Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4
http://www.100md.com
《血液学杂志》
the Departments of Pediatrics, Medical and Molecular Genetics, Biochemistry and Molecular Biology
Microbiology and Immunology, Herman B Wells Center for Pediatric Research
Center for Medical Genomics, Indiana University School of Medicine, Indianapolis, IN.
Abstract
Embryonic stem (ES) cells homozygous for a Shp-2 mutation (Shp-246-110) demonstrate leukemia inhibitory factor (LIF) hypersensitivity and increased LIF-stimulated phosphorylation of signal transducer and activator of transcription (STAT3). We hypothesized that LIF-responsive genes in Shp-246-110 cells would represent potential candidates for molecules vital for ES cell self-renewal. Using microarray analysis, we detected 41 genes whose expression was modified by LIF in Shp-246-110 ES cells. Induction of 2 significantly up-regulated genes, suppressor of cytokine signaling–3 (SOCS-3) and Krüppel-like factor 4 (Klf4), was verified using Northern blotting. ES cells overexpressing SOCS-3 had an increased capacity to differentiate to hematopoietic progenitors, rather than to self-renew. In contrast, ES cells overexpressing Klf4 had a greater capacity to self-renew based on secondary embryoid body (EB) formation. Klf4-transduced d6 EBs expressed higher levels of Oct-4, consistent with the notion that Klf4 promotes ES cell self-renewal. These findings verify the negative role of SOCS-3 on LIF signaling and provide a novel role for Klf4 in ES cell function.
Introduction
The mechanisms that control self-renewal of somatic and embryonic stem (ES) cells are unknown; however, identification of regulating molecules is essential for developing stem cell-based therapeutic strategies. Leukemia inhibitory factor (LIF) promotes murine ES cell self-renewal and maintains pluripotency in culture.1 LIF binds the heterodimeric LIF receptor-glycoprotein 130 (gp130) complex and activates the Jak kinases with recruitment of Shp-2 and signal transducer and activator of transcription (STAT3).2 Mutation of STAT3-interacting tyrosines on gp130 reduces LIF-stimulated phospho-STAT3 and STAT3-DNA–binding activity as well as ES cell self-renewal, providing strong evidence that LIF-stimulated STAT3 activation is critical for ES cell self-renewal.3,4 Mutation of the Shp-2–binding tyrosine on gp130 prolongs STAT3 activation and increases self-renewal, whereas it diminishes Erk activation, indicating that Shp-2 functions to balance LIF-stimulated STAT3 and Erk activation, thus modulating ES cell self-renewal and differentiation, respectively.5
Shp-246-110 ES cells, which are homozygous for an exon 3 deletion from the murine SHP-2 locus,6 have increased sensitivity to LIF and increased self-renewal capacity as assayed by secondary embryoid body (EB) formation, both of which are normalized upon reintroduction of wild-type (WT) Shp-2.7,8 Consistently, Shp-246-110 ES cells manifest increased LIF-stimulated phospho-STAT3 and decreased LIF-stimulated phospho-Erk activity.7
Because LIF-stimulated STAT3 is crucial for ES cell self-renewal, we hypothesized that LIF-induced genes would be fundamental in ES cell self-renewal. Additionally, because LIF-induced phospho-STAT3 levels are higher in Shp-246-110 cells than in WT cells,7 we reasoned that LIF stimulation of Shp-246-110 cells would provide a sensitive system for the detection of STAT3-regulated genes. Therefore, we identified LIF-regulated genes by comparing expression profiles of Shp-246-110 ES cells either unstimulated or stimulated with LIF. The cDNAs for 2 LIF-stimulated genes, which encode suppressor of cytokine signaling-3 (SOCS-3) and the transcription factor, Krüppel-like factor 4 (Klf4), were introduced into WT ES cells for functional analysis.
