Gata2 Is a Direct Target for Smad-Mediated TGFβ Signaling in Hematopoietic Stem/Progenitor Cells

Abstract 1311

Hematopoietic stem cells (HSCs) can give rise to all the different cells of the blood system, and at the same time maintain the stem cell pool throughout the lifetime of the organism. For clinical purposes it would be beneficial to expand the most primitive HSCs in culture without losing their potential. However, this demands increased knowledge regarding the factors that regulate HSC fate decisions, including self-renewal. Transforming growth factor-β (TGFβ) is a potent inhibitor of HSC proliferation in vitro (Batard et al. JCS, 2000; Keller et al. Blood, 1990; Sitnicka et al. Blood, 1996) and the TGFβ signaling pathway has been shown to be activated in HSCs in vivo (Yamazaki et al. Blood, 2009). Together with the observed loss of self-renewal in Smad4 KO HSCs (Karlsson et al. JEM, 2007), this suggests that TGFβ has a role in keeping the HSCs in a dormant state. The mechanisms underlying this effect is largely unknown, even though the cell cycle inhibitor p57 has been demonstrated to play an important role in TGFβ-induced cell cycle arrest of primary human hematopoietic cells (Scandura et al. PNAS, 2004). Murine long-term HSCs (lineage⁻, sca1⁺, ckit⁺ (LSK)CD34⁻) have been reported to express high levels of p57, while short-term HSCs (LSKCD34⁺) do not (Yamazaki et al. EMBO J, 2006). Here, we aim to further identify and functionally validate gene targets of TGFβ signaling in hematopoietic stem/progenitor cells (HS/PCs).

In agreement with earlier studies, quantitative PCR (qPCR) analysis of freshly sorted murine HS/PCs revealed upregulation of p57 (2.8±1.2 fold, P=0.045) following TGFβ (10ng/ml) treatment of LSKCD34⁺ cells. However, p57 expression in LSKCD34⁻ cells was unaffected by TGFβ-treatment. Additionally, TGFβ-induced upregulation of p57 was delayed (5h) and could be effectively diminished by cycloheximide treatment implying that p57 activation is a secondary response to TGFβ. To identify early targets of TGFβ signaling we therefore performed microarray analysis 2h following TGFβ-treatment of Lhx2 cells, a primitive murine hematopoietic cell line with in vivo multi-lineage reconstitution potential (Pinto et al. Blood, 2002) and generated a database of differentially expressed genes involved in HS/PC proliferation and potentially HSC quiescence. Interestingly, we observed upregulation of the transcription factor Gata2, previously described as an important regulator of HSC function (Rodrigues et al. Blood, 2005; Tipping et al. Blood, 2009). To validate this finding in primary cells we performed qPCR analysis on freshly sorted murine HS/PCs and detected an upregulation of Gata2 mRNA (2.0±0.2 fold, P=0.004) upon TGFβ-treatment of LSKCD34⁺ cells, while LSKCD34⁻ cells were unaffected. Importantly, Gata2 upregulation was still observed after cycloheximide treatment, implying that Gata2 is a direct target of TGFβ signaling. Moreover, the robust activation of both Gata2 and p57 gene expression in LSKCD34⁺ cells was not observed in TGFβ-treated Smad4^−/− HS/PCs, demonstrating that TGFβ-induced regulation of these two genes is mediated through canonical TGFβ/Smad signaling.

Since Gata2 has been implicated to have a role in HSC quiescence, we wanted to compare Gata2 mRNA levels in different HS/PC populations, to see if the expression correlated with HSC primitiveness. Murine BM cells were sorted into 3 compartments of cells based on surface markers: LSKCD34⁻ CD48⁻ CD150⁺, LSKCD34⁻ and LSKCD34⁺ cells. qPCR analysis revealed 1.6-fold (P<0.02) higher Gata2 mRNA level in the most primitive LSKCD34⁻ CD48⁻ CD150⁺ cells compared to LSKCD34⁻ cells, and the latter population in turn had 2.9-fold (P<0.001) higher Gata2 level than LSKCD34⁺ cells. The same trend was found when measuring the mRNA level of p57 in these populations. LSKCD34⁻ CD48⁻ CD150⁺ cells had 3.1-fold (P<0.02) higher p57 level compared to LSKCD34⁻ cells, which in turn had 7.4-fold (P<0.03) higher level than LSKCD34⁺ cells. This clearly demonstrates a correlation between Gata2 and p57 expression and primitiveness of HSCs. Together, our results strongly indicate that Gata2 and p57 are mediators of the Smad-mediated TGFβ response in HSCs. We hypothesize that there is a transcriptional network, regulating Gata2, p57 and TGFβ signaling, that has a functional importance in keeping the stem cells in quiescence. We aim to delineate this regulatory circuit and further study its functional relevance in HSC proliferation and self-renewal.