Cell culture and transfections.
Bladder smooth muscle cells (SMC; Cambrex) were cultured in SmGM2-medium (Cambrex), aortic SMC (Cell Applications, San Diego, CA) were cultured in SMC growth medium (Cell Applications), and foreskin fibroblasts were cultured in Dulbecco modified Eagle medium with 10% fetal bovine serum (FBS). To construct Skp2-luc, a 423-bp fragment corresponding to sequence from the 5′ flanking region of the human Skp2 gene (GenBank accession no. NT006576) was inserted between the MluI and BglII sites in the pGL3 promoter (Promega). SMC (~80% confluence on 100-mm tissue culture plates) were transfected with 4 μg of the indicated constructs by using Fugene6 (Roche) according to the manufacturer's protocol.
Collagen matrix assays.
Cells were suspended in 1.4 mg of collagen (Vitrogen; Cohesion Technologies)/ml, and then the collagen was polymerized according to the manufacturer's protocol. Once the cells developed a spindlelike morphology (~6 h), the collagen matrices were either released from or left fixed to the tissue culture plate, and the cells were used in the assays described below.
SMC were cultured in collagen matrices for 24 h, rinsed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 5 min, rinsed with PBS, and incubated with Alexa Fluor 488-phalloidin (Molecular Probes) for 20 min. After a rinse with PBS, the cells were visualized by fluorescence microscopy.
SMC that were cultured in collagen matrices for 24 h were incubated in medium containing 30 μM bromodeoxyuridine (BrdU; Sigma) for an additional 4 h. Cells were isolated from the collagen matrices by incubation in 5 mg of type XI collagenase (Sigma)/ml and analyzed for BrdU uptake by immunofluorescence.
Bladder outlet obstruction.
To create a complete bladder outlet obstruction, the urethras of 8- to 10-week-old anesthetized mice were ligated by using a 4-0 silk suture. Partial bladder outlet obstruction was created by placing a 22-gauge catheter alongside the urethra, tying a 4-0 silk ligature firmly around the urethra and the catheter, and then removing the catheter. At the indicated time points, bladders were excised and snap-frozen in liquid nitrogen or fixed in formalin and stained with hematoxylin and eosin.
SMC were isolated from the collagen matrices by incubation in 5 mg of type XI collagenase (Sigma)/ml and lysed in Triton buffer (0.5% Triton, 10 mM Tris [pH 8.6], 140 mM NaCl, 1.5 mM MgCl2). Cells cultured on tissue culture plates were pelleted and lysed in sodium dodecyl sulfate (SDS) lysis buffer. Murine bladder smooth muscle tissue was snap-frozen, ground to powder, and lysed in SDS lysis buffer. To obtain nuclear lysate from murine bladder smooth muscle tissue, the tissue was excised and chopped into small pieces and then Dounce homogenized in ice-cold homogenization buffer (50 mM Tris-HCl [pH 7.5], 1.5 mM MgCl2, 2 mM β-mercaptoethanol, 200 mM sucrose, 0.1% Triton X-100). To obtain nuclear lysate from cultured SMC, cells were scraped and incubated in homogenization buffer. The nuclei were pelleted, washed in homogenization buffer without Triton, and repelleted. The isolated nuclei were then lysed in SDS lysis buffer. Complete protease inhibitor cocktail (1×; Roche) and phosphatase inhibitor cocktails (Sigma) were present during the complete procedure. For dephosphorylation of NFATc1, human SMC were harvested in 1× passive lysis buffer (Promega) lacking phosphatase inhibitors, and then the lysate was incubated in 1× phosphatase buffer (Invitrogen) in either the presence or the absence of 1 U of calf intestinal alkaline phosphatase (Invitrogen)/μg at 37°C for 1 h. The following antibodies were used: Skp2 (catalog no. 51-1900) and Skp2 (catalog no. 32-3300) from Zymed; p27KIP1 (c-19), p21CIP1 (c-19), PCNA (PC10), β-tubulin (H-235), lamin A/C (N-18), and NFATc3 (F-1) from Santa Cruz; NFATc1 (7A6) from BD Pharmingen; and Erk (catalog no. 9102) and phospho-Erk (catalog no. 9106) from Cell Signaling.
