Die Rolle von HIF prolyl hydroxylase-2 (PHD2) in physiologischen und pathologischen Prozessen in Mäusen
Zusammenfassung der Projektergebnisse
Low oxygen tension (hypoxia) is a characteristic feature during physiological and pathological processes, including wound healing, stem cell maintenance, inflammation and tumor development. The transcriptional response to deprived pO2 is to a large extent regulated by the hypoxia inducible factors (HIF); heterodimeric complexes composed of an oxygen-sensitive HIFα (HIF1 and HIF2) and a constitutive HIFβ subunit. Both HIFα forms can in general actively promote oxygen delivery and adaptive processes to hypoxia such as erythropoiesis, angiogenesis, anaerobic glycolysis and hematopoiesis. ‘Oxygen-sensing’ is therefore indispensable as it enables the cells to instantaneously adapt to this stress situation. The machinery behind this relies on the HIF-prolyl hydroxylases (PHD1-3), enzymes that hydroxylate and, consequently, lead to the inactivation of HIFα in the presence of oxygen. PHD2 is believed to be the crucial oxygen sensor during normoxia and mild hypoxia. In humans, several heterozygous point mutations in the PHD2 gene have been described, which lead to an absolute increase in red blood cell mass. PHD2 is therefore the main HIF prolyl hydroxylase isoform which, next to HIF2α and the von Hippel-Lindau protein (VHL), regulates the expression of EPO and erythropoiesis in humans. In this respect, we recently described a new conditional PHD2 mouse line (CD68:cre-PHD2^f/f) expressing excessive HIF2α-induced EPO production in kidney and brain, extreme hematocrits up to 86%, thrombocytopenia and splenomegaly, but survival of the mice in a HIF1α-dependent manner. Indeed, we demonstrated that simultaneous inactivation of HIF1α and PHD2 leads to early lethality, which we proved to be confined to PHD3-reliant HIF2α-overstabilisation and consequent neurodegeneration in the brain. In the bone marrow (BM), regional hypoxia has been shown to be indispensable to maintain the multipotency and self-renewal capacity of hematopoietic stem cells (HSCs). Moreover, lack of HIF1α in these HSCs results in loss of quiescence. We analyzed in detail the CD68:cre-PHD2^f/f mice (cKO), in which PHD2 is also conditionally ablated in the entire hematopoietic system. In a recent Blood paper we show that very early hematopoietic or multipotent progenitors (MPPs) in the BM of cKO mice have a significantly higher proliferation rate than their WT littermates under steady-state conditions. Furthermore, we discovered that this phenomenon is accompanied by a significant induction of the TGFβ-inhibitor SMAD7 leading to a consequent reduction of the cell cycle inhibitor p21^WAF/CIP1. During severe stress (irradiation/transplantation), loss of PHD2 in this cell compartment resulted in enhanced self-renewal of the quiescent CD34 HSCs and early MPPs. The presence of hypoxic regions has also been detected in solid tumors, already half a century ago. Adaptation to tumor hypoxia is also in these settings commonly orchestrated by the HIFs. We recently demonstrated that loss of PHD2 in different mouse tumor cell lines leads to the transformation of TGFβ from a tumor promoter to a tumor suppressor in a HIF1-independent manner, which resulted in a profound and unexpected reduction in tumor growth in mice. In addition to neoplastic cells, the tumor microenvironment (TME), including stromal cells, endothelial and immune cells play an important role during tumor growth. Hence, we studied the direct effect of PHD2 inhibition in the hematopoietic system in relation to tumor development. Interestingly, we found reduced tumor growth in the CD68:cre-PHD2^f/f mice, which targets the entire hematopoietic system. Using lineage specific PHD2-deficient mice (myeloid, T-cell and B-cell) we discovered that loss of PHD2 in both - myeloid and T-cells - is necessary and sufficient to reduce tumor growth. Skin wound healing is a physiological process that is accompanied by the involvement of different cell types (e.g. inflammatory cells, endothelial cells and keratinocytes) and stages (e.g. clotting, influx of myeloid cells, hyper-proliferation of keratinocytes and angiogenesis). In addition, the skin and especially the wound area are under constant hypoxic stress and it has been suggested that HIFs and PHDs might influence healing of chronic wounds in diabetes mice.(44) In order to investigate the involvement of PHD2 during tissue repair in different cellular compartments, we generated conditional PHD2-deficient mice in keratinocytes, myeloid or endothelial cells and subjected them to experimental skin wounds. Interestingly, we demonstrated that only deletion of PHD2 in keratinocytes leads to a higher migratory rate of these cells during wound closure, which we found to be mediated by an unprecedented HIF1α-related induction of β3-integrin. Moreover, these deficient keratinocytes exhibited also enhanced proliferation in wounds in a TGFβ-dependent manner. In conclusion, we revealed a novel function for PHD2 during wound healing and provided new insights into the molecular mechanisms of tissue repair.
