Cell culture and transfections.
Mv1Lu cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U of penicillin per ml, and 100 μg of streptomycin per ml. The cells were maintained in a humidified incubator with 5% CO2 at 37°C. Transient transfections were performed with Lipofectamine Plus reagent (Gibco BRL, Gaithersburg, Md.) according to the manufacturer's instructions.
Preparation of pancreatic acini and adenoviral infection.
The preparation of pancreatic acini was performed as previously described (43
). Briefly, pancreatic tissue was obtained from male Swiss Webster mice or Smad3−/−
mice and digested with collagenase (100 U/ml) and incubated at 37°C for 45 min with shaking (120 cycles/min). Acini were then mechanically dispersed by trituration of tissue through polypropylene pipettes of decreasing orifice size (3.0, 2.4, and 1.2 mm) and filtration through a 150-μm-pore-size mesh nylon cloth. Acini were purified by centrifugation at 50 × g
for 3 min through a solution containing 4% bovine serum albumin (BSA) and were resuspended in enhanced media that consisted of Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum, 100 U of penicillin per ml, 100 μg of streptomycin per ml, 0.5 mM isobutylmethylxanthine (IBMX), and 0.1 mg of soybean trypsin inhibitor per ml. Cells were maintained in a humidified atmosphere of 5% CO2
in air at 37°C during incubation times. The acinar cells were infected with adenovirus expressing either Smad3 or green fluorescent protein (106
PFU/mg of acinar protein) as described previously (43
In vitro kinase assay for PKA activity.
PKA kinase activity was measured by a PKA kinase activity assay kit (Promega, Madison, Wis.). Mv1Lu cells or acinar cells were treated with TGFβ1 (R & D Systems, Minneapolis, Minn.), washed with phosphate-buffered saline (PBS), and harvested with cold extraction buffer containing 25 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 10 mM β-mercaptoethanol, 1 mg of leupeptin per ml, 1 mg of aprotinin per ml, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Protein concentrations of the crude lysates were quantitated, and equal amounts of protein were added to a reaction mixture containing 40 mM Tris-HCl (pH 7.4), 20 mM MgCl2, 0.1 mg of BSA per ml, 100 mM biotinylated PKA peptide substrate (Kemptide), 3,000 Ci [γ32-P]ATP (Amersham, Arlington Heights, Ill.) per mmol, and 0.5 mM ATP per reaction. The reaction was allowed to proceed for 5 min at 30°C and then terminated by the addition of 2.5 M guanidine hydrochloride. A total of 10 μl of each sample was spotted onto streptavidin-coated disks, washed repeatedly, dried in an oven, and placed in scintillation vials for radioactive counting.
Measurement of cAMP.
Intracellular cAMP levels were measured with a Biotrak cAMP enzyme immunoassay kit (Amersham). Mv1Lu cells were treated with TGFβ or forskolin in the absence and presence of IBMX (100 mM), and the cells were collected and resuspended in PBS with 65% (vol/vol) ethanol. The cell precipitates were centrifuged, the supernatants were drawn off, and the extracts were dried in a vacuum oven. Extracts were resuspended in assay buffer, acetylated, and assayed for cAMP following the instructions supplied by the manufacturer.
Whole-cell lysates were prepared by incubating cells in ice-cold lysis buffer (20 mM Tris [pH 7.8], 2 mM EDTA, 50 mM NaF, 1% Triton X-100, 5 μg of leupeptin per ml, 5 μg of pepstatin per ml, and 0.5 mM PMSF). Cells were sonicated for 8 s and then placed on ice for 15 min. The lysates were then centrifuged at 14,000 × g
for 15 min at 4°C and assayed for protein with the Bio-Rad protein assay reagent. Equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Anti-p21Cip1
antibody and anti-β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif.) were used. Images were visualized with an enhanced chemiluminescence (ECL) detection system (Amersham). For immunoblot analysis of phospho- and total-CREB (cAMP-response element binding protein), nuclear cellular extracts were prepared by the method of Maire as previously described (23
) and anti-phospho-CREB antibody (Upstate Biotechnology, Inc., Lake Placid, N.Y.) and anti-total-CREB antibodies (Santa Cruz Biotechnology) were used.
