NF-κB transcriptional activation by TNFα requires phospholipase C, extracellular signal-regulated kinase 2 and poly(ADP-ribose) polymerase-1.

Primary cultures of mouse cortical astrocytes and microglia were treated with TNFα and suitable signaling pathway modulators (pharmacological and molecular). Outcome measures included calcium imaging, PARP-1 activation status, NF-κB transcriptional activity, DNA damage assesment and cytokine relesease profiling.

Methods Materials PARP-1 inhibitors, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2 h)-isoquinolinone (DPQ) and N -(6-oxo-5,6-dihydrophenanthridin-2-yl)- N , N -dimethylacetamide (PJ34) were obtained from Calbiochem (San Diego, CA) and Inotek Pharmaceuticals (Lexington, MA), respectively. The MAPK inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) was obtained from Tocris Cookson Ltd. (Ellisville, MO). The PLC activator, 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesul fonamide (m-3M3FBS) and PLC inhibitors, 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73112) and tricyclodecane-9-yl-xanthogenate (D609) were obtained from Calbiochem. Cell culture reagents were obtained from Cellgro/Mediatech (Herndon,VI), and all other reagents were from Sigma-Aldrich (St. Louis, MO) except where otherwise stated.

Cell cultures

All animal studies were approved by the San Francisco Veterans Affairs Medical Center animal studies committee and University of Manitoba animal care and use committee, and follow the NIH guidelines for humane care of animals. Cultures were prepared from PARP-1 −/− , and wild-type (wt) mice as described previously [ 14 ]. The PARP-1 −/− mice were descendants of the 129S-Adprtl tmlZqw strain, originally developed by Wang et al. [ 32 ] and obtained from the Jackson Laboratory (Bar Harbor, ME). These mice were outbred for seven generations with wt CD-1 mice, and wt CD-1 mice were used as controls for the PARP-1 −/− mice. Astrocyte cultures were prepared from 1-day old mouse pups of both sexes in 24-well plates as described previously [ 33 ]. At confluency, microglia were harvested by gently shaking and re-plated at density of 5 × 10 5 cells/well on a 24-well plate [ 14 ]. The microglia cultures were subsequently maintained in glia-conditioned medium. The confluent astrocyte cultures were treated with 22 μM cytosine β-D-arabinofuranoside for 2 days to inhibit the proliferation of remaining microglia. The astrocyte cultures were subsequently maintained in Eagle’s minimal essential medium (MEM) supplemented with 3 % fetal bovine serum (FBS). Drug incubations Studies using astrocyte cultures were initiated by replacing the culture medium with a physiologically balanced salt solution (BSS) containing 3.1 mM KCl, 134 mM NaCl, 1.2 mM CaCl 2 , 1.2 mM MgSO 4 , 0.5 mM KH 2 PO 4 , 15.7 mM NaHCO 3 , and 2 mM glucose, pre-equilibrated to pH 7.2 in a 5 % CO 2 atmosphere. Drugs were prepared as concentrated stock solutions in BSS. Experiments were performed at 37 °C in a 5 % CO 2 atmosphere and were terminated by complete medium exchange and replacement with fresh BSS. Studies with microglial cultures were performed identically but used MEM rather than BSS. Immunostaining Fixation and immunostaining of cell cultures was performed as previously described [ 14 ]. For detection of poly(ADP-ribose), incubations were performed with a 1:1000 dilution of rabbit anti-poly(ADP-ribose) (Trevigen, Gaithersburg, MD, Cat# 4336-BPC) at 4 °C for 24 h, followed by incubation with Alexa Fluor 594-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR), 1:500 dilution, for 2 h at room temperature. For evaluation of NF-κB p65 subunit translocation, incubations were performed with a 1:30 dilution of rabbit polyclonal anti- p65 (Cell Signaling Technology, Inc., Danvers, MA, Cat# 3987) at 4 °C for 24 h, followed by incubation with Alexa Fluor 594-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR), 1:500 dilution for 2 h. In some studies, the cells were subsequently incubated with 2 μg/ml propidium iodide for 5 min to obtain nuclear counterstaining. Confocal photomicrographs of astrocytes were obtained with a Zeiss LSM 510 META with a dual-PMT detector and a 32-channel META detector and Zeiss AIM imaging software. Microglia images were obtained with standard epifluorescence microscopy because these cells could not be cultured in a resting state on glass coverslips. Controls prepared in the absence of primary antibody showed no staining under the conditions described (not shown). DNA strand breaks were detected by DNA-polymerase I-mediated biotin-dATP nick translation (PANT) [ 34 ]. Cultures were permeabilized in 1 % Triton X-100 in phosphate buffered saline (PBS) for 30 min, and endogenous peroxidase was quenched with 2 % hydrogen peroxide. The cultures were then incubated for 90 min with the PANT reaction mixture: 5 mM MgCl2, 10 mM 2-mercaptoethanol, 20 μg/ml bovine serum albumin, 30 μM dGTP, 30 μM dCTP, 30 μM dTTP, 1 μM dATP, 29 μM biotinylated dATP, and 40 U/ml of DNA-polymerase I in PBS, pH 7.4. The biotinylated areas of DNA strand breaks were visualized by subsequent incubation with streptavidin-horseradish peroxidase and DAB peroxidase substrate. Controls incubated with PANT reaction mixture without DNA-polymerase I showed no staining. siRNA downregulation of ERK1/2 Astrocytes were incubated with siRNA transfection complexes at day 10 in vitro, at which time they are 95 % confluent. siRNA incubations were performed with RNAiFect reagents (Qiagen Inc., CA) according to the manufacturer’s instructions and as previously described [ 35 ]. The culture medium of each well was replaced with 400 μl of transfection complex mixture containing 1 μg of ERK1 siRNA or 3 μg of ERK2 siRNA and 6 μl of RNAiFect in optiMEM. The siRNA sense sequence for ERK1 was 5′ ACAAGCGCAUCACAGUAGAtt 3′ and for ERK2 was 5′ CAAAGUUCGAGUUGCUAUCtt 3′ (Ambion Inc, TX, USA). Controls were prepared with 3 μg of a mismatch sequence lacking significant homology to any known mouse gene sequences. The siRNA complexes were removed after 6 h and replaced with culture medium. Four days later when cultures were used for experiments, the percentage of siRNA-induced reduction in ERK1 and ERK2 expressions was determined by quantitating the ERK band size in ERK siRNA vs. mismatch siRNA transfected cultures in both ctrl and TNFα conditions.

