PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Hepatology. Author manuscript; available in PMC Nov 1, 2011.
Published in final edited form as:
PMCID: PMC3152209
NIHMSID: NIHMS224851
The transcription factors STAT5A and STAT5B negatively regulate cell proliferation through the activation of Cdkn2b and Cdkn1a expression
Ji Hoon Yu,1 Bing-Mei Zhu,1 Mark Wickre,1 Gregory Riedlinger,1 Weiping Chen,2 Atsushi Hosui,3 Gertraud W. Robinson,1 and Lothar Hennighausen1
1 Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
2 Genomics Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
3 Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
Ji Hoon Yu: yujihoon/at/mail.nih.gov; Bing-Mei Zhu: bingmeiz/at/mail.nih.gov; Mark Wickre: mwickre/at/gmail.com; Gregory Riedlinger: riedlinger/at/mail.nih.gov; Weiping Chen: weipingchen/at/mail.nih.gov; Atsushi Hosui: hosui/at/gh.med.osaka-u.ac.jp; Gertraud W. Robinson: traudl/at/nih.gov; Lothar Hennighausen: lotharh/at/mail.nih.gov
Address Correspondence to: Lothar Hennighausen, Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 8 Center Drive, Room 101, Bethesda, MD 20892-0822, tel. 301-496-2716, fax. 301-480-7312
Although the cytokine-inducible transcription factors STAT5 promote proliferation of a wide range of cell types, there are cell- and context specific cases in which loss of STAT5 results in enhanced cell proliferation. Here we report that loss of STAT5 from mouse embryonic fibroblasts (MEFs) leads to enhanced proliferation, which was linked to reduced levels of the cell cycle inhibitors p15INK4B and p21CIP1. We further demonstrate that growth hormone through the transcription factor STAT5 enhances expression of the Cdkn2b gene and that STAT5A binds to GAS sites within the promoter. We recently demonstrated that ablation of STAT5 from liver results in hepatocellular carcinoma upon CCl4 treatment. We now established that STAT5, like in MEFs, activates expression of the Cdkn2b gene in liver tissue. Loss of STAT5 led to diminished p15INK4B and increased hepatocyte proliferation. This study for the first time demonstrates that cytokines through STAT5 induce the expression of a key cell cycle inhibitor. These experiments therefore shed mechanistic light on the context-specific role of STAT5 as tumor suppressors.
Keywords: STAT5a/b, p15, p21, cell proliferation, liver fibrosis
Signal Transducers and Activators of Transcription (STAT) 5A and 5B are latent transcription factors that are induced by a plethora of cytokines, including growth hormone, prolactin and several interleukins.1 Gene knock-out experiments have revealed context-specific functions of STAT5, that range from the specification, proliferation and survival to differentiation of normal cells.1 Aberrant activation of STAT5 has been detected in a majority of leukemias and many solid tumors, suggesting a critical role in the initiation/progression of these tumors. Notably, the deletion of STAT5 from BCR-ABL induced leukemic cells results in their regression,2 providing evidence that STAT5 is a critical transcription factor in the progression of leukemias. Moreover, transgenic experiments using constitutively active STAT5 mutants have supported this concept.35 In addition to its oncogenic role, a context-specific tumor suppressor function has been associated with STAT5, such as inhibiting expression of NPM1-ALK6 and suppressing STAT3 and TGF-β activity in liver.7
Mice from which the Stat5 locus has been deleted specifically in liver tissue displayed altered metabolic pathways and developed fatty liver (nonalcoholic steatohepatitis).8, 9 Treatment of these mice with CCl4 led to liver fibrosis and tumors suggesting that STAT5 is a tumor suppressor in the context of hepatocytes.7 Aberrant activation of the TGF-β and STAT3 pathways in these mice appears to contribute to the CCl4-induced fibrosis and hetapocellular carcinoma (HCC).7
Evidence is emerging that cytokines through STAT5 regulates the cell cycle by facilitating progression through the G1 phase.1013 This opens the possibility that STAT5 directly controls cell cycle genes and thereby can function either as an oncogene or a tumor suppressor. We have now addressed this question studying mouse embryonic fibroblasts (MEFs) and liver tissue devoid of STAT5. In particular, STAT5-null MEFs were chosen as they exhibit an increased proliferation rate suggesting that in this context STAT5 is a cell cycle suppressor.
