Expression profile of genes regulated by activity of the Na-H exchanger NHE1
© Putney and Barber 2004
Received: 24 March 2004
Accepted: 16 July 2004
Published: 16 July 2004
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© Putney and Barber 2004
Received: 24 March 2004
Accepted: 16 July 2004
Published: 16 July 2004
In mammalian cells changes in intracellular pH (pHi), which are predominantly controlled by activity of plasma membrane ion exchangers, regulate a diverse range of normal and pathological cellular processes. How changes in pHi affect distinct cellular processes has primarily been determined by evaluating protein activities and we know little about how pHi regulates gene expression.
A global profile of genes regulated in mammalian fibroblasts by decreased pHi induced by impaired activity of the plasma membrane Na-H exchanger NHE1 was characterized by using cDNA microarrays. Analysis of selected genes by quantitative RT-PCR, TaqMan, and immunoblot analyses confirmed results obtained from cDNA arrays. Consistent with established roles of pHi and NHE1 activity in cell proliferation and oncogenic transformation, grouping regulated genes into functional categories and biological pathways indicated a predominant number of genes with altered expression were associated with growth factor signaling, oncogenesis, and cell cycle progression.
A comprehensive analysis of genes selectively regulated by pHi provides insight on candidate targets that might mediate established effects of pHi on a number of normal and pathological cell functions.
Intracellular pH (pHi) homeostasis is exquisitely controlled. Variations in pHi both reflect and determine changes in a number of cellular processes, including adhesion, proliferation, metabolism, and programmed cell death. How pHi responds to and regulates distinct cellular processes has primarily been determined by evaluating protein activities. Although effects of pHi on gene expression have been determined in yeast  and bacteria , we know little about how pHi regulates gene expression in metazoan cells.
In metazoan cells pHi homeostasis is maintained by a number of H+ translocating mechanisms, primarily localized at the plasma membrane. In mammalian fibroblasts, a predominant regulator of pHi is the Na-H exchanger, NHE1. NHE1 is an H+ extruder, catalyzing an electroneutral exchange of extracellular Na+ for intracellular H+ and regulating pHi and cell volume homeostasis. NHE1 activity is increased in response to growth factors and oncogenes [3, 4], and increases in NHE1 activity and pHi promote cell cycle progression , increased proliferation [6, 7], and cell survival . NHE1 activity is necessary for a number of cytoskeleton-associated processes including cell shape determination , remodeling of cell-substrate adhesion complexes [6, 9, 10], and directed cell migration [9, 11, 12]. NHE1-dependent increases in pHi also play an essential role in cell transformation and the development of malignant progression [13, 14] and NHE1-deficient cells have a markedly reduced capaCity for tumor growth in vivo .
In this study we used cDNA microarray analysis to determine changes in steady-State gene expression in fibroblasts stably a mutant NHE1 lacking ion translocation activity compared with fibroblasts stably expressing wild-type NHE1. Consistent with a role for NHE1 in cell growth regulation, the unbiased microarray analysis indicated that in the absence of NHE1 activity there are significant changes in the expression pattern of genes related to growth factor signaling, growth and oncogenesis, and DNA synthesis and cell cycle control.
Recent evidence indicates that in addition to the function of NHE1 in ion translocation and pHi homeostasis, the exchanger also acts as a scaffold to assemble signaling complexes and as a plasma membrane anchor for the actin-based cytoskeleton [3, 6]. To selectively impair only ion translocation by NHE1, we engineered an ion translocation-defective NHE1 containing an isoleucine substitution for glutamine 266 (NHE1-E266I). In cells expressing NHE1-E266I, the scaffolding and actin anchoring functions of NHE1 are retained, but ion translocation is absent [5, 6]. Wild-type NHE1 (LAPN cells) and NHE1-E266I (LAPE cells) were stably expressed in NHE1-null LAP1 cells, which are derived from NHE1-expressing Ltk-mouse muscle fibroblasts [16, 17]. As previously reported  NHE1 expression in LAPN and LAPE cells, as determined by immunoblotting, is similar and steady-State pHi in the continuous presence of serum and HCO3 - is ~7.35 for LAPN cells and ~7.10 for LAPE cells. The presence of HCO3 - allowed the function of anion exchangers contributing to pHi homeostasis in the absence of ion translocation by NHE1.
