AbstractThe importance of cholesterol in hair follicle (HF) biology is underscored via links between cholesterol and both the pathogenesis of alopecias and hair growth. Defects in at least one ATP binding cassette (ABC) transporter (ABCA5) alter intracellular cholesterol distribution, leading to congenital hypertrichosis. The underlying mechanisms by which cholesterol levels, and cholesterol transport and compartmentalisation, influence keratinocyte behaviour within HF cell populations is largely unknown. The aim of this study was to characterise the routes of cholesterol transport within the HF and the effects for modulation of cholesterol levels on hair growth and cycling.
Primary keratinocytes isolated from the outer root sheath (ORS) of plucked human HFs were utilised as a model cell system, along with organ cultured human HFs, to examine their responsiveness to cholesterol loading via free cholesterol and depletion via methyl-β-cyclodextrin. Exogenous cholesterol loading in addition to siRNA-mediated knockdown of ABCA5 was performed in ORS keratinocytes. Exploration into cholesterol-sensitive target genes, cholesterol compartmentalisation and cellular behaviour were identified via numerous assays.
Liver X receptor (LXR) agonism demonstrated active regulation of ABCA1 and ABCG1, but not ABCA5 or SCARB1, in human HFs and ORS keratinocytes via qPCR, in addition to LXR regulating cholesterol efflux to apolipoprotein A1 and high-density lipoprotein. ABCA5 colocalised to intracellular organelles with cholesterol loading. Western blotting analysis of ABCA5 revealed additional isoforms at 300 and 400 kDa. The HFs capability to handle excess cholesterol and cholesterol depletion, along with small increases and decreases in Wnt signalling targets, respectively was detected. Methyl-β-cyclodextrin revealed a reduction in cell viability and cellular senescence via β-galactose staining, but no changes in proliferation. Immunofluorescence microscopy in human HF sections revealed differential expression of ABC transporters across the hair cycle. SCARB1 was highly expressed in the dermal papilla basement membrane. Staining for free cholesterol (filipin) revealed prominent cholesterol striations within the basement membrane of the hair bulb. ABCA5 siRNA revealed a dysregulation in cholesterol homeostasis, and partial recovery of cholesterol homeostatic genes with LXR agonist T0901317 was detected. Filipin staining and live BODIPY cholesterol immunofluorescence microscopy revealed reduction of endo-lysosomal cholesterol with ABCA5 knockdown. Analysis of oxysterols via liquid chromatography mass spectrometry revealed significant differences in 25-hydroxycholesterol and 7-β-hydroxycholesterol following cholesterol loading in ORS keratinocytes with ABCA5 knockdown.
The maintenance of cholesterol homeostasis is vital for normal cellular function. As a pre-cursor for steroid hormone synthesis and regulator of signalling pathways associated with HF growth and cycling (i.e. Wnt/β-catenin, Shh), changes in cellular cholesterol could have wide-ranging implications for skin and hair biology. These results demonstrate the capacity of human HFs for cholesterol transport and trafficking. Furthermore, data shown here demonstrate a role for ABCA5 in the intracellular compartmentalisation of free cholesterol in primary HF keratinocytes. Reduced movement of cholesterol to APOA1 could indicate an indirect role for ABCA5 in the delivery of free cholesterol for ABCA1-mediated efflux. Crucially, the loss of normal homeostatic response to excess cholesterol delivery, following ABCA5 knockdown, suggests an impact on LXRmediated transcriptional activity. We therefore speculate that the loss of ABCA5 leads to impaired endo-lysosomal cholesterol transport and thus alterations in signalling pathways such as JAK/STAT and Shh which could lead to alterations in hair growth. Further research should investigate the role of ABCA5 in modulating cholesterol homeostasis with a focus on signalling pathways associated with HF morphogenesis and cycling.
|Date of Award||2023|
|Supervisor||Iain Haslam (Main Supervisor) & Nik Georgopoulos (Co-Supervisor)|