dideoxynucleotide termination sequencing reaction as a size marker adjacent to the primer extension reaction (not shown).

A GC Box Is Necessary for Full Expression in 3T3 Fibroblasts and RAW Macrophages-- To determine the promoter elements required for expression of the c subunit, portions of the L5 clone upstream of the coding region were subcloned in the firefly luciferase expression vector pGL3-basic. All constructs included the entire 5'-untranslated region and were truncated at various points upstream (see Fig. 3A). Fig. 3 shows the activities of these promoter fragments in 3T3 (Fig. 3B, left panel) and RAW 264.7 (right panel) cells. The overall pattern of expression in the two cell types was highly similar; fragments 571 to +158, 145 to +158, and 88 to +158 produced the highest activity. The longest fragment tested, 1.7 kb to +158, was only half as active, suggesting potential negative regulatory elements in the region from 1.7 to 571 kb. However, deletion of regions downstream of 88 produced the most dramatic change in promoter activity levels. As expected, removal of the transcriptional start site (fragment +3 to +158) caused a complete ablation of promoter activity. More significantly, deletion of sequences between 88 and 31 produced greater than 90% loss in promoter activity in both cell types. Inspection of this region for promoter element consensus sequences resulted in identification of a single GC box (GGGGCGGGG) centered at 34 (Fig. 1B). An identical sequence was found in the human gene centered at 36, further suggesting a role for this region in regulation of the c subunit gene (32). Because GC/GT boxes, the cognate binding sequences for the Sp1 family of transcription factors, are often critical components of TATA-less promoters (31), we mutated this site to GGaataaGG and determined activity of the resulting fragment. As illustrated in Fig. 3, construct 88 to +158/mutGC showed activity similar to the construct in which the majority of the GC box was deleted (31 to +158), indicating that this site is a primary element required for basal expression of the c subunit in both 3T3 and RAW 264.7 cells.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Transcriptional activity of the c subunit promoter. A, the deletion constructs used in this experiment are schematized, with the positions of the GC box and the transcriptional start site noted. B, left panel, the relative activities of the promoter constructs transfected into NIH 3T3 cells are shown. Activities were normalized against activity of plasmid pRL-CMV (Promega), which utilizes the cytomegalovirus enhancer/promoter region. B, right panel, activities of the same promoter constructs are shown in RAW 264.7 cells, also normalized against activity of pRL-CMV. For transfections, n  4; the mean ± S.E. are shown. C, nuclei were isolated from 3T3 and RAW cells, and transcription was permitted to proceed in the presence of [32P]UTP. RNA was isolated from these nuclei and equal cpm were hybridized to cDNA probes for the V-ATPase c subunit and the cloning vector pBluescript pKS (bottom). Both 3T3 and RAW cells showed equivalent levels of c subunit transcription.

As shown in Fig. 3B, the activity of the c subunit promoter, relative to the CMV-driven internal standard, was higher in 3T3 cells than in RAW 264.7; however, these data cannot be taken as measurements of absolute activity, because the strength of the CMV promoter varies among cell types. To more accurately measure the relative transcriptional activity of the c subunit promoter in fibroblasts and macrophages, we performed nuclear run-off analysis. Nuclei were prepared from both cell types, and transcription was permitted to proceed in the presence of [³²P]UTP. Equal numbers of radiolabeled RNA were added to membrane strips containing cDNA probes for either the c subunit, or the cloning vector pBluescript II KS (Stratagene, La Jolla, CA) as a negative control. As shown in Fig. 3C, radiolabeled c subunit mRNAs were detected at equal levels in both 3T3 and RAW cells, indicating that c subunit transcription in these cells represents equivalent fractions of overall transcription. Although interpretation of this experiment requires the assumption that the overall levels of RNA synthesis do not differ dramatically between the two cell types, this result further suggests that activity of the c subunit promoter is similar in both fibroblasts and macrophages.