Differential Gene Expression in LAPE Cells Relative to LAPN Cells
Growth Factor/Hormone Signaling
phospholipase C gamma 1
MDK1 (neuronal tyrosine kinase receptor)
TC21 ras-like protein
tyrosine kinase SEK receptor precursor
GC Binding Protein
casein kinase II alpha
FYN tyrosine protein kinase
P2X purinoceptor 3 (ATP receptor)
IRG47 GTP binding-protein
CAP adenylyl cyclase-associated protein
protein-tyrosine phosphatase epsilon precursor
guanine nucleotide binding protein G(K) alpha
brain-derived neurotrophic factor
Grg1 groucho-related gene 1 protein
SOS 2 (ras GEF)
proteinase activated receptor 2, PAR2
receptor of activated protein kinase C (RACK1)
PAK p21-activated kinase
guanine nucleotide binding protein gamma-7
ERF1 EGF-response factor 1
Fgd1 (faciogenital dysplasis) (Cdc42 GEF)
5-HT5B serotonin receptor
chemokine receptor type 4
Emr1 receptor (EGF-7 TM family)
Growth and Oncogenesis
interferon-inducible protein 9–27
testis-specific c-abl protein
MUC18 melanoma-associated antigen
B94 TNF-α-induced early response gene
calpactin I light chain
cell division protein FTSH homolog
ALL-1 zinc finger protein HRX
LAF-4 lymphoid nuclear protein
calpactin I heavy chain
insulin-induced growth response protein CL-6
rearranged mutant c-myb gene
MCF2 Dbl proto-oncogene
HSP 90 alpha
Cell Cycle/DNA Replication
RAD54 DNA-repair gene
14-3-3 protein tau/theta
G1/S-specific cyclin D1
cell division-associated protein BIMB.
proliferating-cell nuclear antigen
CKS-2 cyclin-dep kinases regulatory subunit 2
FLAP endonuclease-1; FEN-1
clip 170 (restin)
septin 2 (NEDD5 PROTEIN)
KIF4 kinesin-like protein
kinesin light chain 1
axonemal dynein heavy chain
septin 4 (BRAIN PROTEIN H5)
myosin regulatory light chain 2, smooth muscle isoform
NF2 neurofibromatosis type 2 isoform I
Cell Adhesion and Extracellular matrix
mast cell protease 5 precursor
neural cell adhesion molecule (NCAM-140)
anti-von Willebrand factor antibody NMC-4 kappa chain
E-selectin ligand-1 (ESL-1)
microfibril associated glycoprotein precursor (MAGP)
integrin beta-5 subunit precursor
type IV collagenase
CMP-sialic acid transporter
AKR voltage-gated potassium-channel (KCNA4)
potassium channel protein NGK2
glucose transporter type 4 insulin-responsive (GT2)
synaptic vesicle amine transporter
retinoic acid-binding protein
HNF-3/Forkhead homolog II
transcription regulatory protein MCP-1 (POU 1)
NfiA2-protein (nuclear factor 1)
transcription factor C1
zinc finger protein 91
winged-helix gene, htlf
retinoid X receptor-beta
HLX homeo box protein
myoblast cell surface antigen
activator 1 37 KD subunit
RNA polymerase II large subunit
DNA-directed RNA polymerase III largest subunit
U1RNA-associated 70-kDa protein
U6 snRNA-associated protein
eukaryotic peptide chain releasing factor GTP-binding subunit
putative ATP-dependent RNA helicase PL10
40S ribosomal protein S10
ubiquitin carboxyl-terminal hydrolase (protease 4)
ribosomal protein L32
elongation factor TS (forms complex with EF-tu)
ubiquitin carboxyl-terminal hydrolase (protease 8)
elongation factor 2 (EF-2)
phenylalanine - tRNA synthetase
elongation factor TU
thioredoxin-dependent peroxide reductase 2
superoxide dismutase 3 (SOD3)
thioredoxin-dependent peroxide reductase 1
glutathione S-transferase, GSTT1
pyruvate kinase M2
acetyl-Coenzyme A acetyltransferase 2
citrate transport protein
phosphorylase B kinase gamma catalytic subunit
Electron Transport and Oxidative Phosphorylation
ATP synthase (subunit D)
NADH-ubiquinone oxidoreductase (complex I)
cytochrome C oxidase VIa
ATP synthase P1precursor (subunit C)