Mice breeding
We generated Stat5f/f;Alb-Cre mice by breeding Stat5f/f mice with Alb-Cre transgenic mice. Stat5f/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), 3 times a week for 12 weeks. For growth hormone (GH) stimulation, mice were injected 2 μg/g body weight of GH by i.p. Four hours after injection they were sacrificed and livers were harvested for analyses.
Isolation of primary MEF cells
Primary MEFs were isolated from day E14.5 Stat5+/+ and Stat5−/−embryos by first mincing the embryos, then digesting in 0.05% trypsin/0.02% 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% FCS. MEFs were maintained in high-glucose DMEM supplemented with 15% FBS, 50 μg/ml streptomycin sulfate,50 units/ml penicillin G sodium, β-mercaptoethanol, and non-essential amino acid in an atmosphere of 5% CO2 at 37°C.
Retrovirus infection
The retroviral-expression vector carrying a wild-type Stat5A gene was based on an MSCV-IRES-GFP backbone (gift from Richard Moriggl, Ludwig-Boltzmann Institute, Vienna, Austria). 293T cells were transfected with the plasmid using FuGENE (Roche, Indianapolis, IN). Supernatants were collected for 48–72 h after transfection and passed through a 0.45-μm filter before freezing at −80°C. For the infection, 106 Stat5/ 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, non fluorescent cells and GFP-expressing cells were isolated using the FACS Vantage (Becton Dickinson, San Jose, CA) and sorted directly into PBS. Sorted MEFs were maintained in Dulbecco’s Modified Eagle medium (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 2.0 array GeneChips (Affymetrix, Santa Clara, CA). Microarray signals were analyzed using the Affymetrix RMA algorithm. Up- and down-regulated genes were selected based on P values less than .05 and fold changes of more than 1.5 or less than 1.5 as assessed by ANOVA using Partek Pro software (Partek, St Louis, MO). The microarray analyses were performed with 3 independent biologic 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.
Cell-cycle analysis
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 PI (5 μg/ml in PBS containing 50 μg/ml RNase A). Cell cycle distribution was determined by FACS analysis using a FACS Calibur (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 min at 70°C and loaded on a NuPAGE 10% Bis-Tris polyacrylamide gel. After electrophoresis in NuPAGE SDS running buffer using the Xcell SureLock Mini-Cell, proteins were transferred to a PVDF 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-βactin 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. Five μm sections 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 min at 125°C. The sections were blocked for 30 min in TBS-T 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 IgG conjugated with a red fluorophore (Alexa Fluor 594; Molecular Probes, Eugene, OR) and goat anti-mouse IgG conjugated with a green fluorophore (Alexa Fluor 488; Molecular Probes, Eugene, OR) for 30 min 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.
Immunoprecipitation analysis
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 RIPA buffer containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 % Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 0.25% NP-40 and 0.5% sodium deoxycholate. The protein A–Sepharose-antibody-antigen complex was collected and washed three times with ice-cold RIPA buffer. The final pellet was resuspended with SDS-sample buffer and boiled for 5 min. 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
ChIP assay was performed as described previously.14 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 min at 37°C. Cells were then harvested and sonicated using the Misonix Sonicator 3000 (Misonix, Farmingdale, NY, USA). Immunoprecipitation was carried out in TE buffer containing protease inhibitors (Sigma, St. Louis, MO). Chromatin was incubated with protein A Dynabeads (Invitrogen, Carlsbad, CA), which were pre-incubated with STAT5A or IgG antibody (R&D Systems, Minneapolis, MN, USA). Immunoprecipitated DNA was eluted and amplified by real-time 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 (GAS sites) within the Socs2 and Cdkn2b genes were as following: For the Socs2 GAS sequence, forward primer 5’-GGAGGGCGGAGTCGCAGGC-3’, reverse primer 5’-GACTTGGCAAGAGTTAACCGTC-3’; the primer sets for Cdkn2b gene were: GAS1, forward 5’-GTTTTGCCGTGATGTCCTTG-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 (cDNA reverse transcription kit; Applied Biosystems, Foster City, CA). Real-time quantification of 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 GAPDH as a housekeeping gene. The primers used were: Cdkn2b, forward 5’-CCCTGCCACCCTTACCAGA-3’, reverse 5’-CAGATACCTCGCAATGTCACG-3’, yielding a 169 bp PCR product; Cdkn1a, forward 5’-GTGGCCTTGTCGCTGTCTT-3’, reverse 5’-GCGCTTGGAGTGATAGAAATCTG-3’, yielding a 126 bp PCR product; GAPDH, forward 5’-AACGACCCCTTCATTGAC-3’, reverse 5’ TCCACGACATACTCAGCAC-3’, yielding a 191bp PCR product.
