Final Report Summary - DISKOMICE (DISKOmice)
1.1 Summary description of the project objectives
The general objective of the DISKOmice project is to study the molecular and cellular mechanisms that allow skeletal tissues to properly differentiate and grow in a hypoxic environment. For this purpose, we focused our investigations on the role of the hypoxia inducible factors (HIFs) in the development of the skeletal elements. The growth plate (GP) and the inner part of the intervertebral disc (IVD), namely the nucleus pulposus (NP), are among the largest avascular structures in vertebrate organisms.
Adaptation to low oxygen tension (hypoxia) is a critical event during development. The HIFs transcription factors are essentials mediators of the homeostatic adaptation to hypoxia by controlling a variety of cellular functions including, survival, proliferation, metabolism regulation and matrix synthesis. In normoxic conditions, von Hippel Lindau protein (VHL), which is an E3 ubiquitin ligase, plays a critical role in the hypoxic signaling pathway by targeting HIFs to the proteasome for degradation.
The better understanding of the hypoxic signaling pathway in the development, aging and associated pathologies of the skeletal tissues will lead to an advanced comprehension of their respective physiopathologies, which will help in defining novel therapeutic targets and innovative repair strategies.
1.2 Description of the work performed since the beginning of the project
In order to establish the role of the HIF pathway, in skeletal development, we used genetically modified mice as our main tool. In particular, to understand whether and how the HIF pathway affects the GP, bone and NP biology in vivo, we specifically deleted the HIF-1alpha, HIF-2alpha and VHL genes in cells of the limb bud mesenchyme and in the notochord using the PRX1-Cre and FOXA2-Cre transgenic mice, respectively. The generated specimens have been carefully analyzed using the state of the art techniques.
1.3 Description of the main results achieved so far
Loss of VHL in the limb bud mesenchyme caused severe dwarfism by altering multiple steps of endochondral bone development. In particular, deletion of VHL in limb bud mesenchyme did not alter the timely differentiation of mesenchymal cells into chondrocytes. However, it caused structural collapse of the cartilaginous growth plate as the result of impaired proliferation, delayed terminal differentiation, and ectopic death of chondrocytes. This phenotype was associated to delayed replacement of cartilage by bone. Notably, loss of HIF-2alpha fully rescued the late formation of the bone marrow cavity in VHL mutant mice, though it did not affect any other detectable abnormality of the VHL mutant growth plate. Conversely, HIF-1alpha was epistatic to VHL, as the growth plate of double mutant lacking both VHL and HIF-1alpha appeared to virtually indistinguishable from the mutant growth plate lacking only HIF-1alpha. Taken together, our findings demonstrate that VHL is a critical regulator of bone morphogenesis, as its loss considerably alters size, shape and overall development of the skeletal elements, with mechanisms that are, at least in part HIF-dependent.
VHL and HIF signaling are also known to play critical role in tumor formation and cancer. To better understand the role of Vhl in the biology of mesenchymal cells, we analyzed mutant mice lacking Vhl in mesenchymal progenitors that give rise to the soft tissues that form and surround synovial joints. Loss of Vhl in mesenchymal progenitors of the limb bud caused severe fibrosis of the synovial joints and formation of aggressive masses with histologic features of mesenchymal tumors. Hif-1α and its downstream target connective tissue growth factor (CTGF) were necessary for the development of these tumors, which conversely still developed in the absence of Epas1, but at lower frequency. Our novel findings in genetically altered mice suggest that activation of the HIF signaling pathway could be an important pathogenetic event in the development and progression of at least a subset of these tumors.
Loss of HIF-1alpha in the notochord did not affect the notochord per se, but it led to the complete disappearance of the nucleus pulposus. In particular, morphologic changes subsequent to the loss of HIF-1alpha in the NP cells could be detected as early as E15.5 and they were followed postnatally, by the progressive disappearance and replacement of the nucleus pulposus with a novel tissue that resembled fibrocartilage. Lineage study and TUNEL assay unequivocally proved that mutant NP cells did not transdifferentiate into chondrocyte like-cells, but they underwent massive cell death, and were completely replaced by a cell population belonging to a cell lineage distinct from the notochordal one. In order to evaluate the functional consequences of HIF-1alpha deletion in the NP, biomechanical testing of mutant IVD was also performed. Loss of the NP cells in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress. These findings are similar to the changes usually observed during human IVD degeneration.
