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

Abstract

Transforming growth factor β (TGFβ) 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 TGFβ-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 TGFβ. Up-regulation of p57 mRNA and protein occurs before TGFβ-induced G 1 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 TGFβ-induced cell cycle arrest in these cells, because two different small interfering RNAs that prevent p57 up-regulation block the cytostatic effects of TGFβ on human hematopoietic cells. Reduction of basal p57 expression by this approach also allows hematopoietic cells to proliferate more readily in the absence of TGFβ. 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 TGFβ mediates its cytostatic effects on human hematopoietic cells and suggests an explanation for the frequent silencing of p57 expression.

A network including TGFβ/Smad4, Gata2, and p57 regulates proliferation of mouse hematopoietic progenitor cells

“…To investigate how TGFβ affects p57 regulation in primary HSPCs over time, we performed a time course qPCR experiment where freshly sorted murine LSKCD34 -cells (HSCs) or LSKCD34 + cells (hematopoietic progenitor cells) were treated with TGFβ and harvested at different time points (Fig 1A). In agreement with earlier studies 13,14 , qPCR analysis of p57 expression revealed a strong upregulation at 5 h (2.8-fold, P=0.045) and 12 h (6.4-fold; P=0.018) following TGFβ treatment, in the hematopoietic progenitor population ( Fig 1B). However, when protein translation was blocked by cycloheximide treatment, TGFβ-induced upregulation of p57 was effectively diminished (<1.3-fold; Fig 1C).…”

“…However, the molecular mechanism underlying TGFβ-induced proliferation arrest is largely unknown. Interestingly, TGFβ has been found to induce expression of the cell cycle inhibitor p57 in HSPCs 13,14 and this activation is crucial for TGFβ-induced cell cycle arrest in vitro 13 HSCs from Smad4 KO mice while p57 expression was significantly reduced. Still, deletion of Gata2 had strong impact on p57 baseline levels in hematopoietic progenitor cells as well as on TGFβ-induced upregulation of p57 and proliferation arrest.…”

“…However, in Lin -cKit + progenitors we observed a significant reduction of p57 and Smad7 mRNA in the Gata2 -/-background compared to littermate controls. 13 Importantly, TGFβ-induced upregulation of p57 was lost in Gata2 -/-progenitors ( Fig 5F) and Smad7 upregulation was significantly impaired (Fig 5G). To address the functional relevance for Gata2 in TGFβ-induced proliferation arrest we cultured lineage-depleted bone marrow cells from Gata2 heterozygous mice for four days in vitro with or without TGFβ.…”

“…The cell cycle inhibitor p57 is important for TGFβ-induced cell cycle arrest of HSPCs and its expression has been shown to be induced by TGFβ in primitive hematopoietic cells 13,14 . To investigate how TGFβ affects p57 regulation in primary HSPCs over time, we performed a time course qPCR experiment where freshly sorted murine LSKCD34 -cells (HSCs) or LSKCD34 + cells (hematopoietic progenitor cells) were treated with TGFβ and harvested at different time points (Fig 1A).…”

“…While the mechanism mediating TGFβ signaling is well characterized, the downstream effector molecules in hematopoietic progenitors are poorly understood. The cell cycle inhibitor p57 has been reported to be transcriptionally activated by TGFβ and to play a critical role in TGFβ-induced cell cycle arrest of hematopoietic progenitor cells 13 and HSCs 14 . Additionally, HSCs have been reported to express high levels of p57, while hematopoietic progenitor cells do not 12,[15][16][17] .…”