Statistics
All statistical analyses were performed using the Student’s t test (2 tailed, unpaired). A P value of 0.05 or less was considered significant.
Increased proliferation rate of STAT5-deficient MEFs
Fibroblasts were isolated from control and Stat5−/− fetuses and their growth curves were established (Fig. 1). While Stat5−/− MEFs from passage 3 displayed a small, but significantly increased proliferation rate when compared to matched controls, the difference was more profound with cells from passage 8 (Fig. 1A) and later passages (Supporting Fig. 1). Notably, after 6 days in culture, the number of passage 8 Stat5−/−MEFs had increased 5-fold, while control MEFs had doubled in number. DNA content profiles demonstrated that the percentage of Stat5−/− MEFs in G0/G1 and S phases was decreased compared to controls, but percentages in the G2/M phase were increased (Fig. 1B).
Figure 1
Figure 1
Cell proliferation in primary Stat5+/+ and Stat5−/− MEFs. (A) Cell growth curve of primary MEFs. Cell viability was determined in Stat5+/+ and Stat5−/− MEFs at the 3rd and 8th passage. All values represent means ± (more ...)
To further explore the molecular basis of this accelerated growth, we performed global gene expression analyses of control and Stat5−/− MEFs (GEO accession number: GSE21861). In addition to the reduced expression of bona fide STAT5 target genes, such as Socs2, we observed a 2-fold and 29-fold reduced expression of Cdkn2b (p15INK4B) and Cdkn1a (p21CIP1), respectively (JAK-STAT Prospector at http://jak-stat.nih.gov). To establish a link between reduced expression of Cdkn2b and Cdkn1a in MEFs and their growth behavior, we analyzed p15INK4B and p21CIP1 mRNA and protein levels in MEFs at passages 3 and 8. While Cdkn2b mRNA levels were reduced in the absence of STAT5 by approximately 80% and 60% at passages 3 and 8, respectively (Fig. 1C), Cdkn1a mRNA levels were reduced by 75% and 50% (Fig. 1D). Western blot analyses confirmed the sharp reduction of p15INK4B and to a lesser extent of p21CIP1 (Fig. 2A). CDK4 levels were slightly increased in the Stat5−/− MEFs. Notably, levels of cyclin D1 and cyclin A, and in particular cyclin B1, were elevated in the absence of STAT5 (Fig. 2B). Absence of a STAT5 signal corroborated the genotype of the cells used. p15INK4B forms an inhibitory complex with CDK4 and immunoprecipitation analyses demonstrated that this complex was greatly reduced in the absence of STAT5 (Fig. 2C), which is in agreement with the reduced p15INK4B levels (Fig. 2A).
Figure 2
Figure 2
Expression of p15INK4B, p21CIP1, cyclin D1, cyclin A and cyclin B1 in primary MEFs. (A, B) Level of p15INK4B, p21CIP1, cyclin D1, cyclin A and cyclin B1 in Stat5+/+ and Stat5/MEFs at 3rd and 8rd passages. Expression of p15INK4B, p21 (more ...)
Expression of Cdkn2b is under STAT5 control in vitro
To determine whether expression of Cdkn2b is under direct GH/STAT5 regulation, control and Stat5−/− MEFs were stimulated with growth hormone (GH) for 4 hours and RNA was analyzed. While Cdkn2b expression was induced 2-fold in control MEFs, no induction was observed in experimental cells, which also exhibited lower baseline levels (Fig. 3A). These experiments demonstrate the STAT5 dependency of Cdkn2b expression. Moreover, we demonstrated that Cdkn1a expression was also under STAT5-dependent GH control (Fig. 3B) supporting and extending earlier findings.15
Figure 3
Figure 3
GH induced expression and STAT5 binding to conserved GAS sites in the Cdkn2b (p15INK4B) gene promoter. (A, B) mRNA expression of Cdkn2b and Cdkn1a in primary MEFs treated with GH. MEFs were treated with GH for 4 hours. Levels of Cdkn2b and Cdkn1a mRNA (more ...)