Study design
ES cell culture and microarray analysis
The Shp-246-110 cell line, IC3, was generated as described.7 IC3 cells were cultured in serum-free, LIF-free media for 6 hours followed by treatment with phosphate-buffered saline (PBS) or LIF (1000 U/mL) for 45 minutes. Total RNA was extracted using QIAamp RNA Mini kit (Qiagen, Valencia, CA) and submitted to the Center for Medical Genomics at the Indiana University School of Medicine for labeling and hybridization to Affymetrix Murine Genome U74A GeneChips (Affymetrix, Santa Clara, CA). LIF stimulation, RNA preparation, and microarray analysis were performed in triplicate and analyzed with MAS5 software (Affymetrix, Santa Clara, CA). The t tests using the signal were performed using CyberT9 for genes present in at least one half of the arrays for either condition.10 Confirmatory Northern blots were conducted as described.11
Retroviral plasmids, ES cell transduction, and in vitro differentiation
The SOCS-3 cDNA and the Klf4 cDNA (from Dr Vincent Yang, Emory University School of Medicine, Atlanta, GA) were epitope-tagged with FLAG at the 5' end, subcloned into the retroviral vector, pMIEG3, in tandem with enhanced green fluorescent protein (EGFP), and confirmed by sequencing. Retroviral supernatants were prepared using Eco-Phoenix packaging cells by the Indiana University Vector Production Facility.
R1 (WT) ES cells were transduced using polybrene (40 mg/mL) over 48 hours. EGFP+ cells were collected using fluorescence-activated cell sorting and subjected to hematopoietic in vitro differentiation assays, as described.7,12 Transduction, sorting, and in vitro differentiation assays were conducted in 2 independent experiments.
Immunoprecipitation and immunoblotting
Proteins from EGFP+ cells were immunoprecipitated as described13 and probed with anti–SOCS-3, anti-Klf4 (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-FLAG (Sigma, St Louis, MO). For Oct-4 detection, 50 μg protein extract was used and probed with anti–Oct-4 (Santa Cruz Biotechnology) or with anti–-actin to control for protein loading. Signals were detected by enhanced chemiluminescence and quantitated using ImageJ (National Institutes of Health, Bethesda, MD).
Results and discussion
To define an appropriate time to evaluate LIF-stimulated gene expression, we determined that c-fos mRNA induction, known to be regulated by LIF-induced STAT3 activation,14,15 was maximal following LIF treatment for 45 minutes (not shown). Total cellular RNA from Shp-246-110 ES cells treated with PBS or LIF 1000 U/mL for 45 minutes was submitted for microarray analysis, which revealed that 41 genes or expressed sequence tags were significantly modified (P < .01, determined by t test on the signal value, Tables S1 and S2; see the Supplemental Tables link at the top of the online article link at the top of the online article on the Blood website). The 2 most significantly up-regulated genes were SOCS3 (increase 34.4-fold; P < .000001) and KLF4 (increase 2.1-fold; P = .000057). Using Northern blot, we examined the effect of LIF on SOCS3 and KLF4 expression in both WT (R1) and Shp-246-110 cells. In the Shp-246-110 cells, SOCS3 expression was increased dramatically and KLF4 expression was increased approximately 2-fold, in agreement with the microarray results (Figure 1A). SOCS3 was similarly induced in the WT cells. However, at baseline, KLF4 expression was elevated in the WT ES cells compared to the Shp-246-110 cells and was not induced by LIF treatment (Figure 1A).
To examine SOCS-3 and Klf4 function within ES cells, each cDNA was introduced into R1 ES cells using retroviral transduction (Figure 1B). Overexpression of SOCS-3 promoted ES cell differentiation to both hemangioblasts and primitive erythroid progenitors, whereas overexpression of Klf4 demonstrated a trend toward reduced differentiation (Figure 1C). Klf4-transduced ES cells retained a high capacity to generate secondary EBs, suggesting that Klf4 promotes ES cell self-renewal in addition to inhibiting ES cell differentiation (Figure 1C). These data support previous studies indicating that SOCS-3 acts as a negative regulator of LIF signaling15-17 and provide a novel role for Klf4 in ES cell differentiation and self-renewal.
Based on the differences in ES cell function induced by SOCS-3 or Klf4 overexpression, we determined the exogenously introduced and total SOCS-3 and Klf4 protein levels achieved in the transduced ES cells. Immunoblotting with anti-FLAG demonstrated that the exogenously introduced SOCS-3 and Klf4 were expressed in the transduced ES cells (Figure 2A). Densitometric measurements demonstrated a 3-fold increase in total SOCS-3 protein and a 6-fold increase in total Klf4 protein compared to cells transduced with vector alone (Figure 2A).