ATP synthase (subunit A)
mitochondrial inner membrane protease subunit 1
BRAIN PROTEIN I47(similar to yeast SEC 17)
PROTEIN TRANSPORT PROTEIN SEC22
anti-DNA immunoglobulin heavy chain IgG
immunoglobulin rearranged kappa chain
complement receptor type 2 precursor (CR2)
thymocyte B cell antigen precursor
immunoglobulin alpha heavy chain
interferon gamma receptor second chain
pre-B cell enhancing factor precursor
interferon beta type 2
anti-DNA immunoglobulin light chain IgG
FK506-binding protein precursor (FKBP-13)
immunoglobulin light chain Fv-fragment
Ig 1B4.B5 heavy chain mRNA for mouse cytochrome c
immunoglobulin-like receptor PIRA1
CD10 neutral endopeptidase (pre-B cell differentiation)
immunoglobulin variable region, heavy chain
immune-responsive gene 1 (Irg1)
anti-DNA immunoglobulin heavy chain IgG
immunoglobulin light chain variable region
immunoglobulin heavy chain variable region
parotid secretory protein
amyloid-like protein 1 precursor
liver receptor homologous protein
beta-hydroxysteroid dehydrogenase type 2
C57BL/6J ob/ob haptoglobin
Swiss Webster demilune cell-specific salivary gland protein
Expression of a number of transcription factor genes acting downstream of growth factor signaling was also differentially regulated in LAPE cells compared with LAPN cells (Fig. 2A). Fra1 and Fra2 are Fos proteins and components of the AP1 transcription factor. They form dimmers with Jun transcription factors to regulate a number of cell processes including differentiation, proliferation, and oncogenic transformation . The regulated expression of two members of the C/EBP (CAAT/enhancer binding protein) family of transcription factors was confirmed. RT-PCR was used to confirm decreased expression of C/EBPδ (Fig. 2C), which dimerizes with C/EBPβ in response to Ras-ERK signaling to regulate adipocyte [29, 30] and epidermal  differentiation. Increased expression of GADD153 (CHOP), which is a transcription factor in the C/EBP family, was confirmed by TaqMan analysis (Fig. 2D). GADD153 dimerizes with other C/EBP isomers to inhibit their binding to C/EBP binding sites in the promoters of a number of genes involved in differentiation and mitogenesis [32, 33]. Expression of GADD153 increases in response to DNA damage , and oxidative stress [35, 36], hence its role in cell cycle progression is also included in Figure 3.
Consistent with LAPE cells having delayed G2/M entry and progression , the array analysis indicated upregulation of genes negatively regulating G2/M, including 14-3-3 θ and Wee1 kinase. Increased expression of Wee1 kinase, which induces inhibitory phosphorylation of Cdc2 on tyrosine 15 , was confirmed by TaqMan analysis (Fig. 3D). Moreover, there was a downregulation of genes associated with promoting cell cycle progression, including CDC25 (Fig 3A) and CKS-2 (Fig. 3A), and involved in chromatin assembly, including histone H3 (Fig. 2A). Although cDNA array analysis of asynchronous LAPE cells did not indicate a change in cyclin B1 expression, we found that with a time-dependent release of cells from a double thymidine block, cyclin B1, as indicated by immunoblotting, was significantly downregulated in LAPE cells compared with LAPN cells (Fig. 3C). Hence, loss of NHE1 activity likely decreases the stability of cyclin B1 protein rather than decreasing cyclin B1 gene expression. Decreased cyclin B1 expression and increased Wee1 kinase expression is consistent with our previous finding that Cdc2 kinase activity is inhibited in LAPE cells compared with LAPN cells .