To determine whether STAT5 directly controls the Cdkn2b gene we scanned the promoter region for GAS motifs. Two conserved GAS motifs were identified at positions −2584, and −1976 (Fig. 3C). ChIP analyses in MEFs confirmed GH-induced STAT5 binding to all two sites (Fig. 3D). Binding to the Socs2 gene promoter served as a positive control. STAT5 binding was also detected at the Cdkn1a gene promoter (data not shown).
Restored growth and Cdkn2b expression upon reestablishing STAT5 expression
Impaired proliferation of Stat5−/− MEFs coincided with reduced expression of the Cdkn1a and Cdkn2b genes suggesting a causal relationship. To establish the link between STAT5A, Cdkn2b expression and cell proliferation kinetics, we reintroduced STAT5A into Stat5−/− MEFs. Stat5−/− MEFs that were complemented with retrovirally-expressed STAT5A displayed reduced cell proliferation as compared to parent Stat5−/− MEFs (Fig. 4A) and exhibited a growth kinetics reminiscent to wild type MEFs. Moreover, the GH-induced expression of Cdkn1a and Cdkn2b was reinstated (Fig. 4B,C) as was Socs2 expression (Fig. 4D). This was also reflected on the protein level (Fig. 4E). While p15INK4B and p21CIP1 levels did not change in Stat5−/− MEFs carrying an empty control retrovirus, ectopic expression of STAT5A resulted in higher levels (Fig. 4E).
Figure 4
Figure 4
Stat5−/− MEFs complemented with transgenic STAT5A results in suppressed cell proliferation. (A) Growth curves of Stat5−/− MEFs infected with an empty (control) retrovirus or with a STAT5A-expressing retrovirus. Cell viability (more ...)
STAT5-dependent regulation of Cdkn1a and Cdkn2b in liver tissue
We discovered earlier that hepatocyte-specific loss of STAT5 led to hepatosteatosis and HCC upon CCl4 treatment.7 H&E staining revealed that hepatosteatosis with accumulation of fat droplets progressed in liver-specific STAT5-null mice (Stat5f/f;Alb-Cre) in the presence of CCl4 (Supporting Fig. 2). To test whether the STAT5-regulated expression of Cdkn1a and Cdkn2b was also occurring in liver tissue, we analyzed the expression of Cdkn1a and Cdkn2b mRNA and the corresponding protein levels in control and liver-specific STAT5-null mice. At 2 months of age, p15INK4B levels were greatly reduced in experimental liver tissue (Fig. 5A) supporting the notion that STAT5 controls expression of this cell cycle regulator. Levels of p21CIP1 were marginally reduced. Like in MEFs, cyclin D1 levels were elevated in STAT5-null livers (Fig. 5A). Further evidence for this was derived from in vivo experiments in which control and experimental mice were injected with GH followed by mRNA analyses. While GH treatment of control mice induced Cdkn2b mRNA levels, no such increase was observed in experimental mice (Fig. 5B). p21CIP1 levels were only slightly reduced in experimental liver tissue. Actin served as a loading control and absence of STAT5 verified the efficient deletion in the liver tissue (Fig. 5A). Immunoprecipitation analyses demonstrated an absence of CDK4-p15INK4B complexes while CDK4-p21CIP1 complexes were reduced to a lesser extent (Fig. 5C).
Figure 5
Figure 5
Expression of p15INK4B and p21CIP1 in liver tissue from Stat5f/f and Stat5f/f;Alb-Cre mice. (A) Protein expression of p15INK4B, p21CIP1 and cyclin D1 was determined by western blotting. (B) mRNA expression of Cdkn2b in Stat5f/f and Stat5f/f;Alb-Cre mice (more ...)
