Autophagy and the hematopoietic niche: a regulatory role for the Forkhead-box transcription factors

Abstract

Two main components of the hematopoietic niche are hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). FOXO transcription factors play a fundamental role in the maintenance of these cells through the regulation of cell cycle and oxidative stress. Other gene expression programs controlled by FOXO include cell death, DNA repair and autophagy. Autophagy, has been found to be a general cellular housekeeping process critical for maintenance of quiescence, self-renewal and differentiation. The work described in this thesis aimed to elucidate the functional importance of autophagy in human HSCs and MSCs and how this is regulated by FOXO. In Chapter 2 we review Forkhead box O transcription factors (FOXOs) and data concerning the role for FOXOs in regulating autophagy. Chapter 3 focuses on the role of autophagy in human umbilical cord blood-derived HSCs. Our data demonstrate a higher autophagic flux in hHSCs compared to myeloid progenitors. We show that autophagy is crucial for hHSCs maintenance by reducing cellular stress and promoting survival. In Chapter 4 we demonstrate that in hMSC, ROS induces phosphorylation of FOXO3A and its translocation to the nucleus. This novel ROS-dependent phosphorylation of FOXO3A at serine-294 is mediated by Jun kinase (JNK). Furthermore, we show that upon H2O2 treatment, activation of FOXO3A in hMSCs results in downregulation of ROS through the activation of autophagy. Finally, our results demonstrate the important role of autophagy in the control of oxidative stress during the osteoblastic differentiation of hMSC. In Chapter 5 we show that FOXP1 is a direct transcriptional target of FOXO acting through a negative feedback loop to suppress FOXO-induced apoptosis. FOXP1 binds to the same genomic enhancer elements as FOXO and modulates the expression of a specific subset of FOXO target genes. Chapter 6 focuses on the development of a highthroughput screen to identify new FOXO3A transcriptional targets genes that could modulate macroautophagy. We identify Copper transporter 2 (CTR2/ SLC31A2) a transmembrane protein that participates in the regulation of copper metabolism, as a novel FOXO3A transcriptional target that modulates LC3 levels. In Chapter 7 the findings described in this thesis are discussed. Taken together, our findings provide novel insight into FOXO biology and may serve as basis for the development of novel approaches involving autophagy activation for the expansion and maintenance of transplantable HSCs. Moreover, they may also serve to consider autophagy modulators as potential therapeutic treatment for diseases such as osteoporosis.