Projektbezogene Publikationen (Auswahl)
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Inhibition of HIF Prolyl Hydroxylase-2 Blocks Tumor Growth in Mice through the Anti-Proliferative Activity of TGFβ. Cancer Res., 1;71(9):3306-16, 2011
Klotzsche-von Ameln, A., Muschter, A., Mamlouk, S., Kalucka, J., Prade, I., Franke, K., Rezaei, M., Poitz, D.M., Breier, G., and Wielockx, B.
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Hypoxia-mediated regulation of stem cell fate. High Alt Med Biol. 13(3):162-8; 2012
Singh R.P., Franke K. and Wielockx B.
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Inhibition of HIF Prolyl Hydroxylase-2 induces MMP expression which unexpectedly leads to tumor growth retardation. Cancer Biol. Ther. 13: 4, 2012
Klotzsche-von Ameln, A., Muschter, A., Breier, G., and Wielockx, B.
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Epithelial HIF Prolyl Hydroxylase-2 (PHD2) negatively regulates skin wound healing in mice. Mol. Cell. Biol., 33(17): 3426-3438, 2013
Kalucka, K., Ettinger, A. Franke, K., Mamlouk, S., Singh, R.P., Muschter, A., Breier, G., Katschinski, D.M., Huttner, W., Weidemann, A. and Wielockx, B.
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Erythropoiesis in Mice and Men - the HIF Pathway in Control. Blood, 122(7): 1122-1128, 2013
Franke, K., Gassmann, M., and Wielockx, B.
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HIF-1α is a Protective Factor in Conditional PHD2 Deficient Mice Suffering from Severe HIF-2α-Induced Excessive Erythropoiesis. Blood, 121(8):1436-1445, 2013
Franke, K., Kalucka, J., Soulafa Mamlouk, S., Singh, R.P., Muschter, A., Weidemann, A., Iyengar, V., Jahn, S., Wieczorek, K., Geiger, K., Muders, M., Sykes, A.M., Poitz, D., Ripich, T., Otto, T., Bergmann, S., Breier, G., Baretton, G., Fong, G., Greaves, D.R., Bornstein, S., Chavakis, T., Fandrey, J., Gassmann, M., and Wielockx, B.
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HIF-prolyl hydroxylase 2 (PHD2) is a critical regulator of hematopoietic stem cell maintenance during steady-state and stress. Blood. 121(26):5158-5166, 2013
Singh, R. P., Franke K., Kalucka J., Mamlouk S., Muschter A., Gembarska A., Grinenko T., Willam C., Naumann R., Anastassiadis K., Stewart A. F., Bornstein S., Chavakis T., Breier G., Waskow C., and Wielockx B.
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Hypoxia-inducible factors as key regulators of tumor inflammation. Int J Cancer 132(12): 2721-2729; 2013
Mamlouk, S. and Wielockx, B.
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Loss of prolyl hydroxylase-2 in myeloid cells and T-lymphocytes impairs tumor development. Int. J. Cancer, 134(4):849-58, 2014
Mamlouk, S., J. Kalucka, R. P. Singh, K. Franke, A. Muschter, A. Langer, C. Jakob, M. Gassmann, G. B. Baretton and Wielockx, B.