Immunohistochemistry was used as an independent means to corroborate the dependence of p15INK4B levels on STAT5. Nuclear p15INK4B was observed in liver tissue of control mice and the levels increased after GH treatment (Fig. 6A,C). In contrast, low expression was observed in the liver-specific STAT5-null mice before and after GH injection (Fig. 6A,C). Strong nuclear STAT5 staining was observed after GH only in the control but not the experimental mice (Fig. 6B). Moreover, the number of nuclei positive for p15INK4b decreased in liver-specific STAT5-null mice (Fig. 6C). Nuclear p15INK4b was observed in liver tissue of control mice in the absence and presence of CCl4 (Fig. 7A,B). In contrast, low expression was observed in the liver-specific STAT5-null mice (Fig. 7A,B). There were no differences in p15INK4B levels in the absence or presence of CCl4 (Fig. 7A,B).
Figure 6
Figure 6
Immunostaining of p15INK4B and STAT5 in Stat5f/f and Stat5f/f;Alb-Cre mice injected with GH. (A) Livers from Stat5f/f and Stat5f/f;Alb-Cre mice were harvested after GH injection and analyzed for p15INK4B expression using immunofluorescence staining with (more ...)
Figure 7
Figure 7
Immunostaining of p15INK4B in Stat5f/f and Stat5f/f;Alb-Cre mice injected with CCl4. (A) Livers from Stat5f/f and Stat5f/f;Alb-Cre mice were harvested after CCl4 injection and analyzed for p15INK4b expression using immunofluorescence staining with anti-p15 (more ...)
CCl4 treatment results in increased cell proliferation
We have recently demonstrated that liver-specific STAT5-null mice develop hepatosteatosis and HCC upon treatment with CCl4.7 A second cohort of mice confirmed that approximately 25% of the CCl4 treated STAT5-null mice developed HCC, while treated control mice remained tumor free (Supporting Fig. 3). To establish whether loss of STAT5 and the sharply reduced levels of p15INK4B correlated with increased cell proliferation, we stained tissue sections for phosphorylated histone H3 as a measure of cell proliferation (Fig. 8A). The number of phospho-Histone H3 positive nuclei in experimental mice was three times higher than in control mice (Fig. 8B). Moreover, CCl4 treatment resulted in a further increase only in the absence of STAT5 (Fig. 8B). In support of this, the number of nuclei positive for PCNA was increased in liver-specific STAT5-null mice (Fig. 9A,B).
Figure 8
Figure 8
Immunostaining of phospho-Histone H3 in Stat5f/f and Stat5f/f;Alb-Cre mice injected with CCl4. (A) Livers from Stat5f/f and Stat5f/f;Alb-Cre mice were harvested after 12 weeks of CCl4 injection and analyzed for phospho-Histone H3 using immunofluorescence (more ...)
Figure 9
Figure 9
Immunostaining of PCNA in Stat5f/f and Stat5f/f;Alb-Cre mice injected with CCl4. (A) Livers from Stat5f/f and Stat5f/f;Alb-Cre mice were harvested after 12 weeks of CCl4 injection and analyzed for PCNA expression using immunofluorescence staining with (more ...)
STAT5’s contribution to cell proliferation and survival has been firmly established.1 For example, upon ablation of STAT5 in mice, mammary luminal epithelial cells fail to proliferate and form alveoli16 and cells from various hematopoietic lineages are absent.1719 Conditional ablation of STAT5 upon the establishment of leukemia results in a complete remission2 further suggesting a role of STAT5 in tumor maintenance. Loss of STAT5A also protects mice from oncogene-induced mammary tumorigenesis2022. Lastly, ectopic expression of a constitutively active STAT5A in mice, cells or transplanted bone marrow cell can induce myeloproliferative disorders3,5,23 and breast cancer.4 These experimental models are in agreement with the findings that many solid tumors and probably all leukemias display constitutively active STAT5. Although the complete set of STAT5 target genes that might control cell proliferation and survival remains elusive, genes encoding cyclin D1, bcl-xL and Akt have been shown to be under STAT5 control.24,25
This study for the first time establishes that STAT5 activates the cell cycle suppressor gene Cdkn2b, and thereby suppresses cell proliferation. This was not only observed in MEFs, but also in liver tissue. The ability of STAT5 to activate expression of the Cdkn2b gene is likely cell- and context-dependent. We have performed global gene expression analyses of several cell lineages and tissue in the presence and absence of STAT5 and a STAT5 regulated expression of the Cdkn2b gene was only observed in MEFs and liver tissue. Expression of the Cdkn2b gene is not only controlled by STAT5 but also by other transcription factors, including FoxO and Oct126,27 suggesting that the context specificity is the result of a combinatorial effect of several transcription factors. Importantly, re-expression of STAT5A in Stat5−/− MEFs led to the reactivation and GH-induced expression of the Cdkn2b and Cdkn1a genes and a concomitant reduced cell proliferation. While this study demonstrates for the first time that expression of the Cdkn2b gene is under the control of STAT5A, other studies have linked STATs to expression of the Cdkn1a gene.19,23,28
The sharp reduction of p15INK4B in STAT5-null liver tissue corresponds to increased phospho-Histone H3 and PCNA staining, indicating increased cell proliferation. Loss of STAT5 in liver tissue combined with a CCl4 insult results in liver tumors and we suggest that the reduction of appropriate p15INK4B levels contributes to these events. In support of this hypothesis, loss of p15INK4B, due to methylation of the Cdkn2b promoter has been detected in patients with hepatocellular carcinoma.