Because the secondary EB assay suggested that Klf4 promotes ES cell self-renewal, we examined the level of Oct-4 protein, which is essential for ES cell self-renewal,18,19 in day-6 EBs. We observed that Oct-4 was significantly elevated (7-fold) in the Klf4-transduced d6 EBs compared to that of vector alone or SOCS-3–transduced d6 EBs (Figure 2B). These results support a role for Klf4 in modulating ES cell self-renewal.
This study has defined a battery of LIF-sensitive genes within murine ES cells that provide potential mediators of ES cell self-renewal and differentiation. Functional evaluation demonstrated a novel role for Klf4 in ES cell differentiation and self-renewal. Klf4 is abundant in epithelial cells of the intestine, colon, skin, and thymus20 and has been implicated as a negative prognosticator in breast cancer21,22 and as a tumor suppressor gene in gastrointestinal malignancies.23,24 Based on the differential effect of LIF-stimulated KLF4 expression in the WT and Shp-246-110 cells, it is possible that altered Klf4 expression partially accounts for the increased self-renewal phenotype in the Shp-246-110 cells. In contrast, we observed that SOCS-3 promotes ES cell differentiation, likely by diminishing the self-renewing effects of LIF. Our findings support the conclusions of others that SOCS-3 is regulated at the transcriptional level by LIF17,25 and functions as a feedback inhibitor on LIF signaling.16,17 These data provide feasible candidate genes for ES cell self-renewal, support a negative role of SOCS-3 on LIF signaling in ES cells, and present a novel role for Klf4 in ES cell function.
Acknowledgements
The authors wish to thank Chris Starkey at the Indiana University Vector Production Facility, Susan Rice and Denessa Luckett at the Indiana University Flow Cytometry Facility, Arliene K. Britt for assistance in manuscript preparation, and Edward Chan for critical review of the manuscript.
Footnotes
Prepublished online as Blood First Edition Paper, September 9, 2004; DOI 10.1182/blood-2004-07-2681.
Supported by F32CA84677 (R.J.C.), RO1HL63169 (M.C.Y.), the Indiana 21st Century Research and Technology Fund (M.C.Y, H.J.E.), and the Indiana Genomics Initiative (M.C.Y., H.J.E.). RJC is a recipient of a March of Dimes Basil O'Connor Starter Scholar Award (5-FY03-136).
The online version of the article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
References
Smith AG, Heath JK, Donaldson DD, et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. 1988;336: 688-690.
Burdon T, Smith A, Savatier P. Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol. 2002;12: 432-438.
Niwa H, Burdon T, Chambers I, Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 1998; 12: 2048-2060.
Matsuda T, Nakamura T, Nakao K, et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J. 1999;18: 4261-4269.
Burdon T, Stracey C, Chambers I, Nichols J, Smith A. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev Biol. 1999;210: 30-43.
Saxton TM, Henkemeyer M, Gasca S, et al. Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp-2. EMBO J. 1997;16: 2352-2364.
Chan RJ, Johnson SA, Li Y, Yoder MC, Feng GS. A definitive role of Shp-2 tyrosine phosphatase in mediating embryonic stem cell differentiation and hematopoiesis. Blood. 2003;102: 2074-2080.
Qu CK, Feng GS. Shp-2 has a positive regulatory role in ES cell differentiation and proliferation. Oncogene. 1998;17: 433-439.
Long AD, Mangalam HJ, Chan BY, Tolleri L, Hatfield GW, Baldi P. Improved statistical inference from DNA microarray data using analysis of variance and a Bayesian statistical framework. Analysis of global gene expression in Escherichia coli K12. J Biol Chem. 2001;276: 19937-19944.
McClintick JN, Jerome RE, Nicholson CR, Crabb DW, Edenberg HJ. Reproducibility of oligonucleotide arrays using small samples. BMC Genomics. 2003;4: 4.
Zhong L, Qing K, Si Y, Chen L, Tan M, Srivastava A. Heat-shock treatment-mediated increase in transduction by recombinant adeno-associated virus 2 vectors is independent of the cellular heat-shock protein 90. J Biol Chem. 2004;279: 12714-12723.
Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G. A common precursor for hematopoietic and endothelial cells. Development. 1998; 125: 725-732.
Shi ZQ, Yu DH, Park M, Marshall M, Feng GS. Molecular mechanism for the Shp-2 tyrosine phosphatase function in promoting growth factor stimulation of Erk activity. Mol Cell Biol. 2000;20: 1526-1536.
