Convergence Of Hypoxia and TGFβ Pathways On Cell Cycle Regulation In Human Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cells reside within specialized hypoxic niches in the bone marrow where they are kept in a relative quiescent state. One of the key pathways activated under low oxygen conditions is the Hypoxia Induced Factor (HIF) pathway. HIF1 and HIF2 act as oxygen sensors that are degraded by the Von Hippel Lindau (VHL) tumor suppressor protein under normoxic conditions but not when oxygen levels are low, resulting in stabilization of the HIF proteins, translocation to the nucleus and initiation of target gene transcription.

Although it has been shown that HIF1 and 2 fulfill essential roles in the regulation of HSC fate, little is known about the mechanisms that are involved. Here, we set out to investigate the effects of hypoxia, HIF1 and HIF2 on gene transcription in human hematopoietic stem/progenitor cells. Cord blood (CB) CD34⁺ cells were cultured under low oxygen conditions (2%), or were lentivirally transduced with constitutively active HIF1(P402/564) and HIF2(P405/531) constructs under normoxic conditions and after 24 hrs transcriptome changes were analyzed by Illumina BeadArray analysis. This provided the possibility to identify common hypoxia-HIF1-HIF2 gene signatures, but also the identification of specific target genes that were exclusively regulated by HIF1, HIF2 or hypoxia. Geneset enrichment analysis (GSEA) using Gene Ontology genesets revealed that overexpression of HIF1 and -2 resulted in significant enrichment for known pathways like “hypoxia induced signaling”, but unexpectedly also for the Transforming Growth Factor beta (TGFβ) pathway. GSEA using a published dataset of TGFβ stimulated CB CD34⁺ cells indeed confirmed a high correlation between hypoxia target genes and TGFβ induced genes. Two of the most significantly upregulated genes in both gene sets were the cyclin dependent kinase inhibitor CDKN1C (p57kip2) and Regulator of G-protein signaling (RGS)1.

q-RT-PCR analysis demonstrated enhanced expression of CDKN1C by hypoxia treatment or HIF overexpression under normoxia in combination with TGFβ stimulation. Although it was demonstrated that CD34⁺cells cultured under hypoxic conditions secreted high levels of latent TGFβ, no rescue of the hypoxia induced cell cycle arrest was demonstrated by knockdown of SMAD4, arguing against direct effects of hypoxia-induced secreted TGFβ on cell cycle quiescence.

RGS1 is a member of the RGS family, involved in the negative regulation of G-protein coupled receptor signaling. Overexpression studies under normoxic conditions in CB CD34⁺cells demonstrated a decrease of SDF1-mediated migration. Furthermore, overexpression of RGS1 attenuated SDF1 and GM-CSF-induced ERK phosphorylation whereas the GM-CSF-induced STAT5 tyrosine phosphorylation was unaffected. These findings indicate that RGS1 can interfere with specific signaling pathways involved in the regulation of cell proliferation and migration.

Analysis of the CDKN1C as well as the RGS1 promoters revealed binding sites for both HIF and SMAD2/3/4 in the proximal part, suggesting that both pathways can indeed converge on the regulation of these important proteins that control cell cycle progression and the response to stimulatory cytokines in human stem/progenitor cells.