RNA was isolated from the cells in the collagen matrices by using TRIzol (Invitrogen) or from cells growing on tissue culture plates and mouse bladder smooth muscle layer using RNeasy (Qiagen). Reverse transcription-PCR (RT-PCR) was performed using RETROscript (Ambion) and PuReTaq Ready-To-Go PCR beads (GE Healthcare) with the following primers: Skp2 sense, 5′-ATGGGATTCCAGCAAGACTTCTGAA-3′; Skp2 antisense, 5′-GCTCAGGGAGGCACAGACAGGA-3′; p27KIP1 sense, 5′-AGCCTGGAGCGGATGGAC-3′; p27KIP1 antisense, 5′-CTTGGGCGTCTGCTCCACA-3′; b-myb sense, 5′-GATGTGCCGGAGCAGAGGGATAG-3′; b-myb antisense, 5′-GTCCATGGCCCCTTGACAAGGTC-3′; β-actin sense, 5′-GTGATGGTGGGCATGGGTCA-3′; β-actin antisense, 5′-TTAATGTCACGCACGATTTCCC-3′; GAPDH sense, 5′-GGTGAAGGTCGGAGTCAACG-3′; and GAPDH antisense, 5′-CAAAGTTGTCATGGATGACC-3′.
Luciferase assays were performed by using a luciferase assay system (Promega). Immunoblots for β-tubulin were performed to confirm that equal numbers of cells were assayed (data not shown).
Actinomycin D treatment.
Human SMC were seeded in collagen matrices as described above. Approximately 6 h after the collagen had polymerized, the media was changed to SmGM-2 containing 1 μg of actinomycin D (Sigma)/ml, and the collagen matrices were either released from or left fixed to the tissue culture plate. At the indicated time points, RNA was isolated for RT-PCR analysis.
Small interfering RNAs (siRNAs) were transfected by using GeneEraser reagent (Stratagene) according to the manufacturer's protocol. The following siRNA sequences were used: scrambled sense, 5′-AUGUAUUGGCCUGUAUUAGUU-3′; scrambled antisense, 5′-CUAAUACAGGCCAAUACAUUU-3′; Skp2(A) sense, 5′-AAGGGAGUGACAAAGACUUUG-3′; Skp2(A) antisense, 5′-CAAAGUCUUUGUCACUCCCUU-3′; Skp2(B) sense, 5′-AAUCUAAGCCUGGAAGGCCUG-3′; Skp2(B) antisense, 5′-CAGGCCUUCCAGGCUUAGAUU-3′; NFATc1(A) sense, 5′-CGUAUGAGCUUCGGAUUGAUU-3′; NFATc1(A) antisense, 5′-UCAAUCCGAAGCUCAUACGUU-3′; NFATc1(B) sense, 5′-GAAACUCCGACAUUGAACUTT-3′; and NFATc1(B) antisense, 5′-TTAGUUCAAUGUCGGAGUUUC-3′. Cells were transfected twice at an interval of 24 h with Skp2 siRNAs and at an interval of 36 h with NFATc1 siRNAs.
Mouse bladder smooth muscle tissue was chopped into small pieces, cross-linked with 1% formaldehyde in 1× PBS, and sonicated. Samples were processed according to the chromatin immunoprecipitation (ChIP) kit manufacturer's protocols (Upstate). DNA was recovered by using the phenol-chloroform method. The immunoprecipitated DNA was subjected to PCR assays with the following primer pairs: Skp2 promoter sense, 5′-CGTCTGGAAGGGACTCAGAAG-3′; Skp2 promoter antisense, 5′-AACCCTCCAGATACCCACAA-3′; β-globin sense, 5′-CCTGCCCTCTCTATCCTGTG-3′; and β-globin sense, 5′-GCAAATGTGTTGCCAAAAAG-3′. The NFATc1 antibody (H-110) was from Santa Cruz.