To determine which metabolic pathways were stressed by low doses of V-ATPase inhibitors, we first determined the dose response to two different inhibitors of the V-ATPase on several cell lines. We wished to find a concentration of each inhibitor that would show an effect on growth, indicating that cells were responding to the stimulus, but which was not so toxic that the response would be prevented or complicated by cells dying during the experiment. The cell lines we tested had IC50s to bafilomycin A that ranged from 10 nM to greater than 10 µM and we chose the moderately sensitive cell line HeLa (IC50
150 nM) for our experiments because its relatively shallow dose response made for more uniform growth inhibition experiments, suggesting that the gene expression experiments would also be more reproducible. We also chose to compare two inhibitors of the V-ATPase that act through distinct mechanisms 
, bafilomycin A (Baf) and phenylsalicylihalamide (LX1077), to control for possible off-target effects of each compound. Concentrations of each compound that inhibited cell growth approximately 25% after 48 hours () were chosen for the gene expression experiments. HeLa cells were cultured overnight and then samples were treated with 15 nM Baf for 6, 12, or 24 hours, or 200 nM LX1077 for 12 and 24 hours, and RNA was harvested and processed for analysis by microarray. As controls, samples of HeLa cells treated for the same interval with 0.1% DMSO (the final concentration of the diluent for Baf or LX1077 in experimental samples), were run in parallel with each experimental condition. Each experimental condition was repeated in an independent experiment conducted on a different day.
Dose-response of HeLa cells to BafA or LX1077.
Two major pathways for uptake of nutrients into cells depend upon the acidity in endosomes supplied by the V-ATPase. Inhibiting the V-ATPase will interfere with the delivery of iron into the cell by preventing the release of iron from transferrin and also prevents uptake of cholesterol by inhibiting the release of LDL from its receptor. Therefore we treated HeLa cells for 6 or 12 hours with deferoxamine (DFO) to chelate iron and separately with medium lacking LDL to mimic inhibiting uptake of exogenous cholesterol through the LDL receptor. Controls were matched samples cultured in normal culture medium. These samples were processed for microarrays exactly as samples treated with V-ATPase inhibitors. In this way we could identify all genes responding by increased transcription when iron was deficient or LDL uptake was blocked. Data was processed by the UT Southwestern DNA Micro Array Core Facility using GCOS software with MAS5 normalization and experimental samples were compared to the DMSO treated samples, or, for DFO or low LDL conditions, samples cultured in normal medium run at the same time to calculate the magnitude of change.
Genes were considered to show significantly increased expression if both samples at one time point with either Baf or LX1077 had a change P value of less than 0.002 and the average of the two samples increased more than 2-fold. In most cases, Baf induced transcription more than LX1077, although the genes responding to Baf also responded to LX1077. The genes that increased transcription earliest are shown in Tables S1
and . Table S1
presents 47 genes that increased expression when HeLa cells were treated with V-ATPase inhibitors or with low cholesterol medium and represent the response to inhibiting the V-ATPase that is due to blocking delivery of exogenous cholesterol. The response of most of these genes is faster in the presence of the V-ATPase inhibitors than it is to medium lacking LDL, perhaps because medium lacking LDL does not block the delivery of cholesterol from LDL particles that are either bound to their receptor at the cell surface or already released into the endocytic pathway at time the medium deficient in LDL was added. The majority of the genes shown in Table S1
function in lipid biosynthetic pathways and 23 are known targets of SREBP1 or 2 
. With the exception of EGLN1 and STARD4, none of the genes that increase transcription early in response to low LDL respond to the iron chelator DFO. EGLN1 encodes an iron-dependent prolylhydroxylase that represses Hif-1α activity under normoxic conditions 
and is not known to be sensitive to the activity of the cholesterol biosynthetic pathway. However, in yeast and mammals there is cross-talk between cholesterol biosynthesis and pathways of adaptation to hypoxia 
. STARD4 encodes a cytosolic cholesterol transfer protein that is an SREBP target 
and also is upregulated by ER stress 
and might be only indirectly upregulated by the iron chelator. Interestingly, increased transcription of INSIG1, encoding a negative regulator of the SREBP transcription factors that control cholesterol biosynthesis, is one of the faster responses to V-ATPase inhibitors. This is possibly a negative feedback mechanism to limit the transcriptional response once lipid metabolic pathways have increased production of endogenous cholesterol 
Genes Increasing Expression Early with V-ATPase Inhibitors and not with DFO or Low LDL.