To determine the identity of factors that bind to the GC box, we performed gel mobility shift assays using nuclear extracts from both 3T3 and RAW 264.7 cells. Fig. 4A shows extracts probed with oligonucleotides encompassing the wild-type c subunit GC box (5'-CCCGTGGGGGCGGGGACAGATC-3'). Three bands, designated A-C, were shifted when extracts from either cell type were used (lanes 2 and 6). Preincubating the nuclear extracts with unlabeled competitor oligonucleotides corresponding to the c subunit GC box (lanes 3 and 7) prior to the addition of probe resulted in loss of all three bands. Similar results were obtained when extracts were preincubated with a GC box containing different flanking sequences (5'-ATTCGATCGGGGCGGGGCGAG-3', obtained from Promega). In contrast, addition of unlabeled competitor oligonucleotides corresponding to a mutated c subunit GC box (5'-CCCGTGGGaataaGGACAGATC-3') did not abolish the bands in question. These data indicate that the proteins responsible for the mobility shift are likely to be related to the Sp1 family of transcription factors.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Gel mobility shift analysis of critical transcription factors in c subunit promoter activity. A, nuclear extracts (in equal amounts) from NIH 3T3 and RAW 264.7 cells were mixed with a 32P-labeled double-stranded oligonucleotide corresponding to the c promoter GC box (see text for details). Three bands, designated A, B, and C, were produced by mixing nuclear extracts from either 3T3 or RAW cells with the probe (lanes 2 and 6). Competition with unlabeled oligonucleotides corresponding to either the c subunit GC box (lanes 3 and 7), or a consensus GC box of different sequence (lanes 4 and 8) resulted in a loss of the three bands. A mutated GC box, however, did not compete with the labeled probe for binding (lanes 5 and 9). The unbound oligonucleotide probe was run off the bottom of the gel in an effort to achieve good separation of bands A and B. No other bands were visible in the gel. B, equal amounts of nuclear extracts from 3T3 and RAW cells were mixed with the same oligonucleotide probe as in Fig. 4A, but reactions were done either in the absence (lanes 1 and 6) or presence (lanes 2-5, 7-10) of supershift-grade antibodies. Addition of an antibody against Sp1 (lanes 2 and 7) shifted band A, whereas addition of antibody against Sp3 (lanes 3 and 8) shifted bands B and C. Antibodies against two forms of transcription factor AP-2, which also recognizes a GC-rich consensus, did not affect the banding pattern.

To positively identify the proteins bound to the c subunit GC box, we performed supershift analysis on nuclear extracts from both 3T3 and RAW 264.7 cells (Fig. 4B). Antibodies against transcription factors Sp1 and Sp3 were included in standard mobility shift reactions, as were antibodies against two members of a different class of transcription factors that also recognize GC-rich sequences, AP-2 and AP-2. Fig. 4B shows that addition of an antibody against Sp1 created a supershifted band, as well as a diminution in the intensity of band with the lowest mobility (A), indicating that the identity of the protein bound in band A is Sp1. Similarly, an antibody against Sp3 created a supershifted band and resulted in decreased intensity of bands B and C. Sp3 is represented by multiple bands due to alternate translational initiation within its coding region (33). In contrast, antibodies against AP-2 family members did not produce supershifted bands.

The c Subunit mRNA Shows Greater Stability in RAW 264.7 Cells Than in NIH 3T3 Cells-- As shown by the above experiments, RAW 264.7 and NIH 3T3 cells utilize the same promoter elements and transcription factors to initiate transcription of the c subunit mRNA. Furthermore, nuclear run-off analysis showed that levels of transcription were similar in both cell types. This was somewhat surprising, given the difference in steady-state levels of c subunit mRNA between RAW and 3T3 cells. We surmised that this disparity might be accounted for by different rates of mRNA decay. A role for stability in control of V-ATPase mRNA levels was further suggested by the finding that V-ATPase mRNAs typically contain long (up to >1 kb), highly conserved 3'-untranslated regions,³ which are often mediators of transcript stability (34).

To investigate this possibility, cells were treated with actinomycin D, an inhibitor of RNA polymerase II, and mRNA levels were examined by Northern analysis. In both cell types, the c subunit mRNA was found to be relatively long-lived, with little to no degradation after 4 h of treatment. However, although the c subunit transcript was decreased by ~75% in 3T3 cells after 24 h of actinomycin D, no change was seen in RAW cells after the same length of time (Fig. 5A). To ensure the activity of actinomycin D in RAW cells, we reprobed the blot with a cDNA corresponding to mouse -actin mRNA, which possesses a half-life that varies from 6 to 26 h, depending on the cell type and growth condition (35, 36). We found a diminution of this mRNA species within 24 h after actinomycin treatment in both 3T3 and RAW cells, indicating that the unchanged expression levels for the c subunit in RAW cells were not due to inactivity of the actinomycin D. Reprobing the blot with a cDNA corresponding to the murine 18 S rRNA controlled for equivalent RNA loading in the gel (lower panel). In two separate experiments, the average half-life of the c subunit mRNA in NIH 3T3 cells was 14 h. We were unable to empirically determine this figure in RAW 264.7 macrophages, because longer treatments with actinomycin D resulted in cell death. However, because we determined that the c mRNA transcription rates in the two cell types are equal and that, at steady-state, RAW cells express 6- to 8-fold more of the transcript than 3T3 cells, their half-lives must also differ by 6- to 8-fold (37). Therefore, the half-life of the mRNA in RAW cells can be estimated at between 3.5 and 4.5 days, values similar to those of other highly stable mRNAs such as -globin (38).