Lentivirus NF-κB reporter gene transfection

The lentivirus NF-κB reporter gene construct (pLenti-kB-dEGFP) has a destabilized enhanced green fluorescent protein (dEGFP) construct under the control of five tandem repeats of κB-enhancer elements specific for p65and a plain TATA box [ 36 ]. Transfections were performed as previously described [ 15 ]. Astrocytes were infected with the Lenti-κB-dEGFP 4–5 days before experiments. EGFP expression was evaluated 1 and 24 h after exposure to TNFα by counting EGFP-expressing cells from five random fields within each culture well. TNFR1 inhibition Cells were incubated with 100 μg/ml of TNFR1 neutralizing antibody (anti-mouse CD120a, Affymetrix eBioscience, San Diego, CA, Cat#16-1202) or control IgG (Armenian hamster IgG, Affymetrix eBioscience, San Diego, CA, Cat#16-4888) in MEM for 4 h prior to starting the experiments.

Calcium imaging

Intracellular calcium levels were measured in astrocytes loaded with Fura-2-AM (Molecular Probes; 3 μM, 60 min). Experiments were carried out in bicarbonate-free BSS at 37 °C and buffered to pH 7.2 with 10 mM PIPES. Calcium-free BSS was prepared by omitting CaCl 2 and adding 2 mM EGTA. The change in dye fluorescence (Fura 340/380 ratio) was measured with aZeiss 200-M inverted microscope outfitted with an ORCA ER II microscope system using Openlab Improvision software. Excitation and emission wavelengths were controlled by Sutter filterwheels and shutters. Baseline values were recorded for 15–20 min prior to drug additions. At the end of each experiment, the calcium ionophore A23187-Br (10 μM) was applied to calibrate the intracellular signal. Data are presented as a change in Fura2 emission (Fura 340/380 ratio), and as a mean change in Fura 340/380 ratio during 15-, 30-, and 60-min intervals from three to four independent experiments. In each independent experiment data were acquired from six to ten cells at 90-s intervals, and at 10-s intervals in neutralizing antibody experiments. The high-affinity dye, Fura-2, was used to maximize sensitivity at low intracellular calcium concentrations, and therefore, the signal is likely saturated and underestimates the differences between Ca 2+ -free and Ca 2+ -containing medium.

