AbstractIntroduction: The regulation of cell fate, such as differentiation to a specialised cell type, continued proliferation, senescence, cell cycle arrest or cell death, is complex and depends on the integration of multiple signals and pathways. The purpose of this PhD has been to examine how ABC transporters, cytokines, and NAD+-dependent signalling influence cell fate in different non-cancer cell models, the potential interplay between these, and the potential therapeutic opportunities of their targeting.
Aims: The aim of this project was to investigate the regulation of cell phenotype and fate by ABC transporter, cytokine, and NAD+-dependent signalling. Four hypotheses were investigated: 1) Human keratinocyte and hair follicle (HF) growth is regulated by the expression and activity of specific ABC transporters and their substrates, and their cross-talk with key inflammatory cytokines, 2) sirtuins as NAD+ dependent protein deacetylases, influence ABCC1 and/or ABCC4 expression, and these ABC transporters, through cAMP signalling effects, can influence sirtuin expression, 3) depletion of NAD+ levels, and decreased NAD+-dependent PARP activity can promote cell senescence, whilst NAD+ precursors can delay the senescent phenotype, and 4) NAD+-dependent SIRT1 is a master regulator of cell fate and suppresses neuronal transdifferentiation of ‘competent’ somatic cells.
Methods: Small molecule inhibitors and RNAi were used to modulate ABC transporters, cytokines, and NAD+-dependent signalling in in vitro and ex vivo HFs. Cell counts, EdU staining, and microscopy were used to monitor proliferation and morphological changes. Senescence was assessed by acidic β-galactosidase staining, and p16 immunoblotting. Gene expression changes were assayed by qPCR, immunoblotting, or immunofluorescence, and protein secretion by ELISA. NAD(H) levels were determined by an enzymatic cycling assay.
Results: ABCC1 inhibition decreased HaCaT keratinocyte proliferation whereas ABCC4 inhibition had minimal impact on keratinocytes or HFs under normal growth conditions. However, small molecule inhibition of ABCC4 rescued HFs from IL-1β induced catagen, potentially elongating the anagen stage of the HF cycle. ABCC1 and ABCC4 inhibition decreased extracellular cAMP and PGE2 secretion, suggesting possible cAMP involvement in HF regression. The relationship between ABCC1/4 and sirtuin expression appeared to be cell context-specific and complex. ABCC1 Inhibition reduced SIRT6 expression whereas SIRT1 inhibition decreased ABCC4 mRNA and cAMP levels. NAD(H) declined in replicatively ‘old’ cells, but PARP activity was sustained potentially due to nuclear NMNAT1 and preferential channelling of NAD+ to the nucleus. Nicotinic acid treatment did not rescue cells from replicative or oxidative stress induced senescence. Inhibiting or silencing SIRT1 induced a neuronal-like phenotype in both epithelial and fibroblast cells, indicative of neuronal transdifferentiation. MITF and TACC3 were investigated as downstream mediators revealing a potential feedback loop. KHS101 also induced a neuronal-like phenotype in ARPE-19 epithelial cells as well as abnormal protein aggregates. However, both KHS101 and SIRT1 induced ‘differentiation’ phenotypes were reversible.
Discussion & Conclusion: These findings suggest a crucial role for ABCC4 in regulating hair follicle growth, potentially mediated through cAMP signalling. ABCC1’s effects and other inflammatory mediators require further investigation. In ‘old’ cells, NAD(H) decline was observed, but PARP activity was sustained, potentially due to NMNAT1 upregulation. The effect of PARP inhibitors on senescence would help to decipher the contribution of PARP versus NAD+ decline to senescence. SIRT1 emerged as a key regulator of cell fate, it’s targeting inducing neuronal-like transdifferentiation. Mechanistic insights and a fuller understanding of transdifferentiation and the roles of SIRT1 and KHS101 are needed. This PhD sheds light on how ABC transporters, cytokines, and NAD+-dependent signalling influence cell fate. ABCC4 has a potential role in hair follicle regulation and the findings regarding NAD+ and senescence provide valuable insights into cell ageing. The influence of SIRT1 and KHS101 on transdifferentiation has implications for neurodegenerative diseases. These results underscore the importance of understanding these mechanisms in the context of cell fate regulation.
|Date of Award
|29 Sep 2023
|Simon Allison (Main Supervisor)