We propose that STAT5 exhibits two distinct inroads into the cell cycle, a generic one observed in many cancer cells and a cell-/context-specific one. Findings described in this study highlight context-specific contributions of STAT5 to cell cycle control, in particular the potential role of STAT5 as a brake. For example, in mammary tissue STAT5 has distinct roles depending on the particular developmental stage. While STAT5 controls progenitor populations during puberty, the presence during early stages of pregnancy is critical for cell proliferation.4,16,29 Upon the completion of the proliferation program, STAT5 is critical for the differentiation of mammary alveolar cells leading to the production of milk.30 Clearly in the context of mammary epithelium, cell proliferation and differentiation occur in different, albeit overlapping, time windows and STAT5 assumes different roles depending on the cell type and hormonal status. Similarly, STAT5 exerts several distinct and unique roles in hematopoietic cell lineages that point to STAT5 as modulators of cell proliferation and differentiation. Like in MEFs, STAT5 can also be a negative regulator of cell proliferation in hematopoietic stem cells (HSCs). A recent study by Wang and colleagues19 demonstrated increased cycling of STAT5-null HSC, followed by reduced survival and a depleted long-term HSC pool. Similar to our study, loss of STAT5 from HSCs also resulted in an increased pool of cells in the S/G2/M phase. While Cdkn1a levels were only slightly reduced in the absence of STAT5A, a more profound reduction of Cdkn1c was observed suggesting transcriptional regulation by STAT5. Notably, we have identified STAT5A binding to the Cdkn1c gene using ChIP-seq technology and expression of the Cdkn1c gene in MEFs was reduced by more than 90% in Stat5−/− MEFs (unpublished). Lastly, STAT5 also appear to contribute to and maintain different physiological states in the liver. Most obvious is the contribution of STAT5 to the differentiation of hepatocytes as measured by the GH-dependent expression of liver-specific genes.9 In hepatocytes the presence of STAT5 is also required to curtail GH-induced activation of STAT3, which in itself is a transforming stimulus.7,8 Finally, in hepatocytes STAT5 also activates the Cdkn2b gene whose product p15INK4B negatively controls the cell cycle. Experiments provided in this study therefore shed light onto yet another mechanism exploited by STAT5 to evoke unique cellular responses.
Table 1
Table 1
Expression of a selected set of genes in Stat5a/b+/+ and Stat5a/b/ MEFs
Supp Fig s1: Supporting figure 1
Cell proliferation in primary Stat5+/+ and Stat5−/− MEFs. (A) Cell growth curve of MEFs. Cell viability was determined in Stat5+/+ and Stat5−/− MEFs at the 13th and 16th passage. All values represent means ± SD from 3 independent experiments. **P < .01 compared with corresponding controls.
Supp Fig s2: Supporting figure 2
Histological analyses of liver tissue from Stat5f/f and Stat5f/f; Alb-Cre mice. (A) Hematoxylin and eosin (H&E) staining of liver sections from STAT5f/f and STAT5f/f;Alb-Cre mice. (B) H&E staining of liver sections from Stat5f/f and Stat5f/f; Alb-Cre mice injected with CCl4 for 12 weeks. In the absence of CCl4, hepatosteatosis was observed only in liver-specific STAT5-null mice (panel A). Exacerbated fat accumulation (arrows) was observed in liver-specific STAT5-null mice upon CCl4 treatment.
