Technical Reference #1610

1610.

Reduction in TRPC4 Expression Specifically Attenuates G-Protein Coupled Receptor-Stimulated Increases in Intracellular Calcium in Human Myometrial Cells Aida Ulloa; Albert Gonzales; Miao Zhong; Yoon-Sun Kim; Jeremy Cantlon; Colin Clay; Cung-Ying Ku; Scott Earley; Barbara Sanborn, Colorado State University, Cell Calcium, 46(1610), (2009)

Abstract:
Canonical transient receptor potential (TRPC) proteins may play a role in regulating changes in intracellularcalcium ([Ca2+]i). Human myometrium expresses TRPC4 TRPC1 and TRPC6 mRNAs in greatestrelative abundance. Contributions of TRPC4 to increases

Keywords:
TRPC channels; Calcium signaling; myometrium; G-protein couples receptor

Materials & Methods:
2.1. Reagents and solutions Primerswere purchased fromIntegrated DNA Technologies Inc. (Coralville IA). SYBR Green I nucleic acid gel stain was purchased from Lonza (Rockland ME). Restriction enzymes were obtained from New England Biolabs Inc. (Beverly MA) or Promega (Madison WI). Fura-2/acetoxymethylester (Fura 2-AM) and Pluronic F127 were obtained from Molecular Probes (Invitrogen Carlsbad CA). Oxytocin thapsigargin ATP Prostaglandin F (PGF)2 and protease inhibitor cocktail were purchased from Sigma–Aldrich (St. Louis MO). 1-Oleoyl-2-acetyl-sn-glycerol (OAG) was obtained from Calbiochem (San Diego CA). Cell culture media and other reagents were obtained from Gibco BRL Invitrogen (Carlsbad CA). TRPC1 rabbit monoclonal antibody and its corresponding blocking peptide were obtained from Epitomics (Burlingame CA). TRPC4 polyclonal antibody and its corresponding blocking peptide were purchased from Alomone Labs (Jerusalem Israel). Anti-HA peroxidase mouse monoclonal antibody was purchased from Roche (Indianapolis IN). Primary polyclonal human PLCB3 antibody and horseradish peroxidase (HRP) conjugated donkey anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz CA). 2.2. Cell culture UtSMC cells derived from nonpregnant human myometrium (Catalog#: CC-2562 Lot# 17590)were purchased fromLonza(Walkersville MD). AD293 cells were obtained from Stratagene (La Jolla CA). UtSMC and AD293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal calf serum 2mM l-glutamine 50 units/ml penicillin and 50 g/ml streptomycin. Immortalized PHM1–41 cells were derived from late-term pregnant human myometrial tissue and retain many morphological and biological characteristics of myometrial smooth muscle cells [101217–19]. PHM1–41 cellswere cultured in the media described above including 0.1mg/ml of G418 sulfate (Gibco BRL Invitrogen Carlsbad CA). PHM1–41 cells were used at passages 20–25 and UtSMC were used at passages 6–10. Myometrial cells were trypsinized and plated in 35mm glass-bottomed dishes (MatTek Ashland MA) for Ca2+-imaging experiments or into 100-mm dishes for RT-qPCR and immunoblot studies. 2.3. Cloning adenovirus construction and adenoviral infection Full-length human TRPC4 alpha cDNA in pcDNA3 was obtained from Dr. J.W. Putney Jr. (NIEHS Raleigh NC). The TRPC4 luciferase reporter (psiTC4) vector was constructed by introducing a NotI restriction site by polymerase chain reaction (PCR) at each end of the TRPC4 cDNA sequence using the Expand High Fidelity PLUS PCR System (Roche Applied Science Indianapolis IN); the resulting product was cloned into the NotI site of the psiCHECK-2 vector (Promega Madison WI). TRPC4 shRNAs were designed using the Dharmacon siDesign Center (Lafayette CO). Sequences were examined for features indicating siRNA functionality [20]. Candidate sequences homologous to other TRPCs other ion channels and common interaction domains were rejected. Selected siRNA duplex sequences were constructed to contain a human pre-microRNA stem sequence (miR-30) which allows efficient processing of the shRNA [21]. A scrambled control sequence