Consistent with increased glycolysis and pyruvate production in LAPE cells, key enzymes favoring ATP production by electron transport and oxidative phosphorylation were increased (Fig. 4A). Increases in NADH-ubiquinone oxidoreductase and cytochrome c oxidase would favor oxidation of NADH. Increases in subunits A, C, and D of ATP synthase would increase endergonic synthesis of ATP. ATP synthase in the inner mitochondrial membrane is a proton translocator, extruding protons into the mitochondrial matrix. Whether decreased cytosolic pH in LAPE cells compared with LAPN cells alters the pH of the mitochondrial matrix and proton-electromotive force powering ATP synthesis remains to be determined. Collectively, the profile of gene expression in LAPE cells suggests equilibrium towards increased glycolytic flux and ATP production.
cDNA array analysis indicated that loss of NHE1 activity in LAPE cells was associated with the regulation of a number of genes involved in cytoskeleton organization, cell adhesion, and extracellular matrix assembly. The regulation of several genes correlates with reported effects of NHE1 activity and pHi on cell shape determination , cell polarity , actin-filament bundling [9, 48], cell-substrate adhesion [9, 10] and cell migration and metastasis [9, 11, 14]. NHE1 acts as an anchor for actin filaments by binding directly members of the ERM (ezrin, radixin, moesin) family of actin binding proteins, and in LAPE cells, expression of NF2 (merlin), a tumor suppressor protein and member of the ERM family, was downregulated. Consistent with a role for NHE1 activity in cell polarity and actin dynamics, loss of NHE1 activity was associated with decreased expression of myosin regulatory light chain, and increased expression of gelsolin (Fig. 5A), a pH-dependent actin severing and capping protein. Although regulation of the microtubule-based cytoskeleton by NHE1 and pHi have previously not been reported, a number of microtubule-related genes, including Clip 170, KIF4, kinesin light chain, and a dynein heavy chain, were upregulated in LAPE cells.
Consistent with NHE1-dependent cell adhesion and migration, LAPE cells had a marked (~26-fold) decrease in type IV collagenase (MMP-9) expression, and zymography confirmed that activity of MMP-9, but not activity of MMP-4, was selectively decreased in LAPE cells compared with LAPN cells (Fig. 5B). Correlating with a decrease in MMP-9 expression, LAPE cells also had similar marked (~24-fold) decrease in the expression of lipocalin 24p3, which was confirmed by TaqMan analysis (Fig. 5C). NGAL (Neutrophil Gelatinase Associated Lipocalin), the human homolog of mouse lipocalin, is covalently bound to MMP-9 and protects MMP-9 from degradation [49, 50]. Lipocalins are transcriptionally regulated by C/EBPβ , which is likely suppressed by decreased expression of C/EBPδ (Fig. 2C) and increased expression of GADD153 (Fig. 2D). Moreover, increased expression of MMP-9  and lipocalins  is associated with tumor cell growth and invasion, which correlates with a role for NHE1 activity in these processes [14, 15].
In summary, global profiling revealed genes regulated by loss of NHE1 activity and decreased pHi. A number of the differentially regulated genes involved in growth factor signaling, cell cycle progression, and cytoskeleton and extracellular matrix remodeling are consistent with previously established roles of NHE1 activity and pHi in mitogenic responses, cell proliferation, and tumor metastasis and invasion. In contrast, some genes, including those regulating carbohydrate metabolism and microtubule dynamics, have previously not been linked to NHE1 activity. An important future direction is to determine primary and secondary effects of gene regulation by NHE1 and of particular interest is whether promoters within the genes differentially regulated in LAPE cells are pH-responsive.