Boeuf H, Hauss C, Graeve FD, Baran N, Kedinger C. Leukemia inhibitory factor-dependent transcriptional activation in embryonic stem cells. J Cell Biol. 1997;138: 1207-1217.
Duval D, Reinhardt B, Kedinger C, Boeuf H. Role of suppressors of cytokine signaling (Socs) in leukemia inhibitory factor (LIF)-dependent embryonic stem cell survival. FASEB J. 2000;14: 1577-1584.
Nicholson SE, Willson TA, Farley A, et al. Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction. EMBO J. 1999;18: 375-385.
Krebs DL, Hilton DJ. SOCS proteins: negative regulators of cytokine signaling. Stem Cells. 2001;19: 378-387.
Nichols J, Zevnik B, Anastassiadis K, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell. 1998;95: 379-391.
Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet. 2000;24: 372-376.
Shields JM, Christy RJ, Yang VW. Identification and characterization of a gene encoding a gut-enriched Kruppel-like factor expressed during growth arrest. J Biol Chem. 1996;271: 20009-20017.
Foster KW, Frost AR, McKie-Bell P, et al. Increase of GKLF messenger RNA and protein expression during progression of breast cancer. Cancer Res. 2000;60: 6488-6495.
Pandya AY, Talley LI, Frost AR, et al. Nuclear localization of KLF4 is associated with an aggressive phenotype in early-stage breast cancer. Clin Cancer Res. 2004;10: 2709-2719.
Dang DT, Chen X, Feng J, Torbenson M, Dang LH, Yang VW. Overexpression of Kruppel-like factor 4 in the human colon cancer cell line RKO leads to reduced tumorigenicity. Oncogene. 2003; 22: 3424-3430.
Zhao W, Hisamuddin IM, Nandan MO, Babbin BA, Lamb NE, Yang VW. Identification of Kruppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer. Oncogene. 2004;23: 395-402.
Auernhammer CJ, Bousquet C, Melmed S. Auto-regulation of pituitary corticotroph SOCS-3 expression: characterization of the murine SOCS-3 promoter. Proc Natl Acad Sci U S A. 1999;96: 6964-6969.(Yanjun Li, Jeanette McCli)
Microbiology and Immunology, Herman B Wells Center for Pediatric Research
Center for Medical Genomics, Indiana University School of Medicine, Indianapolis, IN.
Abstract
Embryonic stem (ES) cells homozygous for a Shp-2 mutation (Shp-246-110) demonstrate leukemia inhibitory factor (LIF) hypersensitivity and increased LIF-stimulated phosphorylation of signal transducer and activator of transcription (STAT3). We hypothesized that LIF-responsive genes in Shp-246-110 cells would represent potential candidates for molecules vital for ES cell self-renewal. Using microarray analysis, we detected 41 genes whose expression was modified by LIF in Shp-246-110 ES cells. Induction of 2 significantly up-regulated genes, suppressor of cytokine signaling–3 (SOCS-3) and Krüppel-like factor 4 (Klf4), was verified using Northern blotting. ES cells overexpressing SOCS-3 had an increased capacity to differentiate to hematopoietic progenitors, rather than to self-renew. In contrast, ES cells overexpressing Klf4 had a greater capacity to self-renew based on secondary embryoid body (EB) formation. Klf4-transduced d6 EBs expressed higher levels of Oct-4, consistent with the notion that Klf4 promotes ES cell self-renewal. These findings verify the negative role of SOCS-3 on LIF signaling and provide a novel role for Klf4 in ES cell function.