was designed by entering the TRPC4- shRNA#4 sequence into the siRNA Sequence Scrambler software (GenScript Piscataway NJ). Oligonucleotides containing TRPC4- shRNA sequences were synthesized (Integrated DNA Technologies Coralville IA) and cloned into the BseRI and BamHI sites downstream of a U6 promoter in the pSHAG vector (provided by G. Hannon Cold Spring Harbor Laboratory NY) producing the pSHTC4sh1– 4 vectors. A modified pAdTrack-RfA(f) plasmid was constructed by introduction of the Reading Frame Cassette A (RfA) from the Gateway Vector Conversion System (Invitrogen Carlsbad CA) into the XbaI/XhoI restriction sites inpAdTrack (ATCC Manassas VA). TRPC4 shRNA adenoviral vector assembly was performed by recombination between the attR sites of pSH-TC4sh1 and the attL sites of pAdTrack-RfA(f) using LR clonase. The pAdT-TC4shRNA cloneswere checked by restriction enzyme digestion and sequence integrity was confirmed by direct sequencing. The pAdT-TC4shRNA clones were linearized with PmeI and subsequently electroporated into Escherichia coli BJ5183 cells according to the manufacturer’s instructions to achieve recombination of the pAdT-TC4shRNAs with the adenoviral backbone plasmid pAdEasy-1 (Stratagene La Jolla CA). The obtained recombined plasmids were linearized with PacI and transfected into AD-293 cells to allowviral synthesis and packaging. The adenoviruses were amplified purified using the BD Adeno-X Maxi Purification kit (Clontech Mountain View CA) and titered by viral particle titration and end-point dilution. Adenoviruses were then used to infect PHM1–41 cells using the enhanced green fluorescent protein (eGFP) marker to identify infected cells. Multiplicity of infection (MOI) of 1000 and 500 were used in PHM1–41 and UtSMC cells respectively resulting in 90–95% infection efficiency as determined by eGFP expression. Cells were used within 72–96 h post-infection and exhibited morphology similar to that of noninfected cells as determined by visual inspection. 2.4. Transfection and electroporation AD293 cells were plated at 1×105 cells/well in 12-well plates. Transient transfectionswere performed 24 h after plating using the GenePORTER2transfection reagent according to the manufacturer’s instructions (Genlantis San Diego CA). Cells were cotransfected with the psiTC4 reporter and the pSH-TC4sh1–4 constructs at a 1:3 molar ratio. Samples were analyzed 48–72 h post-transfection using the Dual-luciferase Reporter Assay (Promega Madison WI). COSM6 cells were plated at 1×105 cells/well in 6-well plates. Transient transfectionswere performed 24 h after plating using the FuGENE 6 transfection reagent (Roche Indianapolis IN) according to manufacturer’s instructions. Cells were cotransfected with the TRPC4 overexpression plasmid and the pSH-TC4sh1 construct at a 1:3 molar ratio. Samples were analyzed 72 h post-transfection. PHM1–41 and UtSMC cells were trypsinized and washed with PBS (137mM NaCl 27mM KCl 100mM Na2HPO4 2mMK2HPO4 pH 7.4). Electroporation was performed using the Basic Smooth Muscle Cell kit (Amaxa Inc. Gaithersburg MD). 2×106 cells were pelleted and resuspended in 100 l Basic Nucleofector solution together with 2 g psiTC4 reporter 1.5 g pSH-TC4sh1–4 and 0.5 g maxGFP vectors and transferred to the provided 2-mm cuvette. Cells were electroporated for 140 V 35ms in the time constant mode using the Gene Pulser Xcell system (Bio-Rad Hercules CA) andwereimmediately dilutedinto 500 l ofpre-warmed culture media. Cells were dispensed (80 l into 3mm×35mm glass-bottomed dishes for Ca2+ imaging experiments and 350 l into a 100-mmdish for RNA analyses) and incubated for 24 h before media was replaced. Samples were analyzed 72–96 h after electroporation. A transfection efficiency of 60% was achieved. 