Convergence of Hypoxia and TGFβ Pathways on Cell Cycle Regulation in Human Hematopoietic Stem/Progenitor Cells

Albertus T J Wierenga,Jan Jacob Schuringa,Edo Vellenga,Zoran Ivanovic

doi:10.1371/journal.pone.0093494

Albertus T J Wierenga, Jan Jacob Schuringa + Show 2 more

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https://doi.org/10.1371/journal.pone.0093494

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Abstract

Although it has been shown that HIF1 and 2 fulfill essential roles within the hematopoietic system and in the regulation of HSC fate, little is currently known about the specific mechanisms that are involved. We identified transcriptome changes induced by hypoxia, constitutively active HIF1(P402/564) and HIF2(P405/531) in human cord blood CD34+ cells. Thus, we were able to identify common hypoxia-HIF1-HIF2 gene signatures, but we also identified specific target genes that were exclusively regulated by HIF1, HIF2 or hypoxia. Geneset enrichment analysis (GSEA) revealed that, besides known pathways associated with “hypoxia-induced signaling”, also significant enrichment for the Transforming Growth Factor beta (TGFβ) pathway was observed within the hypoxia/HIF1/HIF2 transcriptomes. One of the most significantly upregulated genes in both gene sets was the cyclin dependent kinase inhibitor CDKN1C (p57kip2). Combined hypoxia treatment or HIF overexpression together with TGFβ stimulation resulted in enhanced expression of CDKN1C and enhanced cell cycle arrest within the CD34+/CD38− stem cell compartment. Interestingly, we observed that CD34+ cells cultured under hypoxic conditions secreted high levels of latent TGFβ, suggesting an auto- or paracrine role of TGFβ in the regulation of quiescence of these cells. However, knockdown of SMAD4 could not rescue the hypoxia induced cell cycle arrest, arguing against direct effects of hypoxia-induced secreted TGFβ. Finally, the Gα-coupled receptor GTPase RGS1 was identified as a HIF-dependent hypoxia target that dampens SDF1-induced migration and signal transduction in human CD34+ stem/progenitor cells.

Highlights

Hematopoietic stem cells (HSCs) reside within specialized hypoxic niches in the bone marrow microenvironment where they are kept in a relative quiescent state [21,24,26,27,31,34,41]
In order to identify HIF1a, HIF2a and hypoxia target genes in the human hematopoietic stem/progenitor compartment, CD34+ cells were isolated from cord blood and were transduced with constitutively active HIF1a (P405/P564), HIF2a (P405/531) or control vectors and RNA was isolated from GFP+-sorted cells 24 hrs after the last transduction round (Figure 1A)
cord blood (CB) CD34+ cells were cultured under normoxia (21% O2) or hypoxia (1% O2) for 24 hrs followed by RNA isolation (Figure 1A)

Summary

Introduction

Hematopoietic stem cells (HSCs) reside within specialized hypoxic niches in the bone marrow microenvironment where they are kept in a relative quiescent state [21,24,26,27,31,34,41]. In well-oxygenated conditions HIFs are bound by the Von Hippel Lindau (VHL) tumor suppressor protein which recruits an ubiquitin ligase that targets these transcription factors for proteasomal degradation [18]. VHL binding is critically dependent on hydroxylation of proline residues in HIF1 (P405 and P564) and HIF2 (P405 and P531) [40]. The oxygen-sensitive a subunits of HIF1 or HIF2 can heterodimerize with the stable HIF1b (ARNT) subunit that together forms a basic helix-loophelix-PAS (bHLH-PAS) transcriptional regulator that binds to the core sequence RCGTG termed the hypoxia response element (HRE) in promoters of presumed target genes [18,20,28,38]

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