Maximal STAT5-induced proliferation and self-renewal at intermediate STAT5 activity levels

Abstract

The level of transcription factor activity critically regulates cell fate decisions, such as hematopoietic stem cell (HSC) self-renewal and differentiation. We introduced STAT5A transcriptional activity into human HSCs/progenitor cells in a dose-dependent manner by overexpression of a tamoxifen-inducible STAT5A(1*6)-estrogen receptor fusion protein. Induction of STAT5A activity in CD34(+) cells resulted in impaired myelopoiesis and induction of erythropoiesis, which was most pronounced at the highest STAT5A transactivation levels. In contrast, intermediate STAT5A activity levels resulted in the most pronounced proliferative advantage of CD34(+) cells. This coincided with increased cobblestone area-forming cell and long-term-culture-initiating cell frequencies, which were predominantly elevated at intermediate STAT5A activity levels but not at high STAT5A levels. Self-renewal of progenitors was addressed by serial replating of CFU, and only progenitors containing intermediate STAT5A activity levels contained self-renewal capacity. By extensive gene expression profiling we could identify gene expression patterns of STAT5 target genes that predominantly associated with a self-renewal and long-term expansion phenotype versus those that identified a predominant differentiation phenotype.

FIG. 1.

Dose-dependent induction of STAT5 activity using 4-OHT-inducible STAT5A(1*6)-ER fusion proteins. (A) Schematic representation of the retroviral vectors used in this study. (B) Western blot of nuclear extracts of KG1a cells transduced with MiNR1 control or STAT5A(1*6)-ER vectors. Cells were stimulated with 4-OHT in concentrations from 0 to 500 nM for 24 h, and lysates were probed using antibodies against STAT5 and tyrosine 694-phosphorylated STAT5. (C) EMSA using nuclear extracts of MinR1 and STAT5A(1*6)-ER-transduced KG1a cells using a STAT5-specific probe from the beta-casein promoter. Cells were stimulated with 500 nM 4-OHT for 24 h or left unstimulated, and nuclear extracts were prepared. Supershift experiments were performed using antibodies against STAT5. (D) EMSA experiment as described for panel C, except that cells were stimulated with various concentrations of 4-OHT. (E) 293T cells were transiently transfected with MiNR1 or STAT5A(1*6)-ER vectors together with a luciferase reporter containing three STAT5 binding sites in the promoter. Cells were stimulated with various concentrations of 4-OHT for 24 h, and cell lysates were prepared and analyzed for luciferase activity. (F) CB CD34⁺ cells were transduced with STAT5A(1*6)-ER and stimulated with 4-OHT for 24 h, and RNA was isolated. Q-PCR analysis was performed using primers against SOCS2. Q-PCR data were compared to results obtained from gene expression profiling using Illumina BeadChip arrays. (G) CB CD34⁺ cells were stimulated with a cocktail of cytokines (KFT) in a dose-dependent manner. Total cell lysates were prepared, and Western blotting was performed using antibodies against STAT5 and tyrosine 694-phosphorylated STAT5. (H) CB CD34⁺ cells were transduced with wt STAT5A-ER and stimulated with 10 ng/ml IL-3, 400 nM 4-OHT, or both. Total cell lysates were prepared, and Western blotting was performed using antibodies against STAT5 and tyrosine 694-phosphorylated STAT5. (I) CB CD34⁺ cells were stimulated with increasing concentrations of KFT as for panel G or transduced with STAT5A(1*6)-ER or wt STAT5A-ER, after which cells were stimulated with 4-OHT as for panel B (for wt STAT5A-ER, 0, 3.2, 16, 80, 400, and 2,000 nM 4-OHT was used). RNA was isolated, and Q-PCRs were performed using primers against CISH.

FIG. 2.