2.5. mRNA isolation and quantitative real-time RT-PCR Myometrial cell mRNA was isolated 72–96 h post-infection using the RNeasy kit (Qiagen Valencia CA). An on-column DNase digestionwas performed using the RNase-free DNase set according to the manufacturer’s instructions (Qiagen Valencia CA). Quantitative real-time PCR (RT-qPCR) was performed using 100 ng mRNA and the iScript one-step RT-PCR kit with SYBR Green (Bio-Rad Hercules CA) in an iCycler Thermal Cycler (Bio-Rad Hercules CA). Previously designed PCR primers for TRPC1 TRPC3 TRPC5 TRPC6 and TRPC7 [9] and newly designed primers for TRPC4 betaglucuronidase (GUS) plasma membrane Ca2+-ATPase isoform 1 (PMCA1) PMCA4 and sarcoplasmic endoplasmic reticulum Ca2+ ATPase isoform 2 (SERCA2) (Table 1) were used at 500 nM. The PCR conditions were as follows: cDNA synthesis at 50 ◦C for 30 min iScript Reverse-transcriptase inactivation at 95 ◦C for 5min PCR cycling where cDNA was denatured at 95 ◦C for 15 s annealed at 60 ◦C for 30 s and extended at 72 ◦C for 1min. Sequence integrity of RT-qPCR products was verified by direct sequencing. Melting curves for all products showed single peaks. Calculationswere performed using the Ct method [22] where a given RNA was first normalized to GUS in each sample and then expressed relative to the corresponding value in cells infected with empty adenovirus. RT-qPCR products for TRPCs and GUS using 100 ng of RNA for TRPC1 TRPC4 TRPC6 and GUS and 1 g of RNA for TRPC3 TRPC5 and TRPC7 were run in 3% agarose gels in 1× Tris–acetate–EDTA (TAE) electrophoresis buffer (40mM Tris–acetate 1mM EDTA pH 8.0). Gels were then incubated in 1× TAE buffer with SYBR Green I nucleic acid gel stain (1:1000 dilution) for 30min at room temperature with gentle rocking. Bands were visualized by using a Storm imager (Amersham Biosciences). 2.6. Immunoblotting PHM1–41 cells (6×105) were plated in 100-mm dishes in culture medium and infected with adenovirus at an MOI of 1000. Cells were harvested after 72–96 h and whole cell extracts and plasma membrane extracts were prepared. For whole cell extract preparation cells were lysed in modified RIPA buffer (50mM Tris–HCl pH 7.4 150mM NaCl 1% NP-40 (v/v) 1mM EDTA 0.25% Na-deoxycholate (v/v) 0.1% SDS (w/v)) plus protease inhibitor cocktail containing 1.04mM4-(2-aminoethyl)benzenesulfonyl flu- oride hydrochloride (AEBSF) 0.8 M aprotinin 21 M leupeptin 36 M bestatin 15 M pepstatin A 14 M trans-Epoxysuccinyll- leucylamido(4-guanidino)butane (E-64). Lysates were incubated on ice for 10min and sonicated on ice for 3 cycles at 10 s/cycle output setting 3 using aMicro Tip in a Branson Sonifier 250 (Danbury CT). Mixtureswere then centrifuged at 14000×g for 15min at 4 ◦C. The protein concentrations in the supernatants were measured by BCA Protein Assay (Pierce Rockford IL). For plasma membrane preparation a previously validated method [23] was adapted for use with small amounts of material. Cells were lysed in 200 l homogenization buffer (100mM KCl 5mM MgCl2 50mM Tris–HCl 1mM EGTA pH 7.2) plus protease inhibitor cocktail as above and sonicated on ice. Mixtures were centrifuged at 10000×g for 15min at 4 ◦C and the resulting supernatants were centrifuged at 100000×g for 1h at 4◦C in a TLA100.3 rotor. The resulting pellets were dissolved in 10% icecold sucrose in homogenization buffer and layered over a layer of 28% sucrose in homogenization buffer. Samples were centrifuged at 57000×g for 30min at 4 ◦C in a TLS55 swinging bucket rotor. The plasma membrane fractions at the 10–28% sucrose interface were withdrawn and centrifuged at 100000×g for 30min at 4 ◦C. The resulting pelletswere resuspended inmodified RIPA buffer and stored at−80 ◦C. The protein concentrationwas determined by BCA protein assay (Pierce Biotechnology Rockford IL). Cell and membrane extracts were subjected to sodium dodecylsulfate-polyacrylamide electrophoresis in 8% gels and transferred