View larger version (55K): [in this window] [in a new window]   Fig. 5.   Stability of V-ATPase subunit transcripts in fibroblasts and macrophages. A, NIH 3T3 fibroblasts and RAW 264.7 macrophages were treated with actinomycin D for the designated periods of time, and cellular RNA was isolated and prepared for Northern analysis. The RNA blot was probed first with a cDNA corresponding to the V-ATPase c subunit, then stripped and reprobed with cDNAs corresponding to murine -actin and the 18 S rRNA. B, RNA blots such as that shown in A were probed with cDNAs corresponding to various subunits of the V-ATPase. Note the enhanced mRNA stability in RAW cells for all subunits except B2.

To determine whether other V-ATPase subunits showed enhanced stability in RAW 264.7 cells, we performed similar experiments using probes against various V-ATPase subunit mRNAs. Fig. 5B shows total RNA from actinomycin D-treated cells that was hybridized to cDNA probes corresponding to subunits E and F, which are components of all murine V-ATPases, and to three subunit isoforms, B2, a1, and a3. Like the c subunit mRNA, the E, F, and a1 mRNAs showed enhanced stability in RAW 264.7 cells. Subunit a3, which is expressed primarily in cells of the monocyte-macrophage lineage, was not detectable in NIH 3T3 cells under these conditions; nevertheless, the a3 mRNA was highly stable in RAW 264.7 cells. In contrast, the mRNA encoding B2 did not show enhanced stability in the macrophage line, even though steady-state levels were higher in RAW cells than in 3T3 cells. From two independent experiments, the average half-life for the B2 subunit mRNA in 3T3 cells was calculated as 13.9 h, and in RAW 264.7 cells, 14.3 h. Because the differences in steady-state levels of B2 mRNA cannot be accounted for by message stability, this subunit must be transcribed at a greater rate in the macrophage line. These data are consistent with our previous observation that B2 subunit is regulated by transcriptional mechanisms during macrophage differentiation (23).

Although stability of the a1 mRNA is higher in RAW 264.7 cells than in NIH 3T3 cells, both cell types express similar levels of this mRNA (note the "0" time points for a1 in Fig. 5B), indicating that transcriptional controls also may play a role in its expression. Furthermore, the highly tissue-restricted nature of the a3 subunit (12, 13) suggests that specific mediators (such as transcription factors) may be responsible for its expression in macrophages, although our stability data cannot directly address this matter. (We were unable to detect a2 mRNA in macrophage preparations, and a4 is specific to only a few distinct cell types (14).) It is noteworthy that the a1 mRNA (uniquely, out of the V-ATPase mRNAs tested) was not expressed at higher levels in the macrophage cell line than in the fibroblasts. This suggests that increased levels of V-ATPase in macrophages over fibroblasts is accounted for by a3-containing complexes, which may play a specialized role in macrophages, as they do in a related cell type, the osteoclast (18-20). To explore this hypothesis further, we performed immunoblots of a1 and a3 on NIH 3T3 and RAW 264.7 lysates to determine whether protein levels of these subunits mirrored their corresponding mRNA levels. As shown in Fig. 6A, both a3 and B2 proteins were increased in RAW cells over 3T3 cells, as expected from their respective Northern analysis. The a1 subunit protein levels also appeared to be determined by mRNA levels in the two cell types, because immunoblot analysis showed roughly equivalent expression in both cells.

View larger version (80K): [in this window] [in a new window]   Fig. 6.   Expression of a1 and a3 subunits in macrophages. A, equivalent amounts of whole cell extracts from NIH 3T3 or RAW 264.7 cells were run in SDS-PAGE, transferred to membrane, and probed with antisera derived against the a1, a3, or B2 subunits. B, the murine macrophage cell line IC-21 was plated on glass coverslips and immunostained with antibodies against the a1 (top panels) or a3 (bottom panels) subunits. Arrows indicate heavy staining of a1 at the leading edge of the cells, and staining of a3 in tubular patterns that radiate from the nucleus (see text). Bars = 10 µm.