The generation of LAPN and LAPE cells was as previously described . In brief, NHE1-null LAP1 cells developed from parental Ltk-mouse muscle fibroblasts  were used for stable expression of wild-type NHE1 (LAPN cells) or expression of NHE1-E266I containing a single point substitution of glutamate266 for isoleucine that results in complete loss of ion translocation activity (LAPE) [5, 6]. Cells were maintained in DMEM supplemented with 10% FCS in the presence of 25 mM NaHCO3 and 5% CO2. Total RNA was prepared from cells plated for 48 h by using Qiagen's RNeasy® midi kit.RNA. RNA was collected from five independent cell platings and used for five separate DNA array hybridizations.
Total RNA was converted to double-stranded cDNA using the SuperScript Choice system (Gibco BRL), except that HPLC-purified T7-(dT)24 oliomer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG - (dT)24) was used instead of the oligo (dT) or random primers provided with the SuperScript Choice kit. Double-stranded cDNA was collected by ethanol precipitation. Biotinylated cRNA was then generated from the cDNA by an in vitro transcription (IVT) reaction using the ENZO BioArray™ HighYield™ RNA Transcript Labeling Kit. IVT products (cRNA) were collected by using Qiagen's RNeasyR mini kit, then ethanol-precipitated and quantitated. The cRNA was fragmented by alkaline treatment and hybridized to a GeneChip probe array from Affymetrix (Santa Clara, CA). The Affymetrix murine oligonucleotide array (Mu11KSubB) is complementary to ~6,500 murine genes and expressed sequence tags (ESTs). Each gene or EST is represented on the array by 16 - 20 feature pairs. Each feature pair contains a 25-bp oligonucleotide sequence, which is either a perfect match to the gene or a single central-base hommomeric mismatch control.
Affymetrix GeneChip analysis was performed using standard procedures . The expression level of any particular transcript was calculated by subtracting he difference between the fluorescence intensities of the perfect match and mismatch feature pairs and then averaging over the entire probe set (Avg Diff). The Avg Diff value for each transcript was averaged over the five experiments for LAPN and LAPE cells; these average values obtained from five independent hybridizations were then used to calculate fold changes in LAPE cells relative to LAPN cells for each transcript. We did not use comparison algorithms supplied with the Affymetrix software.
Proteins from total cell lysates were separated by SDS-PAGE as previously described  and transferred to PVD nitrocellulose membranes for immunoblotting. Antibodies for immunoblotting included Fyn (NeoMarkers), PLCγ-1 and Erk 1–2 (Cell Signaling), GADD45, FEN1, and cyclin B1 (Santa Cruz Biotechnology), and actin (Sigma).
Confirmation of GeneChip data was accomplished using TaqMan™ chemistry with the ABI 7700 Prism real-time PCR instrument (ABI, Foster City CA). Custom primers specific to the genes of interest were synthesized by Life Technologies and TaqMan™ probes for each gene were synthesized by Integrated DNA Technologies, Inc. The forward and reverse primers for mouse GADD153 (GenBank accession no. X67083) were 5'-GAAACGAAGAGGAAGAATCAAAAAC-3' and 5'-ATCTGGAGAGCGAGGGCTTT-3', respectively, and the probe was 5'-FAM/ACCCTGCGTCCCTAGCTTGGCTGAC/TAM-3', corresponding to an amplicon of 122 bp. The forward and reverse primers for mouse Wee1 kinase (GenBank accession no. NM_009516) were 5'-TTGCTCTTGCTCTCACAGTCGT-3' and 5'-TGGGAAAGCACTTGTGGGAT-3', respectively, and the probe was 5'-FAM/CCTTCCCAGAAATGGAGAGCACTGGC/TAM-3', corresponding to an amplicon of 118 bp. The forward and reverse primers for mouse Glut4 (GenBank accession no. NM_009204) were 5'-TGGCCATCTTCTCTGTGGGT-3' and 