Transforming growth factor beta-induced cell cycle arrest of human hematopoietic cells requires p57KIP2 up-regulation

Abstract

Transforming growth factor beta (TGFbeta) is one of few known negative regulators of hematopoiesis, yet the mechanisms by which it affects cell cycle arrest and stem cell quiescence are poorly understood. Induction of the cyclin-dependent kinase inhibitors, p15INK4b (p15) and p21WAF1 (p21) is important for TGFbeta-mediated cytostasis in epithelial cells but not in hematopoietic cells. Using primary human hematopoietic cells and microarray analysis, we identified p57KIP2 (p57) as the only cyclin-dependent kinase inhibitor induced by TGFbeta. Up-regulation of p57 mRNA and protein occurs before TGFbeta-induced G1 cell cycle arrest, requires transcription, and is mediated via a highly conserved region of the proximal p57 promoter. The up-regulation of p57 is essential for TGFbeta-induced cell cycle arrest in these cells, because two different small interfering RNAs that prevent p57 up-regulation block the cytostatic effects of TGFbeta on human hematopoietic cells. Reduction of basal p57 expression by this approach also allows hematopoietic cells to proliferate more readily in the absence of TGFbeta. p57 is a putative tumor suppressor gene whose expression is frequently silenced by promoter hypermethylation in hematologic malignancies. Our studies identify a molecular pathway by which TGFbeta mediates its cytostatic effects on human hematopoietic cells and suggests an explanation for the frequent silencing of p57 expression.

Figures

Fig. 1.

TGFβ causes G1 cell cycle arrest and up-regulates p57 mRNA and protein. (A) p57 mRNA is rapidly up-regulated by TGFβ in primary human hematopoietic progenitor cells. The average signal for the various CDKI probe sets is shown normalized to the expression before stimulation with TGFβ. (B) Quantitative RT-PCR demonstrates strong up-regulation of p57 mRNA 4 h after exposure of CB-CD34 to TGFβ. There is no statistically significant regulation of other CDKIs by TGFβ (Student's t test). Expression of the indicated mRNA is reported relative to the level of hypoxanthine phosphoribosyltransferase expression. (C) Proliferation rates of M091 cells treated or untreated with TGFβ (200 pM) are shown. (D) Representative cell cycle profiles of M091 cells treated with TGFβ (200 pM) for 24 h, or left untreated (Left). (E) Only p57 mRNA is up-regulated by TGFβ in M091 cells. Expression array data are presented as described for A. (F) Western blot analysis of p57, p27, and p21 protein expression using lysates from M091 cells stimulated with TGFβ for the indicated periods of time. p15 is not detectable by Western blot at any time. α-Tubulin staining was used as a control for protein loading. All error bars represent the standard errors of measurements from three to four independent experiments.

Fig. 2.

TGFβ-induced up-regulation of p57 involves increased transcription of the A3 isoform. (A) M091 cells were pretreated with actinomycin D (lanes 2 and 3) or DMSO alone (lanes 1 and 4) and then stimulated with TGFβ (200 pM) (lanes 3 and 4) for 90 min. The relative expression of p57 mRNA was assessed by RT-PCR. Amplification of hypoxanthine phosphoribosyltransferase mRNA is shown to demonstrate equal RNA input. (B) The organization of the p57 gene is shown (Upper). The translated portion of each exon is colored black if it is invariant or dark gray if it is isoform specific. The 5′ end of the three known isoforms of p57 are presented (A1, A2, and A3) (Lower Left). The locations of the PCR primers used for this analysis are represented by black (the Pan-Isoform Amplimer Set) or gray bars (the Isoform-A1A2 Amplimer Set). Amplification of genomic DNA by these primers yields 381- and 309-bp fragments, respectively. PCR products generated from genomic DNA (lanes 2 and 4) or cDNA (lanes 3 and 5) templates are shown (Lower Right). Only the A3 transcript is detected in CB-CD34 cells (and M091 cells; data not shown).

Fig. 3.

TGFβ induces monoallelic up-regulation of p57 but does not regulate other genes in the imprinted region of chromosome 11p15.5. (A) The genomic organization of a 1-Mb imprinted cluster of genes on chromosome 11p15.5 is shown. Maternally and paternally imprinted genes are represented as pink or blue-filled pentagons, respectively. Nonimprinted genes within the region are shown in black, and those for which the imprinting status is unknown are filled with white. The brackets correspond to the two clusters of genes that are coordinately imprinted and are under the control of independent regulatory elements. (B) The PAPA repeat region was amplified from two individual units of cord blood by using genomic DNA and cDNA made from CB-CD34 cells stimulated with TGFβ for 4 h as templates. Heteroduplexes are seen for the amplified genomic fragments but not when the cDNA is amplified. The doublet seen using genomic DNA but not with the cDNA is due to different allele lengths. (C) p57 mRNA, but not that of other imprinted genes on chromosome 11p15.5, is rapidly up-regulated by TGFβ in CB-CD34. The average signal for the various probe sets is shown normalized to the expression before stimulation with TGFβ. (D) Quantitative RT-PCR analysis of PHLDA2, KCNQ1, IGF2, and CDKN1c (p57) gene expression before and 4 h after exposure of CB-CD34 to TGFβ. Expression of the indicated mRNA is reported relative to the expression of the hypoxanthine phosphoribosyltransferase reference transcript. All error bars represent the standard errors of measurements from three to four independent experiments.

Fig. 4.

Activation of the p57 promoter by TGFβ signaling requires a highly conserved region upstream of the TATA-box. (A) Schematic diagram of the p57 promoter indicating nine canonical Smad-binding elements (CAGAC, represented as small ovals) and the restriction sites used for generating the truncation mutations used for this analysis. Also indicated is a GC-rich region located between -165 and -77 that is highly conserved between the human, rat, and murine p57 genes. (B) The full-length -2191KIP2 reporter was cotransfected into HeLa cells in combination with Smad3, Smad4, and TβR-I(T204D), as indicated. Relative luminescence units were normalized -2191KIP2 reporter activity when cotransfected with pCMV5 alone. (C) The indicated p57 reporter constructs were transfected into HeLa cells with either pCMV5 or TGFβ mediators [Smad3, Smad4, and TβR-I(T204D)]. Relative luminescence units were normalized as described for A. The numbers above each set of bars indicate the fold increase in reporter activity induced by the cotransfection of the TGFβ mediators. (D) The p57 promoter constructs used for these experiments are diagrammed (Left). The indicated p57 reporter constructs were transfected into HeLa cells with either pCMV5 or TGFβ mediators [Smad3, Smad4, and TβR-I(T204D)]. (Right) The fold increase in reporter activity with cotransfection of the TGFβ mediators compared to the reporter activity in the presence of pCMV5. All error bars represent the standard errors of measurements from two to four independent experiments each carried out in triplicate.

Fig. 5.

Up-regulation of p57 is required for the TGFβ-induced cell cycle inhibition of hematopoietic cells. M091 were transfected with siRNA directed against either EGFP or p57 mRNA. (A) The effect of the indicated siRNA on EGFP expression was assessed by flow cytometry by using M091-MIGR1 cells that constitutively express EGFP from the MSCV LTR. Presented is the mean fluorescence normalized to untransfected control cells (not shown). (B) M091 cells were transfected with the indicated siRNA, and split into equal portions, half of which were treated with TGFβ (200 pM) for 4 h before Western blot. The α-tubulin control is shown to demonstrate equal protein loading in all lanes. (C) The fold change in M091 cell numbers after transfection with the indicated siRNA is shown in the presence or absence of TGFβ (200 pM) for 24 h. Error bars represent the standard error of three independent experiments.