Because macrophages and fibroblasts appear to express similar amounts of a1-containing V-ATPases, we surmised that this form of the enzyme may represent a constitutive "housekeeping" form, and that a3-containing complexes might represent a V-ATPase specialized for macrophage-specific functions. We next performed immunostaining of macrophages with antibodies derived against a1 and a3 to determine whether V-ATPases containing these isoforms may reside in different intracellular compartments. RAW 264.7 cells in culture are round and somewhat indistinct in morphology, so these studies were performed on another murine macrophage cell line, IC-21, which exhibits a morphology closer to that of primary macrophages. Fig. 6B shows two typical IC-21 cells stained for a1 (top panels) and two stained for a3 (bottom panels). We previously had noted that staining of macrophages for another V-ATPase subunit, B2, resulted in a tubulovesicular pattern, as well as prominent staining at the leading edge (28). In this study, the a1 and a3 isoforms each appear to compose a subset of the B2 staining. First, a1 staining was most apparent as a punctate, vesicular pattern that was fairly evenly distributed throughout the cytoplasm, and was prominent at the leading edge of motile macrophages (arrows). In contrast, a3 staining was heavy near the nucleus, radiating throughout the cytoplasm in tubular structures (arrows), and was present at the edge of the cells only in a fine, punctate pattern. Although it is not the purpose of this study to define the respective roles of these isoforms in macrophages, these data, coupled with the expression data, suggest that a3-containing complexes account for the bulk of V-ATPases over that required for housekeeping functions in macrophages, and that these complexes may play a specialized role in these cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although all eukaryotic cells require low to moderate levels of V-ATPase to acidify their endocytic networks, specialized, proton-secreting cells such as kidney tubule epithelia, macrophages, and osteoclasts express levels of the enzyme that are greater by an order of magnitude or more. For these cells, amplified levels of the enzyme are required to create a pool that is available for expression at the plasma membrane and other membranous compartments (such as phagosomes) while still maintaining the housekeeping function in the endocytic network. In this study, we compared the promoter activity and mRNA stability of a ubiquitous V-ATPase subunit in both a high expressing and low expressing cell type. This study marks the first comprehensive effort to define the genetic elements required for cell-specific expression levels of the V-ATPase.

Because the V-ATPase is composed of 13 distinct polypeptides in a highly ordered structure that may differ from one tissue to the next, it would seem advantageous to a cell to tightly regulate V-ATPase subunit synthesis to conserve resources. As the cDNAs for V-ATPase subunits have become available, screening of various tissues by Western and Northern blotting showed that mRNA levels reflect those of the translated protein, indicating that subunit levels are controlled at least in part at the level of mRNA expression. Our previous studies suggested further that V-ATPase mRNAs are expressed in the same stoichiometry as V-ATPase subunits (22) and that increases in V-ATPase expression are mediated by coordinated up-regulation of all subunit mRNAs (22, 23). These results underscored the role of mRNA control mechanisms in regulating V-ATPase expression levels and suggested the existence of a universal mechanism that could regulate all subunits in tandem. Our current findings indicate that control of mRNA stability may provide this mechanism. With the exception of the B2 subunit (and possibly a3), all V-ATPase transcripts exhibited increased stability in macrophages, compared with fibroblasts. In addition to the findings presented here using our macrophage model system, we also tested the stability of several V-ATPase subunit mRNAs in a kidney proximal tubule cell line that expresses high levels of the enzyme. We found that the mRNAs similarly showed enhanced stability in the kidney cells (data not shown); thus, this may be a universal mechanism for regulation of the V-ATPase in proton-secreting cells.

Extremely long-lived mRNAs often are associated with differentiated cells that require an accumulation of protein species specific to their function. For example, globin mRNAs accumulate to 95% of the total cellular mRNAs in erythrocytes as a consequence of their extreme stability (t_1/2 > 24 h (38)). Similarly, V-ATPase transcripts can represent a large proportion of cellular mRNAs in proton-secreting cells. Random sequencing of an osteoclast cDNA library showed the V-ATPase c subunit to be one of the two most abundant mRNA species in the cell (24).