lists 64 genes that increased transcription fastest when iron was chelated and also respond to inhibition of the V-ATPase. These are genes in pathways that presumably respond to the inhibition of iron release by transferrin in endosomes when the V-ATPase is inhibited. At least 29 of these genes are known targets of Hif-1α and respond to hypoxia. As one would expect, these genes function in many pathways. Aldolase C is a Hif-1α target 
and is strongly upregulated by V-ATPase inhibitors. Although we did not detect an early increase in aldolase C in cells treated with low LDL medium, aldolase C is also a known target of SREBPs 
. Aldolase C binds to the V-ATPase 
and in yeast is responsible for glucose regulation of V-ATPase activity 
. Since aldolase C responds to hypoxia in lung epithelial cells and to V-ATPase inhibitors in HeLa cells, whereas the more abundant glycolytic enzyme aldolase A does not, it is possible that a major function of aldolase C in response to lack of iron or hypoxia is to regulate the V-ATPase as part of the mechanism to ensure iron homeostasis. Hif-1α is repressed primarily by being hydroxylated on proline by iron-dependent prolyhydroxylases, which targets it for degradation 
. By immunoblot we found that incubating HeLa cells in 15 nM Baf for 1 hr stabilized Hif-1α (data not shown), suggesting that pathways regulated by Hif-1α are among the most sensitive to inhibition of the V-ATPase. Thus, V-ATPase inhibitors provide a most effective means for engaging Hif-1α pathways experimentally.
presents 7 genes that increased transcription in response to V-ATPase inhibitors, but not in HeLa cells treated with low LDL medium or with the iron chelator. These are candidates for pathways that sense the lack of V-ATPase activity independently of the effect on transferrin or LDL. Other than CLCN6, these genes are not strongly upregulated and several of them are involved in the cell cycle and growth control and may be an early part of the growth inhibition that will occur after longer incubation in the inhibitors. CLCN6 is a chloride channel found in neurons and epithelial cells 
, and localizes to late endosomes 
. Chloride channels in endosomes work in concert with V-ATPase and thus CLCN6 upregulation may be a response to loss of late endosome function. SREBF2 (SREBP2) is a major regulator of cholesterol biosynthesis and it is primarily regulated post-transcription by membrane traffic and proteolysis 
. The modest transcriptional increase is likely balanced by the increase in expression of the inhibitor of SREBP activation, INSIG1.
presents the results of taking a less restrictive analysis of the genes that change in HeLa cells treated with 15 nM Baf for 6 hr. All genes that showed a statistically significant change (change P value >0.00025) regardless of the direction of change were analyzed with Ingenuity Pathways to determine which canonical pathways in the Ingenuity Database were enriched in genes that were changed in response to Baf. Pathways that responded significantly to Baf contained very few genes that decreased expression, confirming our assumption that the early response would be increased gene expression. By far the most significantly affected pathways were steroid biosynthesis, followed by glycolysis, and involved genes for the most part also identified by our more restrictive analysis.
Pathways significantly enriched in genes changing expression after 6 h treatment with 15 nM bafilomycin.
After longer intervals of treatment with V-ATPase inhibitors, 52 genes that had increased in response to 12 hours culture in low LDL medium also showed increased expression in cells treated for 24 hours with V-ATPase inhibitors (Table S3
). Eight of the cholesterol responsive genes that were upregulated earlier no longer showed an increase and 15 new cholesterol responsive genes showed increased transcription, with 6 of these encoding proteins involved in lipid biosynthetic pathways. 104 “iron responsive” genes (Table S4
) were increased in cells treated with V-ATPase inhibitors for 24 hours, including a number of genes encoding glycolytic enzymes or repressors of mitochondrial function, transcription factors and proteins regulating cell growth. STXBP1, which encodes the MUNC-18 regulator of membrane fusion, showed modest increase in response to either depleting cholesterol or iron and a stronger increase in cells treated with V-ATPase inhibitors. This is one of the very few genes encoding proteins that regulate membrane traffic that increased in response to inhibiting the V-ATPase (HIP1R, SV2A and OPTN were the others). Sixty two genes increased expression at least two-fold in response to inhibiting the V-ATPase for 24 hours, but did not respond to cholesterol deficient medium or DFO (Table S5
). This set of genes was analyzed using Ingenuity Pathways software to identify metabolic pathways in which genes were significantly upregulated (). By far the most significant response was in genes involved in lipid and steroid biosynthesis.