Maximal proliferative advantage of CB CD34⁺ cells at intermediate STAT5A activity levels while differentiation is most affected at high STAT5A transactivation levels. CB CD34⁺ cells were transduced with MiNR1 control or with STAT5A(1*6)-ER (A) or STAT5A(S710F)-ER (B) vectors and cocultures were initiated on MS5 bone marrow stromal cells in the presence of 4-OHT. Cultures were demidepopulated weekly, and cumulative cell counts of a representative experiment (out of three) are presented. (C) Cells were treated as described for panel A, and cocultures were initiated on MS5 bone marrow stromal cells in the presence of 4-OHT. (D) Cells treated as described for panel A were harvested by weekly demidepopulation and analyzed by FACS for expression of myeloid (CD14, CD15) and erythroid (GPA) antigens. Data from a representative week 2 experiment are shown. (E) Cells treated as described for panel A were harvested at week 2 and analyzed by cytospin and May-Grunwald-Giemsa staining. (F and G) Cells treated as described for panels B and C were harvested by weekly demidepopulation and analyzed by FACS for expression of myeloid (CD14, CD15) and erythroid (GPA) antigens. Data from representative week 2 experiments are shown.

FIG. 3.

(A) Self-renewal of progenitors at intermediate STAT5A activity levels. CB CD34⁺ cells were transduced with MiNR1 control or STAT5A(1*6)-ER vectors and plated in methylcellulose in the presence of 4-OHT. Progenitor numbers were scored 2 weeks later. (B) After scoring, cells were harvested, washed with phosphate-buffered saline, and replated in new methylcellulose to initiate second CFC assays, which were scored 2 weeks after plating (n = 3).

FIG. 4.

CAFC and LTC-IC frequencies are increased at intermediate, but not high, STAT5A activity levels. (A and B) CB CD34⁺ cells were transduced with MiNR1 control or STAT5A(1*6)-ER vectors, and cocultures were initiated on MS5 bone marrow stromal cells at limiting dilutions on 96-well plates in the presence of 4-OHT. Early CAs were enumerated at week 2 (n = 3). (C and D) Stem cell frequencies were determined in LTC-IC assays at limiting dilutions in 96-well plates on MS5 stroma by plating MiNR1- or STAT5A(1*6)-ER-transduced cells at concentrations of 6 to 1,458 cells per well. Cultures were fed weekly with new medium; at week 5, methylcellulose was added, and LTC-IC frequencies were determined 2 weeks later. Data from two representative experiments are shown. (Left) LTC-IC data over a range of plated cell densities without 4-OHT or in the presence of 500 nM 4-OHT. (Right) LTC-IC frequencies over a range of 4-OHT concentrations. (E) Stem cell frequencies were determined in LTC-IC assays similar to those used for panels C and D but with wt STAT5A-ER-transduced cells.

FIG. 5.

Genome-wide changes in gene expression induced by dose-dependent STAT5 signaling. CB CD34⁺ cells were transduced with MiNR1 control or STAT5A(1*6)-ER vectors, and cells were stimulated with 4-OHT for 24 h. RNA was isolated, and genome-wide gene expression patterns were determined on Illumina Sentrix Human-6 BeadChip arrays. The data are gene expression changes in the 20 nM versus 0 nM groups (more than twofold up- or downregulation). Of the 437 genes with changes, 217 were downregulated and 220 were upregulated. The upregulated genes were further subdivided into genes that reached their maximal expression levels at 20 nM (500 nM = 20 nM, ±20%; 138 changes) and genes that were further upregulated in response to further increasing STAT5 activity levels (500 nM > 20 nM, at least 1.2-fold; 60 changes). Gene lists are provided in the supplemental material. A number of gene expression changes were verified by Q-PCR analysis (all Q-PCR analyses are shown in Fig. S1 in the supplemental material). Gene expression changes were annotated according to cellular component, and significant gene ontology (see http://www.geneontology.org/GO.annotation.shtml) cellular component annotations are presented in pie charts.

FIG. 6.

Proposed model for dose-dependent STAT5 signaling in human CD34⁺ cells. Intermediate STAT5 activity levels induce maximal self-renewal and long-term proliferative advantage. High STAT5 activity levels predominantly associate with an induction of erythropoiesis and impaired myelopoiesis. Intermediate STAT5 levels are sufficient to induce maximal levels of growth-promoting oncogenes, such as PIM1 and MAF, or low levels of C/EBPα, as well as maximal levels of membrane-associated genes, such as MUC1, that might mediate the enhanced interaction of human HSCs/progenitor cells with their bone marrow microenvironment. This pattern of gene expression drives long-term expansion and self-renewal. Upon further increases in STAT5 activity, only specific subsets of STAT5 target genes are further enhanced, and these include p21CIP and members of the chemokine family. These negatively affect cell cycle progression and expansion, resulting in a shift toward a differentiation phenotype along the erythroid lineage.