Supp Fig s3: Supporting figure 3
Development of HCC in CCl4-injected Stat5f/f; Alb-Cre mice. (A) Liver of Stat5f/f and Stat5f/f;Alb-Cre mice after 12 weeks of CCl4 injection. (B) Tumor multiplicity, (C) size and (D) incidence in livers of Stat5f/f;Alb-Cre mice that were given CCl4 for 12 weeks. All values represent means ± SD (n = 8–10).
Acknowledgments
This work was supported by the IRP of NIDDK.
1. Hennighausen L, Robinson GW. Interpretation of cytokine signaling through the transcription factors STAT5A and STAT5B. Genes Dev. 2008;22:711–721. [PMC free article] [PubMed]
2. Hoelbl A, Schuster C, Kovacic B, Zhu B, Wickre M, Hoelzl MA, Fajmann S, et al. Stat5 is indispensable for the maintenance of bcr/abl-positive leukaemia. EMBO Mol Med. 2:98–110. [PMC free article] [PubMed]
3. Moriggl R, Sexl V, Kenner L, Duntsch C, Stangl K, Gingras S, Hoffmeyer A, et al. Stat5 tetramer formation is associated with leukemogenesis. Cancer Cell. 2005;7:87–99. [PubMed]
4. Vafaizadeh V, Klemmt P, Brendel C, Weber K, Doebele C, Britt K, Grez M, et al. Mammary Epithelial Reconstitution with Gene-Modified Stem Cells Assigns Roles to Stat5 in Luminal Alveolar Cell Fate Decisions, Differentiation, Involution and Mammary Tumor Formation. Stem Cells [PubMed]
5. Kato Y, Iwama A, Tadokoro Y, Shimoda K, Minoguchi M, Akira S, Tanaka M, et al. Selective activation of STAT5 unveils its role in stem cell self-renewal in normal and leukemic hematopoiesis. J Exp Med. 2005;202:169–179. [PMC free article] [PubMed]
6. Zhang Q, Wang HY, Liu X, Wasik MA. STAT5A is epigenetically silenced by the tyrosine kinase NPM1-ALK and acts as a tumor suppressor by reciprocally inhibiting NPM1-ALK expression. Nat Med. 2007;13:1341–1348. [PubMed]
7. Hosui A, Kimura A, Yamaji D, Zhu BM, Na R, Hennighausen L. Loss of STAT5 causes liver fibrosis and cancer development through increased TGF-{beta} and STAT3 activation. J Exp Med. 2009;206:819–831. [PMC free article] [PubMed]
8. Cui Y, Hosui A, Sun R, Shen K, Gavrilova O, Chen W, Cam MC, et al. Loss of signal transducer and activator of transcription 5 leads to hepatosteatosis and impaired liver regeneration. Hepatology. 2007;46:504–513. [PubMed]
9. Holloway MG, Cui Y, Laz EV, Hosui A, Hennighausen L, Waxman DJ. Loss of sexually dimorphic liver gene expression upon hepatocyte-specific deletion of Stat5a-Stat5b locus. Endocrinology. 2007;148:1977–1986. [PMC free article] [PubMed]
10. Calabrese V, Mallette FA, Deschenes-Simard X, Ramanathan S, Gagnon J, Moores A, Ilangumaran S, et al. SOCS1 links cytokine signaling to p53 and senescence. Mol Cell. 2009;36:754–767. [PubMed]
11. Liang QC, Xiong H, Zhao ZW, Jia D, Li WX, Qin HZ, Deng JP, et al. Inhibition of transcription factor STAT5b suppresses proliferation, induces G1 cell cycle arrest and reduces tumor cell invasion in human glioblastoma multiforme cells. Cancer Lett. 2009;273:164–171. [PubMed]
12. Mallette FA, Gaumont-Leclerc MF, Ferbeyre G. The DNA damage signaling pathway is a critical mediator of oncogene-induced senescence. Genes Dev. 2007;21:43–48. [PubMed]
13. Xiong H, Su WY, Liang QC, Zhang ZG, Chen HM, Du W, Chen YX, et al. Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells. Lab Invest. 2009;89:717–725. [PubMed]
14. Zhu BM, McLaughlin SK, Na R, Liu J, Cui Y, Martin C, Kimura A, et al. Hematopoietic-specific Stat5-null mice display microcytic hypochromic anemia associated with reduced transferrin receptor gene expression. Blood. 2008;112:2071–2080. [PubMed]