toMillipore Immobilon-P transfer membrane (Billerica MA). Immunoblots were probed with primary antibodies against TRPC1 (1:500) and TRPC4 (1:200). Primary polyclonal human PLCB3 (loading control) antibody and HRP-conjugated secondary antibodies were used at 1:2000 dilution. Where indicated antibodies against TRPC4 and TRPC1 were preabsorbed prior to use with the corresponding antigenic peptides overnight at 4 ◦C atmass ratios of 1:1 and 25:1 antigenic peptide:antibody respectively as recommended by the manufacturers. Bands were visualized by enhanced chemiluminescence (ECL) reagent (Amersham Biosciences GE Healthcare Pittsburgh PA). ECL signals were detected using a Storm imager and quantitationwas accomplished by Image- Quant TL software (Amersham Biosciences). 2.7. Measurement of intracellular calcium PHM1–41 and UtSMC cells were plated at 1×105 and 0.4×105 cells/0.8 ml respectively in 35-mm glass-bottomed dishes. Cells were loaded 72–96 h after infection at room temperature for 30 min with 5 M Fura-2-AM and 0.1% Pluronic F-127 in fluorescence buffer (145mM NaCl 5mM KCl 1mM Na2HPO4 0.5mM MgCl2 1mM CaCl2 10mM HEPES 5mM glucose pH 7.4). Cells were then washed twice in the same buffer and incubated an additional 45 min at room temperature to allow for Fura-2 ester hydrolysis. Immediately prior to assay cells were placed in Ca2+- free fluorescence buffer (buffer containing 100 M EGTA but no CaCl2) and changes in fluorescence were measured at 340 and 380nm excitation and 510 nm emission wavelengths in an ImCyt2 imaging system (Intracellular Imaging Inc. Cincinnati OH). Cells co-expressing eGFPwere identified using an eGFP/fluorescein excitation filter (485 nm). Electroporated or virally infected cells did not display nonspecific membrane leaks as evidenced by the fact that no significant increases in [Ca2+]i were observed following addition of 1mM CaCl2 if cells were not exposed to stimuli eliciting SRCE. Changes in Ca2+ were observed in 20–45 cells/dish. 2.8. Electrophysiological recordings PHM1–41 cells exposed to adenoviral constructs expressing empty vector or TC4sh1 sequences were cultured on glass coverslips for >72 h prior to patch clamp experiments. Cultured cellswere placed into a recording chamber (SA-OLY Warner Instruments) at room temperature. Cell-attached recordingswere performed using an AxoPatch 200B amplifier equipped with an Axon CV 203BU headstage (Axon Instruments). Recording electrodes (resistance 3–5M ) were pulled polished and coated with wax to reduce capacitance. Currents were filtered at 1 kHz digitized at 40 kHz and stored for subsequent analysis. Clampex and Clampfit versions 10.2 (Axon Instruments) were used for data acquisition and analysis respectively. Pipette potential was clamped at −40mV and all recordings were performed at room temperature (22 ◦C). The bath solution was 125.4mM NaCl 20mM tetraethylammounium chloride (TEA) 0.1mMMgCl2 5mMHEPES 11mMglucose 1 MCaCl2 and 100 nM nifedipine. The pipette solution contained 135mM CsCl 2.5mM MgCl2 10mM HEPES 10mM glucose 10mM EGTA and 10 M paxilline (pH 7.2). Cell-attached channel activity was recorded before and after addition of oxytocin (100 nM). 2.9. Data analyses Data are presented as mean±S.E.M. andwere analyzed by t-test or one-way ANOVA and Tukey’s test as appropriate using Prism software (GraphPad Software La Jolla CA) software. Changes in intracellular Ca2+ were analyzed using numerical analyses software (CalciumComp) developed by an engineer consultant (K.J. Bois Fort Collins CO). CalciumComp aligns the initial [Ca2+]i peaks removes noise and calculates the extent of the increase [Ca2+]i (peak height) and integrated area under the [Ca2+]i transient curve. Data obtained using this method agreedwithin 95% with that analyzed bymanual methods

Microscopic Technique
NA

Cell Type(s)
UtSMC