Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Obesity accelerates hair thinning by stem cell-centric converging mechanisms.

Obesity is a worldwide epidemic that predisposes individuals to many age-associated diseases, but its exact effects on organ dysfunction are largely unknown1. Hair follicles-mini-epithelial organs that grow hair-are miniaturized by ageing to cause hair loss through the depletion of hair follicle stem cells (HFSCs)2. Here we report that obesity-induced stress, such as that induced by a high-fat diet (HFD), targets HFSCs to accelerate hair thinning. Chronological gene expression analysis revealed that HFD feeding for four consecutive days in young mice directed activated HFSCs towards epidermal keratinization by generating excess reactive oxygen species, but did not reduce the pool of HFSCs. Integrative analysis using stem cell fate tracing, epigenetics and reverse genetics showed that further feeding with an HFD subsequently induced lipid droplets and NF-κB activation within HFSCs via autocrine and/or paracrine IL-1R signalling. These integrated factors converge on the marked inhibition of Sonic hedgehog (SHH) signal transduction in HFSCs, thereby further depleting lipid-laden HFSCs through their aberrant differentiation and inducing hair follicle miniaturization and eventual hair loss. Conversely, transgenic or pharmacological activation of SHH rescued HFD-induced hair loss. These data collectively demonstrate that stem cell inflammatory signals induced by obesity robustly represses organ regeneration signals to accelerate the miniaturization of mini-organs, and suggests the importance of daily prevention of organ dysfunction.

Stem cell competition orchestrates skin homeostasis and ageing.

Stem cells underlie tissue homeostasis, but their dynamics during ageing-and the relevance of these dynamics to organ ageing-remain unknown. Here we report that the expression of the hemidesmosome component collagen XVII (COL17A1) by epidermal stem cells fluctuates physiologically through genomic/oxidative stress-induced proteolysis, and that the resulting differential expression of COL17A1 in individual stem cells generates a driving force for cell competition. In vivo clonal analysis in mice and in vitro 3D modelling show that clones that express high levels of COL17A1, which divide symmetrically, outcompete and eliminate adjacent stressed clones that express low levels of COL17A1, which divide asymmetrically. Stem cells with higher potential or quality are thus selected for homeostasis, but their eventual loss of COL17A1 limits their competition, thereby causing ageing. The resultant hemidesmosome fragility and stem cell delamination deplete adjacent melanocytes and fibroblasts to promote skin ageing. Conversely, the forced maintenance of COL17A1 rescues skin organ ageing, thereby indicating potential angles for anti-ageing therapeutic intervention.

Genotoxic stress abrogates renewal of melanocyte stem cells by triggering their differentiation.

Somatic stem cell depletion due to the accumulation of DNA damage has been implicated in the appearance of aging-related phenotypes. Hair graying, a typical sign of aging in mammals, is caused by the incomplete maintenance of melanocyte stem cells (MSCs) with age. Here, we report that irreparable DNA damage, as caused by ionizing radiation, abrogates renewal of MSCs in mice. Surprisingly, the DNA-damage response triggers MSC differentiation into mature melanocytes in the niche, rather than inducing their apoptosis or senescence. The resulting MSC depletion leads to irreversible hair graying. Furthermore, deficiency of Ataxia-telangiectasia mutated (ATM), a central transducer kinase of the DNA-damage response, sensitizes MSCs to ectopic differentiation, demonstrating that the kinase protects MSCs from their premature differentiation by functioning as a "stemness checkpoint" to maintain the stem cell quality and quantity.

Mitf is required for T cell maturation by regulating dendritic cell homing to the thymus.

Thymic dendritic cells (DCs) promote immune tolerance by regulating negative selection of autoreactive T cells in the thymus. How DC homing to the thymus is transcriptionally regulated is still unclear. Microphthalmia-associated transcription factor (Mitf) is broadly expressed and plays essential roles in the hematopoietic system. Here, we used Mitf-mutated mice (Mitfvit/vit) and found enlargement of the thymus and expansion of CD4/CD8 double-positive T cells. Mitf was highly expressed in a subset of thymic DCs among the hematopoietic system. Genetic mutation or pharmacological inhibition of Mitf in DCs decreased the expression levels of Itga4, which are critical molecules for the homing of DCs to the thymus. Further, inhibition of Mitf decreased thymic DC number. These results suggest a pivotal role of Mitf in the maintenance of T cell differentiation by regulating the homing of DC subsets within the thymus.

Label-free quality control and identification of human keratinocyte stem cells by deep learning-based automated cell tracking.

