We generated Stat5f/f;Alb-Cre mice by breeding Stat5f/f mice with albumin/cyclic adenosine monophosphate response element (Alb-Cre) transgenic mice.14Stat5f/f and Alb-Cre transgenic mice were on a mixed background. Only 10- to 18-week-old male mice were used in the experiments unless otherwise indicated. We treated the animals humanely and performed procedures according to the protocol approved by the Animal Use and Care Committee at the National Institute of Diabetes and Digestive and Kidney Diseases.
Liver Fibrosis Induced by CCl4.
Hepatic fibrosis in mice was induced by intraperitoneal (i.p.) injection with 2 mL/kg body weight of 10% CCl4 (Sigma, St. Louis, MO) dissolved in olive oil (Sigma, St. Louis, MO), three times a week for 12 weeks. For GH stimulation, mice were i.p. injected with 2 μg/g body weight GH. They were sacrificed 4 hours after injection and livers were harvested for analyses.
Isolation of Primary MEF Cells.
Primary MEFs were isolated from embryonic day 14.5 (E14.5) Stat5+/+ and Stat5−/− embryos by first mincing the embryos, then digesting in 0.05% trypsin/0.02% ethylene diamine tetraacetic acid (EDTA) for 30 minutes at 37°C, pelleting the tissue, and resuspending in growth medium consisting of Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum. MEFs were maintained in high-glucose DMEM supplemented with 15% fetal bovine serum, 50 μg/mL streptomycin sulfate, 50 U/mL penicillin G sodium, β-mercaptoethanol, and nonessential amino acid in an atmosphere of 5% CO2 at 37°C.
The retroviral-expression vector carrying a wild-type Stat5A15 gene was based on an MSCV-IRES-GFP (murine stem cell virus–internal ribosome entry site–green fluorescent protein) backbone (gift from Richard Moriggl, Ludwig-Boltzmann Institute, Vienna, Austria). The 293T cells were transfected with the plasmid using FuGENE (Roche, Indianapolis, IN). Supernatants were collected for 48-72 hours after transfection and passed through a 0.45-μm filter before freezing at −80°C. For the infection, 106Stat5−/− MEFs were seeded on a 10-cm culture dish and infected the next day with retrovirus in the presence of 8 μg/mL polybrene. After infection, nonfluorescent cells and GFP-expressing cells were isolated using the FACS Vantage (Becton Dickinson, San Jose, CA) fluorescence-activated cell sorter (FACS) and sorted directly into phosphate-buffered saline (PBS). Sorted MEFs were maintained in DMEM supplemented as described above.
Affymetrix Microarray Analysis.
Primary MEFs derived from Stat5+/+ or Stat5−/− embryos were cultured to passage 13. After starvation for 5 hours, MEFs were stimulated with GH (1 μg/mL) for 2 hours. Unstimulated samples were used as control. Total RNAs were prepared by using a RNeasy Plus Mini Kit (Qiagen, Valencia, CA). RNA quality was verified using an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA). Microarray analyses were performed using Affymetrix Mouse Genome 430 version 2.0 array GeneChips (Affymetrix, Santa Clara, CA). Microarray signals were analyzed using the Affymetrix RMA algorithm. Up-regulated and down-regulated genes were selected based on P < 0.05 and fold-changes of more than 1.5 or less than 1.5 as assessed by analysis of variance using Partek Pro software (Partek, St. Louis, MO). The microarray analyses were performed with three independent biological sample sets. Microarray data have been deposited in Gene Expression Omnibus (GEO; accession number: GSE21861).
Analysis of Cell Proliferation.
The proliferation of cells was determined by a Trypan blue dye exclusion assay. In brief, primary Stat5+/+ and Stat5−/− MEFs (1 × 105 cells/well) were seeded on tissue culture plates and cultured in high-glucose DMEM. The number of viable cells was counted after 2, 4, and 6 days. MEFs were harvested with trypsin-EDTA. The cell suspension was loaded onto a hemocytometer (1:1) with the dye Trypan blue, which is taken up by dead cells. Both viable and dead cells were counted, from which both the percentage of dead cells and total cell number were calculated.
Primary Stat5+/+ and Stat5−/− MEFs were washed twice with PBS and fixed for 30 minutes at −20°C in 70% ethanol. Total DNA was stained with propidium iodide (5 μg/mL in PBS containing 50 μg/mL ribonuclease A). Cell cycle distribution was determined by FACS analysis using a FACSCalibur (Becton Dickinson, San Jose, CA). Data are presented as a percentage of viable cells remaining in the respective cell cycle phase.
Antibodies, Immunoblotting, and Immunostaining.