Metabolic functions of genes increasing expression after 24 hours in response to inhibitors of V-ATPase, but not responding to low cholesterol or iron chelation.
As one would expect, if both increases and decreases in gene expression are considered, after 24 hours in Baf there is a complicated response involving a number of pathways that control growth and response to stress (Table S6
). The most significant of these is the NRF2-mediated oxidative stress response, suggesting that inhibiting the V-ATPase increases reactive oxygen species. As important components of the mitochondrial respiration pathway require iron, and inhibition of oxidative phosphorylation can produce ROS, changes in NRF2 mediated pathways could be a secondary consequence of inhibiting iron import from transferrin.
Our original intent in pursuing these studies was to investigate the causes of differential response to V-ATPase inhibitors seen in cancer cells. Because the major immediate responses to inhibition of the V-ATPase were to the lack of iron or cholesterol, we investigated if these were responsible for the hypersensitivity of some cancer cells to V-ATPase inhibitors (). We measured the dose-response of cervical cancer derived HeLa, melanoma derived cell lines SK-Mel-5 and SK-Mel-28, non-small cell lung cancer line H1299, breast cancer MCF-7 and Morris Hepatoma (MH) cells to Baf in the presence or absence of 150 µM iron citrate. Baf had very similar IC50 concentrations for HeLa, MCF7 and H1299. Both MH and SK-Mel-5 cells were 10-fold more sensitive than HeLa, and SK-Mel-28 were highly insensitive (). For Hela, SK-Mel-5 and SK-Mel-28 we also tested the effect of adding cholesterol delivered into cells with methy-β-cyclodextrin, which adds cholesterol directly to the plasma membrane 
. Adding iron to the medium made HeLa cells 20-fold more resistant to growth inhibition caused by 20 to 200 nM bafilomycin, but provided little protection to the cytotoxic effects observed at higher concentrations (). Cholesterol was protective for HeLa cells, but not for the Baf-sensitive SK-Mel-5 cells or Baf resistant SK-Mel-28 (). This indicates that the cytostatic effects of V-ATPase inhibitors in HeLa cells are largely due to the inhibition of iron and cholesterol uptake, but the cytotoxic effects are due to an alternative, and as yet unknown, function. Supplementing H1299, MCF7 or the more sensitive SK-Mel-5 melanoma cell line with iron protected them at lower concentrations of Baf similar to HeLa cells (). In fact, MCF7 or SK-Mel-5 cells treated with iron and bafilomycin had a dose response remarkably similar to HeLa cells treated with bafilomycin alone, suggesting that these cell lines were particularly sensitive to inhibition of iron uptake. Neither iron nor cholesterol had any protective effect on the highly resistant cell line SK-Mel-28 or the quite sensitive MH cell line.
Iron supplement desensitizes cancer cell lines to bafilomycin.
Cholesterol supplement does not desensitize SK-Mel-5 cells to bafilomycin.
To investigate the mechanism of cytotoxicity induced by Baf in SK-Mel-5 and HeLa cells, we treated cells of each line with 0 to 1000 nM Baf for 24 hours and then investigated the cleavage of PARP or caspase 3 as signs for the induction of apoptosis (). In both cell lines Baf induced apoptosis after 24 hours; however, the concentration required was over 20-fold lower for SK-Mel-5 than HeLa cells, very similar to the differences in lethal doses for the two cell lines. Thus, the cytotoxicity resulting from inhibiting the V-ATPase in the hypersensitive cell line SK-Mel-5 is through induction of apoptosis.
