Oxidative Repression of NHE1 Gene
Expression Involves Iron-mediated Caspase Activity
Alan P. Kumar1*, Michelle Ker Xing Chang2*, Larry Fliegel3, Shazib Pervaiz4,5,
Marie-Véronique Clément2,5
* Both authors contributed equally to this work
1
National University Medical Institutes, 2Department of Biochemistry, 4Department of
Physiology, Yong Loo Lin School of Medicine, 5NUS Graduate School for Integrative
Sciences and Engineering, National University of Singapore and 3Department of
Biochemistry, University of Alberta, Canada.
Address correspondence to:
Marie-Véronique Clément, The Yong Loo Lin School of Medicine Department of
Biochemistry National University of Singapore 8 Medical Drive, MD7 Singapore 117
597, Tel: (65) 6516-7985; Fax:(65) 6779-1453; E-Mail:bchmvc@nus.edu.sg
Running title: Oxidative repression of NHE1
Supplementary Data:
Materials and Methods
Reagents and antibodies
Beta mercaptoethanol (ME), H2O2, diamide, sodium formate, crystal violet, DMSO,
DMTU, desferioxamine (DFO), ferric chloride (FeCl3), propidium iodide, actinomycin D,
staurosporine, and protease inhibitors were obtained from Sigma-Aldrich. Cell culture
medium and fetal bovine serum (FBS) were purchased from Hyclone. Caspase-specific
tetra-peptide inhibitors and pan caspase inhibitor were purchased from R&D Systems.
Primary antibodies: NHE1 (Chemicon); procaspase-3, and -6 antibodies (Cell Signaling);
-actin (Sigma); secondary antibodies: peroxidase-conjugated goat anti-rabbit antibody
was obtained from DakoCytomation. Peroxidase-conjugated goat anti-mouse antibody
was from Pierce.
Cell culture
Rat muscle cell line, L6 stably transfected with full-length 1.1kb of the mouse NHE1
promoter, inserted 5’ to the luciferase reporter gene (designated pXP1.1MP cells) were
maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
FBS, 2mM L-glutamine, 0.25mg/ml geneticin (GIBCO) and 1mM gentamicin sulfate
(BioWhittaker). NIH3T3 and MCF-7 cells were maintained in RPMI with 10% FBS, rat
myocardium H9c2 cells in DMEM with 10% FBS, and normal human fibroblasts
(IMR90) in MEM supplemented with vitamins, essential and non-essential amino acids.
Caspase activity assay
Cells were lysed using 1x Cell Lysis Buffer (BD Pharmingen). Cell lysate was added to
equal volume of 2X Reaction Buffer (10mM HEPES, pH7.4, 2mM EDTA, 6mM DTT,
10mM KCl and 1.5mM MgCl2) supplemented with protease inhibitors (1mM
phenylmethylsulfonyl fluoride (PMSF), 10µg/ml aprotinin, 10µg/ml pepstatin A,
20µg/ml leupeptin), and 50µM caspase substrate (AG Scientific). Samples were
incubated at 37ºC for 1.5h in the spectrofluorometer and AFC fluorescence measured
(excitation: 400nm, emission: 505nm). Caspase activity was normalized to protein
amounts and expressed as RFU/µg total protein. Protein concentration was determined
using the Coomasie Plus Protein Assay Reagent Kit (Pierce).
Crystal violet assay
Culture medium was removed, washed with 1ml of 1xPBS per well. Five hundred
microlitres of crystal violet solution was added per well and incubated for 10min at 25 oC
in the dark. Crystal violet solution was then removed, washed with tap water several
times. Plates were left to dry overnight. One milliliter of 1xPBS/1%SDS solution was
added per well and left overnight to ensure complete lysis. Absorbance was then
measured at 595nm using a plate reader.
DNA fragmentation assay
Cell death was determined by staining cells with propidium iodide (PI) to assess DNA
fragmentation. Cells were fixed with 75% ethanol, resuspended in 0.5ml PI (10µg/ml) in
the presence of RNAse A (0.25mg/ml). After 30min incubation at 37ºC in the dark,
stained cells were analyzed by flow cytometry. Cells were excited at 488nm and emission
at 610nm.
Western blot analysis
Whole cell lysates were prepared with RIPA lysis buffer containing 10mM Tris-HCL
pH7.4, 30mM NaCl, 1mM EDTA, 1% Nonidet P-40, supplemented with 1mM Na3VO4,
1µg/ml leupeptin, 1µg/ml pepstatin A, 1µg/ml aprotinin and 1mM PMSF before use.