To further investigate the role of hypoxia in the nucleus pulposus physiology, we have taken advantage of the FOXA2 Cre; HIF-1alphaf/f mouse model we have generated and described previously. We hypothesize that NP cells adapt to their hypoxic niche through modulation of autophagy. In various cell types, hypoxia is known to induce autophagy in a HIF-1α-dependent fashion, however little is known about hypoxic regulation of autophagy in NP cells. Our study, for the first time shows that NP cells regulate autophagy in a non-canonical fashion independent of mTOR and HIF-1α signaling.
1.4 Expected final results and their potential impact and use
Understanding skeletal tissue biology and especially IVD has become one of the hottest topics in the regenerative medicine field as low back pain has a prevalence of 80% in the general population. In addition to health concern it represents one of the major socio-economic burdens in industrialized countries.
The DISKO mice project allowed the introduction of new notions such as the unique origin of the NP demonstrated by the specific inactivation of HIF-1 alpha in the cells of the notochord. The fact that the loss of HIF-1alpha leads to a dramatic reduction of PGK suggests an important role of HIF-1alpha in the metabolism regulation. HIF pathway could play a major role in the control of the energy consumption at the cell level.
In addition to providing us with fundamental knowledge about the role of the HIF pathway in cell survival, proliferation, differentiation and metabolism, the original FOXA2 Cre; HIF-1 alphaf/f mouse model, generated during this project, mimics the IVD degeneration occurring in human with a progressive disappearance of the NP structure. This mouse model could therefore represent a valuable tool in order to develop new cell-based tissue repair strategies and to identify new molecular targets.
The notions of HIF-1/HIF-2 complementarity and redundancy have still to be explored to address the following questions:
- How hypoxia can be considered as a physiological signal in the process of cell differentiation and organogenesis in the IVD context?
- What are the molecular mechanisms involved in the IVD development and ageing?
- To what extent these molecular mechanisms could represent potential therapeuthic targets?
The obtained and validated results, in addition to original concepts (and questions to be answered) developed during the DISKOmice project will consolidate or bring us to a leadership position in this research field. This valuable asset will allow us to compete on the international scene for the obtainment of future additional financial support, in order to pursue and develop long-term research projects.
Project websites:
http://www.odontologie.univ-nantes.fr/62918803/0/fiche___pagelibre/
https://www.univ-nantes.fr/59987093/0/fiche___pagelibre/&RH=1350647014953
The general objective of the DISKOmice project is to study the molecular and cellular mechanisms that allow skeletal tissues to properly differentiate and grow in a hypoxic environment. For this purpose, we focused our investigations on the role of the hypoxia inducible factors (HIFs) in the development of the skeletal elements. The growth plate (GP) and the inner part of the intervertebral disc (IVD), namely the nucleus pulposus (NP), are among the largest avascular structures in vertebrate organisms.
Adaptation to low oxygen tension (hypoxia) is a critical event during development. The HIFs transcription factors are essentials mediators of the homeostatic adaptation to hypoxia by controlling a variety of cellular functions including, survival, proliferation, metabolism regulation and matrix synthesis. In normoxic conditions, von Hippel Lindau protein (VHL), which is an E3 ubiquitin ligase, plays a critical role in the hypoxic signaling pathway by targeting HIFs to the proteasome for degradation.
The better understanding of the hypoxic signaling pathway in the development, aging and associated pathologies of the skeletal tissues will lead to an advanced comprehension of their respective physiopathologies, which will help in defining novel therapeutic targets and innovative repair strategies.
1.2 Description of the work performed since the beginning of the project
In order to establish the role of the HIF pathway, in skeletal development, we used genetically modified mice as our main tool. In particular, to understand whether and how the HIF pathway affects the GP, bone and NP biology in vivo, we specifically deleted the HIF-1alpha, HIF-2alpha and VHL genes in cells of the limb bud mesenchyme and in the notochord using the PRX1-Cre and FOXA2-Cre transgenic mice, respectively. The generated specimens have been carefully analyzed using the state of the art techniques.
1.3 Description of the main results achieved so far
Loss of VHL in the limb bud mesenchyme caused severe dwarfism by altering multiple steps of endochondral bone development. In particular, deletion of VHL in limb bud mesenchyme did not alter the timely differentiation of mesenchymal cells into chondrocytes. However, it caused structural collapse of the cartilaginous growth plate as the result of impaired proliferation, delayed terminal differentiation, and ectopic death of chondrocytes. This phenotype was associated to delayed replacement of cartilage by bone. Notably, loss of HIF-2alpha fully rescued the late formation of the bone marrow cavity in VHL mutant mice, though it did not affect any other detectable abnormality of the VHL mutant growth plate. Conversely, HIF-1alpha was epistatic to VHL, as the growth plate of double mutant lacking both VHL and HIF-1alpha appeared to virtually indistinguishable from the mutant growth plate lacking only HIF-1alpha. Taken together, our findings demonstrate that VHL is a critical regulator of bone morphogenesis, as its loss considerably alters size, shape and overall development of the skeletal elements, with mechanisms that are, at least in part HIF-dependent.