Introduction
The mechanisms that control self-renewal of somatic and embryonic stem (ES) cells are unknown; however, identification of regulating molecules is essential for developing stem cell-based therapeutic strategies. Leukemia inhibitory factor (LIF) promotes murine ES cell self-renewal and maintains pluripotency in culture.1 LIF binds the heterodimeric LIF receptor-glycoprotein 130 (gp130) complex and activates the Jak kinases with recruitment of Shp-2 and signal transducer and activator of transcription (STAT3).2 Mutation of STAT3-interacting tyrosines on gp130 reduces LIF-stimulated phospho-STAT3 and STAT3-DNA–binding activity as well as ES cell self-renewal, providing strong evidence that LIF-stimulated STAT3 activation is critical for ES cell self-renewal.3,4 Mutation of the Shp-2–binding tyrosine on gp130 prolongs STAT3 activation and increases self-renewal, whereas it diminishes Erk activation, indicating that Shp-2 functions to balance LIF-stimulated STAT3 and Erk activation, thus modulating ES cell self-renewal and differentiation, respectively.5
Shp-246-110 ES cells, which are homozygous for an exon 3 deletion from the murine SHP-2 locus,6 have increased sensitivity to LIF and increased self-renewal capacity as assayed by secondary embryoid body (EB) formation, both of which are normalized upon reintroduction of wild-type (WT) Shp-2.7,8 Consistently, Shp-246-110 ES cells manifest increased LIF-stimulated phospho-STAT3 and decreased LIF-stimulated phospho-Erk activity.7
Because LIF-stimulated STAT3 is crucial for ES cell self-renewal, we hypothesized that LIF-induced genes would be fundamental in ES cell self-renewal. Additionally, because LIF-induced phospho-STAT3 levels are higher in Shp-246-110 cells than in WT cells,7 we reasoned that LIF stimulation of Shp-246-110 cells would provide a sensitive system for the detection of STAT3-regulated genes. Therefore, we identified LIF-regulated genes by comparing expression profiles of Shp-246-110 ES cells either unstimulated or stimulated with LIF. The cDNAs for 2 LIF-stimulated genes, which encode suppressor of cytokine signaling-3 (SOCS-3) and the transcription factor, Krüppel-like factor 4 (Klf4), were introduced into WT ES cells for functional analysis.
Study design
ES cell culture and microarray analysis
The Shp-246-110 cell line, IC3, was generated as described.7 IC3 cells were cultured in serum-free, LIF-free media for 6 hours followed by treatment with phosphate-buffered saline (PBS) or LIF (1000 U/mL) for 45 minutes. Total RNA was extracted using QIAamp RNA Mini kit (Qiagen, Valencia, CA) and submitted to the Center for Medical Genomics at the Indiana University School of Medicine for labeling and hybridization to Affymetrix Murine Genome U74A GeneChips (Affymetrix, Santa Clara, CA). LIF stimulation, RNA preparation, and microarray analysis were performed in triplicate and analyzed with MAS5 software (Affymetrix, Santa Clara, CA). The t tests using the signal were performed using CyberT9 for genes present in at least one half of the arrays for either condition.10 Confirmatory Northern blots were conducted as described.11
Retroviral plasmids, ES cell transduction, and in vitro differentiation
The SOCS-3 cDNA and the Klf4 cDNA (from Dr Vincent Yang, Emory University School of Medicine, Atlanta, GA) were epitope-tagged with FLAG at the 5' end, subcloned into the retroviral vector, pMIEG3, in tandem with enhanced green fluorescent protein (EGFP), and confirmed by sequencing. Retroviral supernatants were prepared using Eco-Phoenix packaging cells by the Indiana University Vector Production Facility.
R1 (WT) ES cells were transduced using polybrene (40 mg/mL) over 48 hours. EGFP+ cells were collected using fluorescence-activated cell sorting and subjected to hematopoietic in vitro differentiation assays, as described.7,12 Transduction, sorting, and in vitro differentiation assays were conducted in 2 independent experiments.
Immunoprecipitation and immunoblotting
Proteins from EGFP+ cells were immunoprecipitated as described13 and probed with anti–SOCS-3, anti-Klf4 (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-FLAG (Sigma, St Louis, MO). For Oct-4 detection, 50 μg protein extract was used and probed with anti–Oct-4 (Santa Cruz Biotechnology) or with anti–-actin to control for protein loading. Signals were detected by enhanced chemiluminescence and quantitated using ImageJ (National Institutes of Health, Bethesda, MD).