15. Matsumura I, Ishikawa J, Nakajima K, Oritani K, Tomiyama Y, Miyagawa J, Kato T, et al. Thrombopoietin-induced differentiation of a human megakaryoblastic leukemia cell line, CMK, involves transcriptional activation of p21(WAF1/Cip1) by STAT5. Mol Cell Biol. 1997;17:2933–2943. [PMC free article] [PubMed]
16. Yamaji D, Na R, Feuermann Y, Pechhold S, Chen W, Robinson GW, Hennighausen L. Development of mammary luminal progenitor cells is controlled by the transcription factor STAT5A. Genes Dev. 2009;23:2382–2387. [PubMed]
17. Kimura A, Rieger MA, Simone JM, Chen W, Wickre MC, Zhu BM, Hoppe PS, et al. The transcription factors STAT5A/B regulate GM-CSF-mediated granulopoiesis. Blood. 2009;114:4721–4728. [PubMed]
18. Ohmori K, Luo Y, Jia Y, Nishida J, Wang Z, Bunting KD, Wang D, et al. IL-3 induces basophil expansion in vivo by directing granulocyte-monocyte progenitors to differentiate into basophil lineage-restricted progenitors in the bone marrow and by increasing the number of basophil/mast cell progenitors in the spleen. J Immunol. 2009;182:2835–2841. [PMC free article] [PubMed]
19. Wang Z, Li G, Tse W, Bunting KD. Conditional deletion of STAT5 in adult mouse hematopoietic stem cells causes loss of quiescence and permits efficient nonablative stem cell replacement. Blood. 2009;113:4856–4865. [PubMed]
20. Humphreys RC, Hennighausen L. Signal transducer and activator of transcription 5a influences mammary epithelial cell survival and tumorigenesis. Cell Growth Differ. 1999;10:685–694. [PubMed]
21. Humphreys RC, Hennighausen L. Transforming growth factor alpha and mouse models of human breast cancer. Oncogene. 2000;19:1085–1091. [PubMed]
22. Miermont AM, Parrish AR, Furth PA. Role of ER{alpha} in the differential response of Stat5a loss in susceptibility to mammary preneoplasia and DMBA-induced carcinogenesis. Carcinogenesis [PMC free article] [PubMed]
23. Wierenga AT, Vellenga E, Schuringa JJ. Maximal STAT5-induced proliferation and self-renewal at intermediate STAT5 activity levels. Mol Cell Biol. 2008;28:6668–6680. [PMC free article] [PubMed]
24. Brockman JL, Schroeder MD, Schuler LA. PRL activates the cyclin D1 promoter via the Jak2/Stat pathway. Mol Endocrinol. 2002;16:774–784. [PubMed]
25. Creamer BA, Sakamoto K, Schmidt JW, Triplett AA, Moriggl R, Wagner KU. Stat5 promotes survival of mammary epithelial cells through transcriptional activation of a distinct promoter in Akt1. Mol Cell Biol [PMC free article] [PubMed]
26. Katayama K, Nakamura A, Sugimoto Y, Tsuruo T, Fujita N. FOXO transcription factor-dependent p15(INK4b) and p19(INK4d) expression. Oncogene. 2008;27:1677–1686. [PubMed]
27. Hitomi T, Matsuzaki Y, Yasuda S, Kawanaka M, Yogosawa S, Koyama M, Tantin D, et al. Oct-1 is involved in the transcriptional repression of the p15(INK4b) gene. FEBS Lett. 2007;581:1087–1092. [PubMed]
28. Olthof SG, Fatrai S, Drayer AL, Tyl MR, Vellenga E, Schuringa JJ. Downregulation of signal transducer and activator of transcription 5 (STAT5) in CD34+ cells promotes megakaryocytic development, whereas activation of STAT5 drives erythropoiesis. Stem Cells. 2008;26:1732–1742. [PubMed]
29. Cui Y, Riedlinger G, Miyoshi K, Tang W, Li C, Deng CX, Robinson GW, et al. Inactivation of Stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation. Mol Cell Biol. 2004;24:8037–8047. [PMC free article] [PubMed]
30. Hennighausen L, Robinson GW. Information networks in the mammary gland. Nat Rev Mol Cell Biol. 2005;6:715–725. [PubMed]