Western blotting

Western blots were prepared and quantified as described [ 14 ]. Membranes were incubated with 1:1000 dilutions of rabbit anti-ERK1/2 or anti phospho-ERK1/2 (Cell Signaling Technology, Beverly, MA, Cat# 9102 and 4376), or with a 1:500 dilution of mouse monoclonal anti-poly(ADP-ribose) (Trevigen, Gaithersburg, MD, Cat# 4335-MC). After washing, the membranes were incubated for 2 h with peroxidase-conjugated anti-rabbit or anti-mouse IgG (Vector Laboratories, Burlingame, CA) diluted 1:7500. The protein bands were visualized using ECL TM WB Detection kit (Amersham-Pharmacia Biotech) and X-OMAT AR film (Kodak) or ChemiDoc MP imaging system (BioRad). To quantify protein loading, the membranes were re-probed with mouse monoclonal anti β-actin at a 1:10,000 dilutions, followed by peroxidase-conjugated anti-mouse IgG (1:10,000 dilutions). Controls performed in the absence of primary or secondary antibodies showed no signal (data not shown). Band densities were quantified with the NIH Image J program. Site-directed PARP-1 mutations The mutant PARP-1 constructs were prepared as described [ 35 ]. In short, human PARP-1 cDNA (hPARP-1, BC03754 from NIH Mammalian Gene Collection) was corrected to match the human genomic sequence at amino acid residue 762 (from alanine to valine) using Site-Directed Mutagenesis kit (Stratagene). Single (S372E) or double mutants (S372A and T373A) of these constructs were then generated and sequence-verified. PARP-1 −/− astrocytes were transfected 2 days before experiments with the PARP-1 constructs by replacing the culture medium with 300 μl of transfection complex mixture containing 0.5 μg of DNA and 1 μl of Lipofectamine 2000 (Invitrogen) in OptiMEM. The medium was replaced with culture medium 8 h after the initiation of transfection and cells were used for experiments 40 h later. The transfection efficacy of this method has been established previously by western blots detecting PARP-1 expression with a polyclonal antibody (ALX-210-302, Alexis/Enzo Life Sciences, Plymounth Meeting, PA) [ 35 ].

Cytokine assay

Cytokines were analyzed in 50-μl aliquots of cell culture medium using a Milliplex mouse multiplex immunoassay bead system according to the manufacturer’s instructions (Millipore) [ 37 ]. Samples were assayed in duplicate, and the fluorescent signal corresponding to each cytokine was measured with a BioPlex 200 system (BioRad, Hercules, CA) in parallel with known standards. Cytokine concentrations measured from culture medium were normalized to the protein content of each well as determined by the bicinchoninic assay [ 38 ].

Statistics

For cell culture studies, each “n” denotes an independent experiment, where each experiment is from a separate cell culture preparation and comprised of 3–4 parallel treatments per condition. Results are presented as a means ± standard error. Statistical significance was evaluated by one-way ANOVA followed by the Student-Newman-Keuls multiple comparison test. p values below 0.05 were considered significant.

Materials PARP-1 inhibitors, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2 h)-isoquinolinone (DPQ) and N -(6-oxo-5,6-dihydrophenanthridin-2-yl)- N , N -dimethylacetamide (PJ34) were obtained from Calbiochem (San Diego, CA) and Inotek Pharmaceuticals (Lexington, MA), respectively. The MAPK inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) was obtained from Tocris Cookson Ltd. (Ellisville, MO). The PLC activator, 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesul fonamide (m-3M3FBS) and PLC inhibitors, 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73112) and tricyclodecane-9-yl-xanthogenate (D609) were obtained from Calbiochem. Cell culture reagents were obtained from Cellgro/Mediatech (Herndon,VI), and all other reagents were from Sigma-Aldrich (St. Louis, MO) except where otherwise stated.