Results and discussion
To define an appropriate time to evaluate LIF-stimulated gene expression, we determined that c-fos mRNA induction, known to be regulated by LIF-induced STAT3 activation,14,15 was maximal following LIF treatment for 45 minutes (not shown). Total cellular RNA from Shp-246-110 ES cells treated with PBS or LIF 1000 U/mL for 45 minutes was submitted for microarray analysis, which revealed that 41 genes or expressed sequence tags were significantly modified (P < .01, determined by t test on the signal value, Tables S1 and S2; see the Supplemental Tables link at the top of the online article link at the top of the online article on the Blood website). The 2 most significantly up-regulated genes were SOCS3 (increase 34.4-fold; P < .000001) and KLF4 (increase 2.1-fold; P = .000057). Using Northern blot, we examined the effect of LIF on SOCS3 and KLF4 expression in both WT (R1) and Shp-246-110 cells. In the Shp-246-110 cells, SOCS3 expression was increased dramatically and KLF4 expression was increased approximately 2-fold, in agreement with the microarray results (Figure 1A). SOCS3 was similarly induced in the WT cells. However, at baseline, KLF4 expression was elevated in the WT ES cells compared to the Shp-246-110 cells and was not induced by LIF treatment (Figure 1A).
To examine SOCS-3 and Klf4 function within ES cells, each cDNA was introduced into R1 ES cells using retroviral transduction (Figure 1B). Overexpression of SOCS-3 promoted ES cell differentiation to both hemangioblasts and primitive erythroid progenitors, whereas overexpression of Klf4 demonstrated a trend toward reduced differentiation (Figure 1C). Klf4-transduced ES cells retained a high capacity to generate secondary EBs, suggesting that Klf4 promotes ES cell self-renewal in addition to inhibiting ES cell differentiation (Figure 1C). These data support previous studies indicating that SOCS-3 acts as a negative regulator of LIF signaling15-17 and provide a novel role for Klf4 in ES cell differentiation and self-renewal.
Based on the differences in ES cell function induced by SOCS-3 or Klf4 overexpression, we determined the exogenously introduced and total SOCS-3 and Klf4 protein levels achieved in the transduced ES cells. Immunoblotting with anti-FLAG demonstrated that the exogenously introduced SOCS-3 and Klf4 were expressed in the transduced ES cells (Figure 2A). Densitometric measurements demonstrated a 3-fold increase in total SOCS-3 protein and a 6-fold increase in total Klf4 protein compared to cells transduced with vector alone (Figure 2A).
Because the secondary EB assay suggested that Klf4 promotes ES cell self-renewal, we examined the level of Oct-4 protein, which is essential for ES cell self-renewal,18,19 in day-6 EBs. We observed that Oct-4 was significantly elevated (7-fold) in the Klf4-transduced d6 EBs compared to that of vector alone or SOCS-3–transduced d6 EBs (Figure 2B). These results support a role for Klf4 in modulating ES cell self-renewal.
This study has defined a battery of LIF-sensitive genes within murine ES cells that provide potential mediators of ES cell self-renewal and differentiation. Functional evaluation demonstrated a novel role for Klf4 in ES cell differentiation and self-renewal. Klf4 is abundant in epithelial cells of the intestine, colon, skin, and thymus20 and has been implicated as a negative prognosticator in breast cancer21,22 and as a tumor suppressor gene in gastrointestinal malignancies.23,24 Based on the differential effect of LIF-stimulated KLF4 expression in the WT and Shp-246-110 cells, it is possible that altered Klf4 expression partially accounts for the increased self-renewal phenotype in the Shp-246-110 cells. In contrast, we observed that SOCS-3 promotes ES cell differentiation, likely by diminishing the self-renewing effects of LIF. Our findings support the conclusions of others that SOCS-3 is regulated at the transcriptional level by LIF17,25 and functions as a feedback inhibitor on LIF signaling.16,17 These data provide feasible candidate genes for ES cell self-renewal, support a negative role of SOCS-3 on LIF signaling in ES cells, and present a novel role for Klf4 in ES cell function.
Acknowledgements
The authors wish to thank Chris Starkey at the Indiana University Vector Production Facility, Susan Rice and Denessa Luckett at the Indiana University Flow Cytometry Facility, Arliene K. Britt for assistance in manuscript preparation, and Edward Chan for critical review of the manuscript.
Footnotes
Prepublished online as Blood First Edition Paper, September 9, 2004; DOI 10.1182/blood-2004-07-2681.
Supported by F32CA84677 (R.J.C.), RO1HL63169 (M.C.Y.), the Indiana 21st Century Research and Technology Fund (M.C.Y, H.J.E.), and the Indiana Genomics Initiative (M.C.Y., H.J.E.). RJC is a recipient of a March of Dimes Basil O'Connor Starter Scholar Award (5-FY03-136).