The V-ATPase promoters characterized so far share the property of initiating transcription from TATA-less promoters. These promoters are often associated with "housekeeping" genes; that is, those encoding products that are essential to eukaryotic cell survival. Although the V-ATPase fits this definition, there is a great degree of promoter variability among the subunits studied. The c subunit gene contains the simplest arrangement of promoter elements, with an Inr box and a Sp1-binding site about 30 bp upstream. In contrast, promoters of the B2 and B1 isoforms are arranged in a more complicated organization. The B2 gene contains a TATA-less promoter that is characterized by a high G+C content and lacks an Inr sequence (23), an arrangement often found in genes regulated by the cell cycle (39). Moreover, the activator elements of the B2 promoter (overlapping Sp1 and AP-2 sites) reside downstream of the site for transcription initiation (23, 40). The B1 gene, also lacking a TATA box, contains consensus Inr sequences and is marked by long GA/CT sequences proximal to the potential start sites.⁴

Expression of the B2 isoform in macrophages is unusual among V-ATPase subunits examined in this and previous studies perhaps because of its association with the cytoskeleton and potential role in intracellular trafficking (15, 16, 23). Here, we showed that, unlike other subunits tested, B2 mRNA did not show enhanced stability in macrophages over fibroblasts. This result is consistent with previous work in which we examined the regulation of several V-ATPase subunits during monocyte-to-macrophage differentiation (23). During this process, overall V-ATPase content increased about 4-fold. However, nuclear run-off analysis showed that transcriptional controls mediated this increase only for B2, but not other subunits, which were regulated by post-transcriptional mechanisms. Thus, in macrophages, B2 appears to be primarily under transcriptional control.

Although mRNA stability may be mediated by sequences throughout the transcript, it is most often mediated by portions of the 3'-untranslated region (3'-UTR). Although many eukaryotic genes possess 3'-UTRs of less than 100 bp, V-ATPase subunit genes (with a few exceptions) contain extremely long 3'-UTRs of up to more than 1 kb. In addition, these regions tend to be more highly conserved than the 3'-UTRs of many eukaryotic genes, with regions of over 85% identity between human and rodent sequences.³ Thus, V-ATPase 3'-UTRs are likely candidates to mediate the extreme stability of mRNAs in macrophages and other cell types. Additionally, in osteoclasts, a cell type that targets V-ATPase to an apical ruffled membrane, V-ATPase mRNAs were shown to be targeted toward this membrane domain during the cell's active, polarized state (41). Targeting of mRNAs in such a manner universally has been attributed to sequences in 3'-UTRs that contain signals for localization mediated by cytoskeletal components (42), and such interactions may modulate mRNA half-life. Therefore, the 3'-UTRs of V-ATPase mRNAs may contain a wealth of genetic information that mediates expression of this functionally diverse enzyme.

Macrophages express greater levels of all subunit mRNAs and proteins tested in this study, with the exception of the a1 subunit. Levels of a1 in macrophages were equivalent to those found in fibroblasts, which appear to utilize the V-ATPase only for constitutive, housekeeping functions. These results suggest that the additional V-ATPases of macrophages contain the a3 subunit and may be utilized for a function specific to macrophage physiology. A close relative of macrophages, the osteoclast, appears to use a3-containing V-ATPases to shuttle between intracellular stores and the plasma membrane, as required for regulated proton extrusion and resorption of bone (18-20). The differences in distribution between a1 and a3 (as shown in Fig. 6B) strongly suggest differential functions for these two forms in macrophages, most likely relating to their roles in immunity and proton secretion. Further experiments on this subject should provide significant insights into specialized membrane domains of the macrophage.

	ACKNOWLEDGEMENTS

We thank Drs. Jan Mattsson and Xiao-Song Xie for their kind gifts of antibodies, Dr. Stephen Gluck for contributions made to early portions of this work, and Dr. Jill Rafael for the use of microscopy facilities.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF366932.

^¶ To whom correspondence should be addressed: Dept. of Physiology and Cell Biology, The Ohio State University, 302 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. Tel.: 614-688-3585; Fax: 614-292-4888; E-mail: lee.2076@osu.edu.

Published, JBC Papers in Press, January 10, 2002, DOI 10.1074/jbc.M111959200

² GenBank^TM accession number AF366932.

³ B. S. Lee, personal observation.

⁴ R. D. Nelson, personal communication.

	ABBREVIATIONS

The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; CMV, cytomegalovirus; Inr, initiator element; UTR, untranslated region.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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