Protein concentration was determined for each sample and equal amounts of protein were
warmed at 37°C in the water bath for NHE1 protein, boiled for 5min for other proteins,
with 1xSDS sample buffer and resolved by 8% or 15% SDS-PAGE. For the detection of
caspases, whole cell lysates were prepared with CHAPS lysis buffer [50 mM Pipes/ HCl
(pH 6.5), 2 mM EDTA, 0.1% Chaps, 20 μg/ml Leupeptin, 10 μg/ml Pepstatin A, 10
μg/ml Aprotinin] supplemented with 1mM PMSF and 5mM DTT. The cells were lysed
by three freeze-thraw cycles. Lysates were spun at 12,000 r.p.m, 4°C for 5 minutes and
the supernatant (cell extract) fraction was transferred to a new tube. Protein concentration
was determined for each sample and 50 μg of protein from each sample was boiled at
95°C for 5min, mixed with 1xSDS sample buffer and resolved by 15% SDS-PAGE.
Thereafter, proteins were transferred onto nitrocellulose membrane, blocked for 1 hour at
RT with 5% non-fat milk, and incubated overnight at 4ºC with the primary antibody.
After probing with secondary antibody for 1h at 25oC, protein bands were detected by
using the Supersignal West Pico Chemiluminescence (Pierce). -actin antibody was used
as a loading control.
Luciferase reporter assay
NHE1 promoter activity was assessed with a single-luciferase assay kit (Promega).
Briefly, feeding medium was removed from the wells, washed once with 1x PBS, and
lysed with ice-cold 100µl of reporter lysis buffer. Ten microlitres of cell lysate was then
added to 50µl of luciferase substrate solution. Bioluminescence generated was measured
using a Sirius luminometer (Berthold). The luminescence readings obtained were
normalized to the protein content of the corresponding cell lysate.
CAT ELISA
The levels of CAT protein were quantified using a CAT antigen capture enzyme-linked
immunosorbent assay (ELISA) (Roche Molecular Biochemicals). All CAT quantitations
were normalized to the protein concentration of the cell extract, as determined using the
Coomasie Plus Protein Assay Reagent Kit (Pierce).
Reporter plasmid constructs
Luciferase reporter plasmid constructs: pXP-1.1MP, pXP-0.9MP, pXP-0.5MP, pXP0.2MP, pXP-0.18MP, pMP+AP2, pMP-AP2, pMP(MUT)AP2, and empty vector pXP1
were kindly provided by Dr. Larry Fliegel, Department of Biochemistry, University of
Alberta, Canada
12
.pUCSS-CAT reporter plasmid constructs: -1374/+16, -850/+16, -
654/+16, -252/+16, -92/+16, and empty vector pUCSS-CAT were kindly provided by Dr.
Alexey Kolyada, Dept of Medicine, Tufts University School of Medicine, Boston, USA
25
.
DNA transfection
Cells were transfected using CalPhost Mammalian transfection kit (Clonetech) for 15h.
Cells were then washed twice with PBS and culture media added for another 24h.
Cotransfection with the Renilla plasmid (Clonetech) was used to assess transfection
efficiency in dual-luciferase reporter assay (Promega).
RNA interference for silencing caspases 3 and 6
Small interfering RNA (siRNA) inhibition of endogenous caspase-3 and -6 was achieved
using custom designed siRNA that target the respective DNA sequences (Ambion). A
control siRNA (non-homologous to any known gene sequence) (Qiagen) was used as a
negative control. Cells were transfected with siRNA using the CalPhos Mammalian
Transfection kit.
RNA isolation and NHE1 mRNA determination by Real-Time PCR
Total RNA was isolated from cells by TRIZOL reagent (Invitrogen) as described by
manufacturer’s instructions with a DNAse treatment step incorporated into the protocol.
Each RT reaction contains 2.5g of total RNA, 1X RT buffer, 5mM MgCl2, 425M each
of dNTPs, 2M random hexamers, 0.35U/l RNase inhibitor, 1.1U/l MultiScribe™
reverse transcriptase and made up to 10l with sterile water. RT reaction was carried out
at 37oC for 1h. Five microlitres of the 10µl cDNA reaction volume was used in realtime
quantitative PCR using ABI PRISM 7500 (Applied Biosystems). Normalization was to
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for human RNA and 18S RNA for
mouse and rat RNA. Fluorescence was measured with the Sequence Detection Systems
2.0 software. PCR was performed in multiplex (both target and endogenous control coamplified in the same reaction) with distinct fluorescent dyes. The sequences for primers
(300nM) and probe (200nM) for mouse NHE1 used in this study are as follows: mouse
NHE1, forward (5-TGC CTC ATG AAG ATA GGT TTC CA-3), reverse (5- AGC AGC
CCC ACT ACG ATC AG-3), and probe (5-FAM-CAC CAT CTC AAG CAT CGT CCC
GGA-TAMRA-3).
Primers
and
probe
for
human
glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), rat NHE1, human NHE1, and 18S RNA were purchased as
kits from Applied Biosystems (Assays on Demand).
Cell Morphology
The morphology of the cells was analyzed using the Olympus digital camera (C4040ZOOM, 4.1 mega pixels) attached to the light microscope (Olympus CK2) at 200X
magnification.