Role of p16ink4a and bmi-1 in oxidative stress-induced premature senescence in human dental pulp stem cells
- Autores: Cristina Mas Bargues
- Directores de la Tesis: José Viña Ribes (dir. tes.), Juan Gambini Buchón (codir. tes.), Consuelo Borrás Blasco (codir. tes.)
- Lectura: En la Universitat de València ( España ) en 2017
- Idioma: español
- Tribunal Calificador de la Tesis: Manuel Serrano Marugán (presid.), Federico Pallardó Calatayud (secret.), Niki Chondrogianni (voc.)
- Programa de doctorado: Programa de Doctorado en Fisiología por la Universitat de València (Estudi General)
- Materias:
- Texto completo no disponible (Saber más ...)
- Resumen
Human mesenchymal stem cells (MSCs) have a therapeutic potential in tissue engineering and regenerative medicine. Human dental pulp stem cells (hDPSCs) have proven to be a good source for cell therapy as pulp tissue is easily available from teeth after extraction without ethical issues. Cell therapy requires a large number of cells, thus, an in vitro expansion step is required before implantation. Long-term in vitro culture entails the inconvenience of senescence following a certain number of passages, thereby losing hDPSCs stemness properties and regenerative potential. Currently, the in vitro culture of MSCs is carried out under ambient oxygen tension (21% pO2). However, the local oxygen tension varies between 3-6% pO2 within the organism depending on the vascularization of the tissue and its metabolic activity. Mimicking physiological conditions in cell culture experiments is a useful tool to gain knowledge. Thus, the aim of this study was to compare long-term in vitro culture of hDPSCs under ambient (21% pO2) and physiological (3% pO2) oxygen partial pressure, and to determine whether hyperoxia can alter the physiology and affect the senescence of normal adult stem cells.
First, we investigated if ambient oxygen tension could induce oxidative stress in hDPSCs during long-term culture. Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species (ROS) and the biological ability of a system to readily detoxify the intermediates and to repair the resulting damage. Oxidative stress is known to produce damage to biomolecules such as DNA, carbohydrates, lipids and proteins. Therefore, we assessed ROS levels, mitochondrial membrane potential, protein and lipid oxidation, as well as antioxidant gene expression during long-term hDPSCs in vitro culture. Our data revealed increased ROS levels, protein carbonylation and lipid oxidation, and a disruption of the mitochondrial membrane potential in the cells that were cultured under ambient oxygen tension. Furthermore, these cells showed an upregulation of the expression of manganese superoxide dismutase, catalase and glutathione peroxidase genes, suggesting an increased antioxidant defence to withstand increasing ROS production. Thus, ambient oxygen tension induces oxidative stress in hDPSCs in vitro culture.
Senescence was described as an irreversible state of cell cycle arrest, which is accompanied by morphological alterations, reduced proliferation rate, apoptosis resistance and increased expression of senescence-associated β-galactosidase activity and p16INK4a. The CDKN2A gene, also known as the INK/ARF locus, encodes both p16INK4a and p14ARF, which are cell cycle regulators. The p16INK4a protein inhibits cyclin D-dependent CDK4 and CDK6 to prevent phosphorylation of the retinoblastoma protein (pRb). The hypophosphorylated form of pRb sequesters E2F transcription factors, preventing them from coordinately activating a suite of genes required for DNA replication. The p14ARF protein binds to the MDM2 E3 ubiquitin ligase to prevent p53 polyubiquitylation and to facilitate p53 activation. In turn, the p53 transcription factor regulates an extensive group of genes that are commonly induced by cellular stress leading to apoptosis. We then determined some senescence characteristics in hDPSCs cultured under both oxygen tension conditions. Cells that were cultured under ambient oxygen tension rapidly began to show enlarged and flattened phenotypes and decreased their regenerative potential as they only reached 15 passages, while those cells that were cultured under physiological oxygen tension reached 25 passages and preserved a “younger” phenotype. Accordingly, 21% pO2 was accompanied by increased β-galactosidase activity, increased expression of p16INK4a and reduced expression of p14ARF. Taken together, our results suggest that oxidative stress induces a premature senescence of hDPSCs cultured under ambient oxygen tension.
Another characteristic that is not included in the definition of stem cell senescence, but should be taken into consideration, is the loss of stemness properties. hDPSCs are adult stem cells that express pluripotency-related genes. Those genes are the four transcription factors: OCT4, SOX2, KLF4 and c-MYC, abbreviated to, “OSKM”, and they are involved in the induction and maintenance of pluripotency. hDPSCs cultured at 3% pO2 showed high levels of OCT4 and SOX2 at early stages, but their expression was downregulated as passages accumulated. However, at advanced passages, these cells upregulated the KLF4 and c-MYC expression. On the other hand, hDPSCs cultured at 21% pO2 showed a downregulation of all four OSKM factors along passages. Taken together, our data suggest that ambient oxygen tension entails a loss of the stemness properties in long-term in vitro culture.
Finally, we aimed to investigate a possible relation between p16INK4a and OSKM expression in hDPSCs. A well described upstream regulator of the INK/ARF locus is BMI-1. Through repression of target gene expression, the BMI-1 protein regulates a myriad of cellular processes critical for cell growth, cell fate decision, development, senescence, apoptosis, and self-renewal of stem cells. Thus, we tested whether BMI-1 regulates hDPSCs fate while modulating p16INK4a and OSKM gene expression. To this end, we first analysed BMI-1 protein expression in hDPSCs in long-term culture under either oxygen pressure percentage. Our results show that hDPSCs cultured at 3% pO2 retained constant levels of BMI-1 along passages. However, hDPSCs cultured at 21% pO2 showed higher BMI-1 protein levels at early stages, which rapidly plummeted as passages accumulated. These data suggest that ambient oxygen tension accelerated BMI-1 protein degradation. We next proceeded to silence BMI-1 gene expression in hDPSCs cultured under ambient oxygen tension by siRNA transfection. Knocked-down hDPSCs exhibited the same amount of BMI-1 protein as hDPSCs cultured under physiological oxygen tension. Interestingly, following transfection, p16INK4a expression was not altered, but OCT4 and SOX2 expression levels were upregulated.
Taken together, we can conclude that in vitro culture carried out under ambient oxygen tension causes an oxidative stress-induced premature senescence and a loss of the stemness properties of hDPSCs. Moreover, BMI-1 levels should be kept in a balance that allows normal stem cell proliferation, while preventing stem cell senescence thereby maintaining proper stem cell homeostasis.
