With age, the epidermis becomes hypoplastic and hypoproliferative. Hypoproliferation due to aging has been associated with decreased stem cell (SC) self‐renewal in multiple murine tissues. The fate of SC self‐renewal divisions can be asymmetric (one SC, one committed progenitor) or symmetric (two SCs). Increased asymmetric SC self‐renewal has been observed in inflammatory‐mediated hyperproliferation, while increased symmetric SC self‐renewal has been observed in cancers. We analyzed SC self‐renewal divisions in aging human epidermis to better understand the role of SCs in the hypoproliferation of aging. In human subjects, neonatal to 78 years, there was an age‐dependent decrease in epidermal basal layer divisions. The balance of SC self‐renewal shifted toward symmetric SC self‐renewal, with a decline in asymmetric SC self‐renewal. Asymmetric SC divisions maintain epidermal stratification, and this decrease may contribute to the hypoplasia of aging skin. P53 decreases in multiple tissues with age, and p53 has been shown to promote asymmetric SC self‐renewal. Fewer aged than adult ALDH+CD44+ keratinocyte SCs exhibited p53 expression and activity and Nutlin‐3 (a p53 activator) returned p53 activity as well as asymmetric SC self‐renewal divisions to adult levels. Nutlin‐3 increased Notch signaling (NICD, Hes1) and DAPT inhibition of Notch activation prevented Nutlin‐3 (p53)‐induced asymmetric SC self‐renewal divisions in aged keratinocytes. These studies indicate a role for p53 in the decreased asymmetric SC divisions with age and suggest that in aged keratinocytes, Notch is required for p53‐induced asymmetric SC divisions.
We previously described the effects and behaviour of the main circulating active androgen testosterone (Testo) in an androgen-sensitive sebocyte cell line. Testosterone and DHT readily induce androgen receptor (AR) translocation, expression of target genes and finally a lipogenic differentiation program. DHEA is not significantly transformed into Testo, had no significant androgenic (agonist), nor anti-androgenic (antagonist) effect by itself in the tested conditions. Here we analysed the activity of reference anti-androgens (Finasteride, Dutasteride, Cyproterone acetate), both at the level of their potential targets (5-alpha-reductase, AR translocation, induced transcripts) and in the functional lipid accumulation assay. As expected, because the 5-alpha-reductase type 1 predominates in this model, Dutasteride was found 100-fold more active than Finasteride in inhibiting DHT production. Cyproterone acetate (CypAc) did not interfere with Testo metabolism. Regarding the effects on testosterone or DHT early-induced gene RASD1(1), CypAc was fully active in the 10 nM range, while Dutasteride and Finasteride were active only over the micromolar range, this when Testo was the inducer. CypAc did not interfere with DHT-induced AR translocation, suggesting that its activity targets the intra-nuclear transcription process. Regarding the diversity of the metabolite profile, and the diversity of the mechanisms it was important to test the potential inhibitors in a functional assay relevant of the biological effects of interest. In the validated 7 days lipid accumulation assay (1), CypAc and Dutasteride were found the most potent, but active concentrations were in the micromolar range. We further used this assay to evaluate other/new potential anti-androgens, as well as miscellaneous compounds that could interfere with androgen-induced lipid accumulation in sebocytes. (1) Barrault et al. (2015) J Steroid Biochem Mol Biol ;152:34-44.
While proliferation is decreased in aged tissues, no change in SC number was detected by our group or Bickenbachs. Lechler et al. examined types of SC divisions. Divisions perpendicular to the basement membrane were 8% in single-layered embryonic epidermis, 66% in multilayer epidermis and 85% in adult mice. In humans there is a decrease in cells positive for the proliferation marker Ki67. Our aim was to quantify mitoses of basal keratinocytes in vivo, and examine the types of division, over the human lifespan. We quantified divisions in the basal layer in neonatal (0-7 days), adult (30-45 years) and aged (70-80 years) skin. Asymmetric segregation of polarity proteins is a defining characteristic of SCs. We visualized division orientation in the basal layer using gtubulin (spindle) and NUMA (polarity protein) in biopsies. Because of the limited divisions seen in vivo, we also quantified division type in keratinocytes from freshly obtained biopsies, in vitro, using Numb segregation to identify asymmetric SC divisions. The number of divisions/10cm of basal layer was significantly decreased with age [neonatal 7.6AE2 (n¼5) vs. adult 3.2AE0.5 (n¼4) vs. aged 1.5AE 0.4 (n¼8), p¼0.001]. Both perpendicular (asymmetric) and parallel (symmetric) divisions were decreased with age. No significant change in the proportion of perpendicular (asymmetric) SC divisions was detected (neonatal 41.7AE14.4% vs. adult 27.5AE15.9% vs. aged 36.8AE8%). Similar results were found in vitro [neonatal 29.1AE7.4% (n¼11) vs. adult 32.2% (n¼ 2) vs. aged 31.2AE11.6% (n¼ 7)]. We conclude that epidermal basal cell divisions decrease over the human lifespan such that the number of divisions in 70+ year olds is 20% of that in neonates. However, the ratio of asymmetric/symmetric divisions remains unchanged. These findings could reflect a decrease in SC number, an increase in SC quiescence, and/or longer cell cycle duration in human epidermis.
Global regulation of gene expression is essential for appropriate epidermal stratification. One set of global regulators is the histone deacetylase (HDAC) family; HDACs repress transcription by removing acetyl groups from histones. HDAC inhibitors are used to treat cutaneous malignancies, but broadly inhibit all class I HDAC family members and have side effects. Delineating the activities of individual class I HDACs in the skin will be important for the development of improved therapeutics. To address this, we are using loss of function genetic analyses in mice. We find that epidermal deletion of Hdac3 during embryonic life results in failure to form a functional barrier and perinatal lethality; this phenotype is distinct from that caused by deletion of Hdac1/2 but partially overlaps with the effects of loss of the transcription factor KLF4, suggesting a possible interaction of HDAC3 and KLF4. Transcriptional profiling of Hdac3 and Klf4 embryonic mutant epidermis reveals statistically significant overlap in dysregulated genes, including epidermal differentiation and danger response genes. ChIP-seq experiments identify statistically significant overlap in sites bound by HDAC3 and KLF4, and proximity ligation assays reveal direct HDAC3/KLF4 interaction in suprabasal cells. In vitro data suggest that HDAC3 functions in a complex with NCoR/SMRT. To determine whether HDAC3 function is dependent on interaction with NCoR/SMRT in the epidermis in vivo, we have deleted the genes coding for these proteins. Ncor/Smrt double mutants recapitulate the epidermal barrier defect and gene expression changes observed in Hdac3 mutant epidermis. Intriguingly, total HDAC3 protein is severely decreased in Ncor/Smrt mutant epidermis, demonstrating that NCoR/SMRT function to stabilize HDAC3 in keratinocytes. Together, our data indicate that HDAC3 has distinct functions from HDAC1/2 and associates with KLF4 and NCoR/SMRT to repress gene expression in the developing epidermis.
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