The online version of the article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
References
Smith AG, Heath JK, Donaldson DD, et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. 1988;336: 688-690.
Burdon T, Smith A, Savatier P. Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol. 2002;12: 432-438.
Niwa H, Burdon T, Chambers I, Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 1998; 12: 2048-2060.
Matsuda T, Nakamura T, Nakao K, et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J. 1999;18: 4261-4269.
Burdon T, Stracey C, Chambers I, Nichols J, Smith A. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev Biol. 1999;210: 30-43.
Saxton TM, Henkemeyer M, Gasca S, et al. Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp-2. EMBO J. 1997;16: 2352-2364.
Chan RJ, Johnson SA, Li Y, Yoder MC, Feng GS. A definitive role of Shp-2 tyrosine phosphatase in mediating embryonic stem cell differentiation and hematopoiesis. Blood. 2003;102: 2074-2080.
Qu CK, Feng GS. Shp-2 has a positive regulatory role in ES cell differentiation and proliferation. Oncogene. 1998;17: 433-439.
Long AD, Mangalam HJ, Chan BY, Tolleri L, Hatfield GW, Baldi P. Improved statistical inference from DNA microarray data using analysis of variance and a Bayesian statistical framework. Analysis of global gene expression in Escherichia coli K12. J Biol Chem. 2001;276: 19937-19944.
McClintick JN, Jerome RE, Nicholson CR, Crabb DW, Edenberg HJ. Reproducibility of oligonucleotide arrays using small samples. BMC Genomics. 2003;4: 4.
Zhong L, Qing K, Si Y, Chen L, Tan M, Srivastava A. Heat-shock treatment-mediated increase in transduction by recombinant adeno-associated virus 2 vectors is independent of the cellular heat-shock protein 90. J Biol Chem. 2004;279: 12714-12723.
Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G. A common precursor for hematopoietic and endothelial cells. Development. 1998; 125: 725-732.
Shi ZQ, Yu DH, Park M, Marshall M, Feng GS. Molecular mechanism for the Shp-2 tyrosine phosphatase function in promoting growth factor stimulation of Erk activity. Mol Cell Biol. 2000;20: 1526-1536.
Boeuf H, Hauss C, Graeve FD, Baran N, Kedinger C. Leukemia inhibitory factor-dependent transcriptional activation in embryonic stem cells. J Cell Biol. 1997;138: 1207-1217.
Duval D, Reinhardt B, Kedinger C, Boeuf H. Role of suppressors of cytokine signaling (Socs) in leukemia inhibitory factor (LIF)-dependent embryonic stem cell survival. FASEB J. 2000;14: 1577-1584.
Nicholson SE, Willson TA, Farley A, et al. Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction. EMBO J. 1999;18: 375-385.
Krebs DL, Hilton DJ. SOCS proteins: negative regulators of cytokine signaling. Stem Cells. 2001;19: 378-387.
Nichols J, Zevnik B, Anastassiadis K, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell. 1998;95: 379-391.
Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet. 2000;24: 372-376.
Shields JM, Christy RJ, Yang VW. Identification and characterization of a gene encoding a gut-enriched Kruppel-like factor expressed during growth arrest. J Biol Chem. 1996;271: 20009-20017.
Foster KW, Frost AR, McKie-Bell P, et al. Increase of GKLF messenger RNA and protein expression during progression of breast cancer. Cancer Res. 2000;60: 6488-6495.
Pandya AY, Talley LI, Frost AR, et al. Nuclear localization of KLF4 is associated with an aggressive phenotype in early-stage breast cancer. Clin Cancer Res. 2004;10: 2709-2719.
Dang DT, Chen X, Feng J, Torbenson M, Dang LH, Yang VW. Overexpression of Kruppel-like factor 4 in the human colon cancer cell line RKO leads to reduced tumorigenicity. Oncogene. 2003; 22: 3424-3430.
Zhao W, Hisamuddin IM, Nandan MO, Babbin BA, Lamb NE, Yang VW. Identification of Kruppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer. Oncogene. 2004;23: 395-402.
Auernhammer CJ, Bousquet C, Melmed S. Auto-regulation of pituitary corticotroph SOCS-3 expression: characterization of the murine SOCS-3 promoter. Proc Natl Acad Sci U S A. 1999;96: 6964-6969.(Yanjun Li, Jeanette McCli)