Mv1Lu cells were treated with 100 pM TGFβ for indicated time periods. Cells were then lysed by sonicating for 5 s in 1 ml of detergent-free lysis buffer (PBS, 5 mM EDTA, 0.02% sodium azide), 10 mM iodoacetamide, 1 mM PMSF, and 2 μg of leupeptin per ml at 4°C. The lysates were cleared by microcentrifuging for 15 min at 16,000 × g at 4°C. Antibody-conjugated beads were prepared by combining 1 μg of polyclonal antibodies with 30 μl of a 50% protein A-Sepharose bead slurry in 0.5 ml of ice-cold PBS for 1 h at 4°C in a tube rotator and then were washed two times with 1 ml of lysis buffer. The antibodies used for immunoprecipitation were rabbit polyclonal anti-PKA RIβ and RIIα and anti-PKA Cα subunit antibodies (Santa Cruz Biotechnology). Cell lysate (500 μg) was incubated with the prepared beads and 10 μl of 10% BSA overnight at 4°C. The beads were washed four times with washing buffer (50 mM Tris-HCl [pH 7.4], 300 mM NaCl, 5 mM EDTA, 0.02% sodium azide, 0.1% Triton X-100) and one time with ice-cold PBS. Proteins were revealed after SDS-PAGE and Western blotting with the following antibodies: mouse anti-Flag antibody (Sigma, St. Louis, Mo.) and rabbit polyclonal antibodies to Smad4, Smad3, PKA RIβ and RIIα, and PKA Cα (Santa Cruz Biotechnology). Images were visualized by using the ECL detection system.
In vitro binding and GST pull-down assays.
Glutathione S-transferase (GST)-labeled constitutively active Smad3 (Smad3D) fusion protein and GST-Smad4 protein were produced in Escherichia coli and purified by using a bulk GST purification module (Amersham). One microgram of purified GST, GST-Smad3, or GST-Smad4 protein was immobilized on glutathione Sepharose beads and added to 1 μg of purified recombinant PKA RIIα protein in PBS supplemented with 10% BSA as a nonspecific competitor. After incubation for 1 h at 4°C, the samples were washed four times with PBS, resolved by SDS-PAGE, and blotted with anti-PKA RIIα. The same membrane was stripped and blotted with anti-Smad4 and anti-Smad3 antibodies. Images were visualized by using the ECL detection system.
PKA holoenzyme assay.
A PKA RIIα2
holoenzyme was formed and purified by sucrose gradient centrifugation as described previously (10
) by using 8 μg of purified PKA Cα protein and twofold excess of purified PKA RIIα protein. Briefly, the purified proteins were incubated for 10 min at 4°C and then were loaded on the top of a 13-ml 5 to 20% sucrose (in 100 mM NaCl) gradient centrifugation column. The centrifugation was performed at 100,000 × g
for 22 h. The fraction with peak cAMP-dependent kinase activity was considered as purified PKA holoenzyme. The kinase activity assay was performed as described above. The activities of RIIα2
were measured in the presence of 100 nM cAMP, a 1 μM concentration of purified Smad3D protein, a 1 μM concentration of purified Smad4 protein, or a combination of the Smad3D and Smad4 proteins, each at a concentration of 1 μM.
Nuclear extracts were prepared and used for electrophoretic mobility shift assays (EMSAs) as previously described (34
). Nuclear protein (5 μg) was incubated with gel shift binding buffer [10 mM HEPES, 10% glycerol, 1 mM dithiothreitol, 1 mg of poly(dI-dC) per 10 ml, and 5 mg of BSA per 10 ml] and a CREB oligonucleotide probe labeled with [γ-32
P]ATP by T4 polynucleotide kinase. The oligonucleotide probes were provided by a gel shift assay system (E3300; Promega). The reaction was allowed to proceed for 30 min at room temperature. For cold competition experiments, the extract was preincubated for 30 min with 50-fold molar excess of unlabeled CREB oligonucleotide. For the antibody supershift assay, 1 μg of anti-CREB antibody was incubated with the nuclear extracts for 30 min at room temperature prior to the addition of labeled probe. Reactions were analyzed on a 10- by 12-cm, 0.75-mm thick, nondenaturing, 4% acrylamide gel. Gels were transferred to Whatman paper on a gel dryer, exposed to a Bio-Rad GS-250 screen overnight, and then analyzed on a Bio-Rad molecular imager.
Cell proliferation was measured by using a CellTiter 96 AQ nonradioactive cell proliferation assay (Promega). Briefly, cells were plated in 96-well plates at a density of 2,000 cells/well in 100 μl of medium. Cells were allowed to grow up to 5 days; then combined MTS [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-phenozine methosulfate solution (20 μl/well) was added. After incubation for 2 h at 37°C in a humidified 5% CO2 atmosphere, the absorbance was measured at 490 nm by using an enzyme-linked immunosorbent assay plate reader. Data presented represent the average of three wells in one experiment which was repeated twice.