Signal integration between notch and hypoxia : insights into development and disease

Oxygen is essentially required for aerobic metabolism in most eukaryotic organisms. Therefore, it is striking that a number of tissues develop, and are maintained at low oxygen (hypoxic) conditions in the body. For instance, the human embryo is located in a hypoxic environment. At the cellular level, hypoxia activates a signaling pathway, governed by hypoxia inducible factor (HIF-1α). Hypoxic adaptation leads to induced erythropoiesis and angiogenesis, as well as a switch to glycolytic metabolism. In addition, hypoxia also promotes the undifferentiated cell state in various stem and precursor cell populations. One of the principal signaling mechanisms that controls development, is Notch signaling, an evolutionarily conserved pathway involved in cell fate decisions. In this thesis, I have investigated the interplay of hypoxia with the Notch signaling pathway. Here we showed that Notch signaling is required for hypoxia-mediated reduction of progenitor cell differentiation (Paper I). Hypoxia blocked neuronal and myogenic differentiation in a Notch-dependent manner. Furthermore, the Notch intracellular domain (ICD) interacted with HIF-1α, and HIF-1α was recruited to Notch-responsive promoters upon Notch activation during hypoxic conditions. Taken together, these data provide molecular insights into how reduced oxygen levels control the cellular differentiation status and demonstrate a role for Notch in this process. Hypoxia signaling pathway is frequently involved in several adult pathologies, including cardiovascular disease and cancer. Therefore, it is interesting from at therapeutic prospective to investigate whether the link between hypoxia and Notch signaling also synergize during tumor development. Specifically, hypoxia induces epithelial-mesenchymal transition (EMT), which leads to increased migration of tumor cells, and clinically associates with an aggressive tumor phenotype. However, the molecular mechanisms linking hypoxia to EMT in tumors are poorly understood. In Paper II, we report that Notch signaling is critically required to convert a hypoxic stimulus into EMT in tumor cells. Inhibition of Notch signaling abrogated the hypoxia-induced EMT and reduced cell migration. Furthermore, Notch positively regulated the level of the EMT inducer Snail-1 in two distinct ways: through direct transcriptional control of Snail-1 and indirectly, through increased lysyl oxidase expression. In sum, these data provide support for a hypoxia-Notch-EMT link in tumor cells. The hypoxia inducible transcription factors (HIFs) are regulated at the level of protein stability and transcriptional activity in an oxygen-dependent manner by prolyl and asparaginyl hydroxylation, respectively. Both these families of hydroxylating enzymes use oxygen as co-substrates, and thus are oxygen sensors in the cell. In the presence of oxygen, the hydroxylase Factor inhibiting HIF-1α (FIH-1) targets a conserved asparaginyl residue within the C-terminal transactivation activation domain (C-TAD) of HIF-α, leading to repression of HIF-mediated transcription by abrogating recruitment of cofactors. In Paper III, we identify Notch as a novel substrate for hydroxylation by FIH-1, providing yet another interaction point between hypoxia and Notch signaling. Notch ICD was hydroxylated at an asparaginyl residue within its ankyrin repeat region. In addition, FIH-1 interacted with Notch, and enforced expression of FIH-1 resulted in abrogated Notch activity. Furthermore, FIH-1 increased differentiation of myocytes, in keeping with the inhibitory effect of FIH on Notch signaling. These data show that FIH-1 interacts with Notch ICD, and is capable of reducing Notch activity. To more closely investigate the temporal and spatial aspects of Notch signaling, we designed a fluorescent reporter assay that allows Notch activation to be followed in real time in individual cell (Paper IV). We generated a reporter construct composed of 12 CSL-binding motifs linked to two fluorescent proteins. After transfection into cells, the reporters rapidly responded to various forms of Notch activation, including ligand activation of full-length Notch receptors. Finally, we used this assay to gain insights into the level of Notch signaling in CNS progenitor cells in culture and in vivo. To conclude, we have identified a crosstalk between hypoxia and Notch signaling. Our results show that there is a functional relationship between Notch, hypoxia and FIH in the control of progenitor cell differentiation. In addition, we have shown that the hypoxia-Notch link also occurs in tumors, and is specifically important for hypoxia-induced EMT. LIST OF PUBLICATIONS This thesis is based on the following papers: I. Maria V. Gustafsson, Xiaowei Zheng, Teresa Pereira, Katarina Gradin, Shaobo Jin, Johan Lundkvist, Jorge L. Ruas, Lorenz Poellinger, Urban Lendahl and Maria Bondesson. Hypoxia requires Notch signaling to maintain the undifferentiated cell state. Developmental Cell (2005) 9; 617-628. II. Cecilia Sahlgren, Maria V. Gustafsson, Shaobo Jin, Lorenz Poellinger and Urban Lendahl. Notch signaling is required for hypoxia-induced epithelialmesenchymal transition in tumor cells.