Both SK-Mel-5 cells and MCF7 cells were more sensitive to Baf than HeLa cells and this difference in sensitivity was abolished by supplementing the culture medium with iron (). Thus we investigated the expression of proteins involved in iron uptake, storage or efflux in these cell lines and HeLa cells (). SK-Mel-5 cells expressed less transferrin receptor than either of the other two cell lines and very low levels of DMT1, the transporter responsible from moving iron across the endosome membrane to the cytoplasm. Remarkably, SK-Mel-5 cells expressed high levels of the iron efflux transporter, ferroportin, which transports iron across the plasma membrane and out of the cell. Ferroportin was not detected in either of the other two cell lines. SK-Mel-5 cells expressed less ferritin than HeLa cells, consistent with having low levels of cytoplasmic iron. Taken together, this pattern of expression suggested that SK-Mel-5 cells were actively maintaining relatively low cytoplasmic iron levels. In contrast, MCF7 cells had levels of transferrin receptor similar to HeLa cells, increased expression of DMT1 and very low levels of the iron storage protein, ferritin. This is a pattern of expression consistent with a cell that has low cytoplasmic iron levels and has upregulated iron transport mechanisms in response. Thus, SK-Mel-5 cells and MCF7 cells, although both hypersensitive to Baf due to inhibition of iron uptake, differed in the molecular mechanisms underlying this vulnerability.
The expression of proteins involved in iron transport in SK-Mel-5 cells indicates a decrease in iron stores.
The differences in expression of transferrin receptor and ferroportin in SK-Mel-5 cells were not due to an inability to regulate these proteins in response to iron. When SK-Mel-5 cells or HeLa cells were treated with DFO to chelate iron, both upregulated expression of transferrin receptor (). In response to DFO, transferrin receptor expression in SK-Mel-5 cells was very similar to that in HeLa, indicating that mechanisms for expressing the receptor in response to iron were intact in SK-Mel-5 cells. DFO treatment decreased the expression of ferroportin and ferritin in SK-Mel-5 cells, as one would expect for a cell striving to maintain cellular iron levels. Thus, mechanisms for regulating these proteins by iron were also intact in SK-Mel-5 cells. When SK-Mel-5 cells were treated with Baf, they also increased expression of the transferrin receptor and decreased ferritin. However, ferroportin expression was significantly increased. Since chelating iron decreased ferroportin expression in SK-Mel-5 cells, this response was due to some other effect of inhibiting the V-ATPase.
Iron regulation of transferrin receptor, ferroportin and ferritin is intact in SK-Mel-5 cells.
A possible reason for a cell to actively depress cytoplasmic iron levels would be to protect against the generation of reactive oxygen species (ROS) by iron. To investigate if SK-Mel-5 cells might be producing more ROS, we labeled SK-Mel-5 and HeLa cells with dyes that increase fluorescence in the presence of ROS. Cells were photographed with identical camera settings and fluorescence intensities of cells were measured using Image J software. SK-Mel-5 cells produced significantly more ROS than HeLa cells, measured with both dyes ().
Since SK-Mel-5 cells appeared to have low levels of intracellular iron and were hypersensitive to Baf, which would further decrease iron uptake, we investigated the sensitivity of SK-Mel-5 cells to the iron chelator DFO (). As expected, SK-Mel-5 cells were hypersensitive to DFO compared to HeLa cells. In fact, DFO was only cytostatic for HeLa cells, whereas it was cytotoxic for SK-Mel-5. Adding exogenous iron to the cell culture medium of SK-Mel-5 or HeLa cells had no effect on cell growth (data not shown).
SK-Mel-5 cells are hypersensitive to DFO.
Three observations suggested that ferroportin expression might be induced by ROS in SK-Mel-5 cells. SK-Mel-5 cells had both increased ROS production and increased ferroportin expression. One of the major pathways induced by Baf treatment of HeLa cells for 24 hr was the antioxidant response mediated by NRF2, suggesting that Baf treatment could increase ROS. Baf induced rather than repressed ferroportin expression in SK-Mel-5 cells, whereas lowering iron levels with DFO decreased ferroportin expression. If ferroportin expression was induced by ROS, then treating SK-Mel-5 cells with the anti-oxidant N-acetylcysteine (NAC) would be expected to decrease ferroportin expression. When SK-Mel-5 cells were treated for 48 hrs with increasing concentrations of NAC, ferroportin expression was decreased (). Taken together, our data suggest that SK-Mel-5 cells are vulnerable to inhibition of iron uptake because they maintain low iron reserves, perhaps to protect themselves from generating toxic levels of ROS.
Ferroportin expression in SK-Mel-5 cells is decreased by the antioxidant NAC.