VHL and HIF signaling are also known to play critical role in tumor formation and cancer. To better understand the role of Vhl in the biology of mesenchymal cells, we analyzed mutant mice lacking Vhl in mesenchymal progenitors that give rise to the soft tissues that form and surround synovial joints. Loss of Vhl in mesenchymal progenitors of the limb bud caused severe fibrosis of the synovial joints and formation of aggressive masses with histologic features of mesenchymal tumors. Hif-1α and its downstream target connective tissue growth factor (CTGF) were necessary for the development of these tumors, which conversely still developed in the absence of Epas1, but at lower frequency. Our novel findings in genetically altered mice suggest that activation of the HIF signaling pathway could be an important pathogenetic event in the development and progression of at least a subset of these tumors.
Loss of HIF-1alpha in the notochord did not affect the notochord per se, but it led to the complete disappearance of the nucleus pulposus. In particular, morphologic changes subsequent to the loss of HIF-1alpha in the NP cells could be detected as early as E15.5 and they were followed postnatally, by the progressive disappearance and replacement of the nucleus pulposus with a novel tissue that resembled fibrocartilage. Lineage study and TUNEL assay unequivocally proved that mutant NP cells did not transdifferentiate into chondrocyte like-cells, but they underwent massive cell death, and were completely replaced by a cell population belonging to a cell lineage distinct from the notochordal one. In order to evaluate the functional consequences of HIF-1alpha deletion in the NP, biomechanical testing of mutant IVD was also performed. Loss of the NP cells in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress. These findings are similar to the changes usually observed during human IVD degeneration.
To further investigate the role of hypoxia in the nucleus pulposus physiology, we have taken advantage of the FOXA2 Cre; HIF-1alphaf/f mouse model we have generated and described previously. We hypothesize that NP cells adapt to their hypoxic niche through modulation of autophagy. In various cell types, hypoxia is known to induce autophagy in a HIF-1α-dependent fashion, however little is known about hypoxic regulation of autophagy in NP cells. Our study, for the first time shows that NP cells regulate autophagy in a non-canonical fashion independent of mTOR and HIF-1α signaling.
1.4 Expected final results and their potential impact and use
Understanding skeletal tissue biology and especially IVD has become one of the hottest topics in the regenerative medicine field as low back pain has a prevalence of 80% in the general population. In addition to health concern it represents one of the major socio-economic burdens in industrialized countries.
The DISKO mice project allowed the introduction of new notions such as the unique origin of the NP demonstrated by the specific inactivation of HIF-1 alpha in the cells of the notochord. The fact that the loss of HIF-1alpha leads to a dramatic reduction of PGK suggests an important role of HIF-1alpha in the metabolism regulation. HIF pathway could play a major role in the control of the energy consumption at the cell level.
In addition to providing us with fundamental knowledge about the role of the HIF pathway in cell survival, proliferation, differentiation and metabolism, the original FOXA2 Cre; HIF-1 alphaf/f mouse model, generated during this project, mimics the IVD degeneration occurring in human with a progressive disappearance of the NP structure. This mouse model could therefore represent a valuable tool in order to develop new cell-based tissue repair strategies and to identify new molecular targets.
The notions of HIF-1/HIF-2 complementarity and redundancy have still to be explored to address the following questions:
- How hypoxia can be considered as a physiological signal in the process of cell differentiation and organogenesis in the IVD context?
- What are the molecular mechanisms involved in the IVD development and ageing?
- To what extent these molecular mechanisms could represent potential therapeuthic targets?
The obtained and validated results, in addition to original concepts (and questions to be answered) developed during the DISKOmice project will consolidate or bring us to a leadership position in this research field. This valuable asset will allow us to compete on the international scene for the obtainment of future additional financial support, in order to pursue and develop long-term research projects.
Project websites:
http://www.odontologie.univ-nantes.fr/62918803/0/fiche___pagelibre/
https://www.univ-nantes.fr/59987093/0/fiche___pagelibre/&RH=1350647014953