5'-ATTGGCTAGGCCCATGAGG-3', respectively, and the probe was 5'-FAM/TATGCTGGCCAACAATGTCTTGGCC/TAM-3', corresponding to an amplicon of 138 bp. The forward and reverse primers for mouse 24p3 (GenBank accession no. W13166) were 5'-GGCAGCTTTACGATGTACAGCA-3' and 5'-TCTGATCCAGTAGCGACAGCC-3', respectively, and the probe was 5'-FAM/CATCCTGGTCAGGGACCAGGACCAG/TAM-3', corresponding to an amplicon of 111 bp. For each gene, PCR was conducted in triplicate with 50 μl reaction volumes of 1x PCR buffer A (Applied Biosystems, Foster City, CA), 2.5 mM MgCl2, 0.4 μM each primer, 200 μM each dNTP, 100 nM probe and 0.025 u/μl Taq Gold (ABI, Foster City CA). For each experiment, a large master mix of the above components was made and aliquoted into each optical reaction tube. Each primer/probe set (5 - 10 μl) was then added, and PCR conducted using the following cycle parameters: 95°C 12 min × 1 cycle, (95°C 20 sec, 60°C 1 min) × 40 cycles. Data analysis was carried out using sequence detection software that calculates the threshold cycle (Ct) for each reaction which is used to quantitate the amount of starting template in the reaction. A difference in Ct values (ΔCt) was calculated for each gene by taking triplicate Ct values from three reactions and subtracting the mean Ct of the triplicates for the control gene, GAPDH, for each cDNA sample at the same concentration. An additional difference in Ct values (ΔCt) was calculated for each gene by taking the triplicate ΔCt values for each gene in the mutant LAPN1-E266I cells and subtracting the mean ΔCt of the triplicates for the wild-type LAPN cells. The relative expression levels were calculated as = 2 -ΔΔCt .
Relative quantitative RT-PCR was preformed using QuantumRNA™ 18S internal standards from Ambion, Inc. (Austin TX) that included 18S Primers and Competimers™. By optimizing the assay and choosing an appropriate18S Primer:Competimer ratio for each sample, the 18S signal was reduced to the same linear range as that identified empirically for the gene specific product. The amplicon for the 18S primers was 315 bp. Custom primers specific to the genes of interest were synthesized by Life Technologies. The forward and reverse primers for calpactin I light chain (GenBank accession no. M16465) were 5'-GTGGACAAAATAATGAAGGAC-3' and 3'-ACAAGAAGCAGTGGGGCAGAT-5', respectively, corresponding to an amplicon of 222 bp. The forward and reverse primers for CEBPδ (GenBank accession no. NM_007679.1) were 5'-ATACCTCAGACCCCGACAGCG-3' and 3'-CAAAAGTCTGTCGGAAATGTC-5', respectively, corresponding to an amplicon of 220 bp. Total RNA was isolated from LAPN1 and LAPN1-E266I cells in four separate experiments, using Qiagen's RNeasy® midi kit. Reverse transcription of total RNA from each sample was carried out using random decamers and the RETROscript™ kit from Ambion. RT reactions were then subjected to PCR using the gene specific primers above (final concentration of 0.4 uM each), the appropriate 18S
Primer:Competimer ratio and 10uCi/ul [α-32P]dCTP for labeling. PCR was conducted using the following cycle parameters: (94°C 30 sec, 57°C 30 sec, 72°C 30 sec) × 21 cycles for CEBPδ and 19 cycles for calpactin I light chain. Empirically derived 18S Primer:Competimer ratios were 1:18 for CEBPδ and 2:8 for calpactin I light chain.
Activity of type IV collagenase (MMP9) was determined by zymography, as previously described .
Ezrin, radixin, moesin
Na-H exchanger type 1
Neutrophil Gelatinase Associated Lipocalin
type IV collagenase
We thank members of the Zena Werb laboratory at UCSF for assistance with zymography for MMP-9, and Lauren Ellis for help in preparing the manuscript. This work was supported by National Institutes of Health grant GM47413.
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