In brief, primary MEFs were lysed by adding NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA). Cell lysates were heat-denatured for 10 minutes at 70°C and loaded on a NuPAGE 10% Bis-Tris polyacrylamide gel. After electrophoresis in NuPAGE sodium dodecyl sulfate running buffer using the Xcell SureLock Mini-Cell, proteins were transferred to a polyvinylidene fluoride membrane according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). The rabbit polyclonal anti-STAT5 (N-20), anti-STAT5 (C-17), anti-p15 (K-18), anti-p21 (C-19), anti–cyclin D1 (HD-11), anti–cyclin A (H-432), anti–cyclin B1 (H-433), anti-Cdk4 (C-22), and anti–β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used for probing western blots. Immunohistochemistry was performed using standard procedures. In short, liver tissues were removed and fixed in 10% neutral buffered formalin and embedded in paraffin wax. Sections (5 μm) were prepared for hematoxylin and eosin (H&E) staining and immunofluorescence analyses. After deparaffinization, antigen unmasking was performed in a Decloaking chamber (Biocare Medical, San Diego, CA) using BORG Decloaker Solution (Biocare Medical, San Diego, CA) for 5 minutes at 125°C. The sections were blocked for 30 minutes in Tris-buffered saline with Tween-20 containing 3% goat serum. Primary antibodies used in this study included rabbit anti-STAT5 (N-20), rabbit anti–phospho-STAT5 (Tyr694) (Cell Signaling Technology, Beverly, MA), rabbit anti-p15 (K-18) (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti–phospho-histone H3 (Ser-10) (Upstate Biotechnology, Lake Placid, NY), in addition to mouse anti-PCNA (DAKO Cytomation, Carpinteria, CA) and mouse anti–β-catenin (BD Transduction Laboratories, San Jose, CA). For double-labeling immunofluorescence analyses, sections exposed to a pair of primary antibodies were incubated in a 1:400 dilution of goat anti-rabbit immunoglobulin G (IgG) conjugated with a red fluorophore (AlexaFluor 594; Molecular Probes, Eugene, OR) and goat anti-mouse IgG conjugated with a green fluorophore (AlexaFluor 488; Molecular Probes, Eugene, OR) for 30 minutes at room temperature. Images were obtained with a Retiga Exi camera on a Olympus BX51 microscope (Olympus America, Center Valley, PA) using Image-Pro 5.1 software. For quantitation, three images taken with the 40× objective were counted per mouse. Three mice from each experimental group were evaluated.
Cellular proteins were extracted from primary Stat5+/+ and Stat5−/− MEFs. Protein fractions were incubated overnight with an anti-CDK4 antibody and protein A–sepharose beads at 4°C in radioimmunoprecipitation assay buffer containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.25% Nonidet P-40, and 0.5% sodium deoxycholate. The protein A–sepharose antibody–antigen complex was collected and washed three times with ice-cold radioimmunoprecipitation assay buffer. The final pellet was resuspended with sodium dodecyl sulfate sample buffer and boiled for 5 minutes. This preparation was subjected to western blot analysis with the anti-p15 or anti-p21 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Chromatin Immunoprecipitation Assay.
Chromatin immunoprecipitation (ChIP) assay was performed as described.16 In brief, after starvation for 5 hours, primary Stat5+/+ MEFs were stimulated with GH for 45 minutes. Unstimulated samples were used as controls. MEFs were cross-linked in 1.5% formaldehyde for 15 minutes at 37°C. Cells were then harvested and sonicated using the Misonix Sonicator 3000 (Misonix, Farmingdale, NY). Immunoprecipitation was carried out in Tris-EDTA buffer containing protease inhibitors (Sigma, St. Louis, MO). Chromatin was incubated with protein A Dynabeads (Invitrogen, Carlsbad, CA), which were preincubated with STAT5A or IgG antibody (R&D Systems, Minneapolis, MN). Immunoprecipitated DNA was eluted and amplified by real-time polymerase chain reaction (PCR) using a 7900 HT fast real-time PCR system (Applied Biosystems, Foster City, CA) and analyzed using SDS2.3 Software (Applied Biosystems, Foster City, CA). Sequence-specific primers used for amplification of the putative STAT5 binding sites (interferon-gamma–activated sequence [GAS] sites) within the Socs2 (suppressor of cytokine signaling 2) and Cdkn2b (cyclin-dependent kinase inhibitor 2b) genes were as following: For the Socs2 GAS, forward primer 5′-GGAGGGCGGAGT CGCAGGC-3′, reverse primer 5′-GACTTGGCAAGA GTTAACCGTC-3′; the primer sets for Cdkn2b gene were: GAS1, forward 5′-GTTTTGCCGTGATGTCC TTG-3′, reverse 5′-ATCGCACTGCTTCGTGTAAC-3′; GAS2, forward 5′-GACAGGCATTGTCCAAGACA-3′, reverse 5′-GTGCCACATTCTCCCACTTT-3′.
RNA Isolation and Quantitative Real-Time PCR Analysis.
Total RNA was isolated from primary Stat5+/+ MEFs, Stat5−/− MEFs, and liver tissues using Trizol reagent (Invitrogen, Carlsbad, CA). One μg amounts of RNA were reverse transcribed (complementary DNA reverse transcription kit; Applied Biosystems, Foster City, CA). Real-time quantification of messenger RNA (mRNA) transcript levels was performed using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Real-time PCR was carried out using an ABI-Prism 7900HT (Applied Biosystems, Foster City, CA). Individual PCRs were performed in triplicate on samples using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. The primers used were: Cdkn2b, forward 5′-CCCTGCCA CCCTTACCAGA-3′, reverse 5′-CAGATACCTCGCA ATGTCACG-3′, yielding a 169–base pair (bp) PCR product; Cdkn1a, forward 5′-GTGGCCTTGTCGCT GTCTT-3′, reverse 5′-GCGCTTGGAGTGATAGAAA TCTG-3′, yielding a 126-bp PCR product; GAPDH, forward 5′-AACGACCCCTTCATTGAC-3′, reverse 5′ TCCACGACATACTCAGCAC-3′, yielding a 191-bp PCR product.
All statistical analyses were performed using the Student t test (two-tailed, unpaired). A P value of 0.05 or less was considered significant.