Stem cell-based products have clinical and industrial applications. Thus, there is a need to develop quality control methods to standardize stem cell manufacturing. Here, we report a deep learning-based automated cell tracking (DeepACT) technology for noninvasive quality control and identification of cultured human stem cells. The combination of deep learning-based cascading cell detection and Kalman filter algorithm-based tracking successfully tracked the individual cells within the densely packed human epidermal keratinocyte colonies in the phase-contrast images of the culture. DeepACT rapidly analyzed the motion of individual keratinocytes, which enabled the quantitative evaluation of keratinocyte dynamics in response to changes in culture conditions. Furthermore, DeepACT can distinguish keratinocyte stem cell colonies from non-stem cell-derived colonies by analyzing the spatial and velocity information of cells. This system can be widely applied to stem cell cultures used in regenerative medicine and provides a platform for developing reliable and noninvasive quality control technology.

Inhibition of PAX3 by TGF-beta modulates melanocyte viability.

The protein encoded by paired-box homeotic gene 3 (PAX3) is a key regulator of the microphthalmia-associated transcription factor (Mitf) in the melanocyte lineage. Here, we show that PAX3 expression in skin is directly inhibited by TGF-beta/Smads. UV irradiation represses TGF-beta in keratinocytes, and the repression of TGF-beta/Smads upregulates PAX3 in melanocytes, which is associated with a UV-induced melanogenic response and consequent pigmentation. Furthermore, the TGF-beta-PAX3 signaling pathway interacts with the p53-POMC/MSH-MC1R signaling pathway, and both are crucial in melanogenesis. The activation of p53-POMC/MSH-MC1R signaling is required for the UV-induced melanogenic response because PAX3 functions in synergy with SOX10 in a cAMP-response element (CRE)-dependent manner to regulate the transcription of Mitf. This study will provide a rich foundation for further research on skin cancer prevention by enabling us to identify targeted small molecules in the signaling pathways of the UV-induced melanogenic response that are highly likely to induce naturally protective pigmentation.

Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF.

The genomic organization of the CDK2 gene, which overlaps the melanocyte-specific gene SILV/PMEL17, poses an interesting regulatory challenge. We show that, despite its ubiquitous expression, CDK2 exhibits tissue-specific regulation by the essential melanocyte lineage transcription factor MITF. In addition, functional studies revealed this regulation to be critical for maintaining CDK2 kinase activity and growth of melanoma cells. Expression levels of MITF and CDK2 are tightly correlated in primary melanoma specimens and predict susceptibility to the CDK2 inhibitor roscovitine. CDK2 depletion suppressed growth and cell cycle progression in melanoma, but not other cancers, corroborating previous results. Collectively, these data indicate that CDK2 activity in melanoma is largely maintained at the transcriptional level by MITF, and unlike other malignancies, it may be a suitable drug target in melanoma.

Topical drug rescue strategy and skin protection based on the role of Mc1r in UV-induced tanning.

Ultraviolet-light (UV)-induced tanning is defective in numerous 'fair-skinned' individuals, many of whom contain functional disruption of the melanocortin 1 receptor (MC1R). Although this suggested a critical role for the MC1R ligand melanocyte stimulating hormone (MSH) in this response, a genetically controlled system has been lacking in which to determine the precise role of MSH-MC1R. Here we show that ultraviolet light potently induces expression of MSH in keratinocytes, but fails to stimulate pigmentation in the absence of functional MC1R in red/blonde-haired Mc1r(e/e) mice. However, pigmentation could be rescued by topical application of the cyclic AMP agonist forskolin, without the need for ultraviolet light, demonstrating that the pigmentation machinery is available despite the absence of functional MC1R. This chemically induced pigmentation was protective against ultraviolet-light-induced cutaneous DNA damage and tumorigenesis when tested in the cancer-prone, xeroderma-pigmentosum-complementation-group-C-deficient genetic background. These data emphasize the essential role of intercellular MSH signalling in the tanning response, and suggest a clinical strategy for topical small-molecule manipulation of pigmentation.

Intracrine activity involving NAD-dependent circadian steroidogenic activity governs age-associated meibomian gland dysfunction.

Canonically, hormones are produced in the endocrine organs and delivered to target tissues. However, for steroids, the concept of tissue intracrinology, whereby hormones are produced in the tissues where they exert their effect without release into circulation, has been proposed, but its role in physiology/disease remains unclear. The meibomian glands in the eyelids produce oil to prevent tear evaporation, which reduces with aging. Here, we demonstrate that (re)activation of local intracrine activity through nicotinamide adenine dinucleotide (NAD+)-dependent circadian 3ß-hydroxyl-steroid dehydrogenase (3ß-HSD) activity ameliorates age-associated meibomian gland dysfunction and accompanying evaporative dry eye disease. Genetic ablation of 3ß-HSD nullified local steroidogenesis and led to atrophy of the meibomian gland. Conversely, reactivation of 3ß-HSD activity by boosting its coenzyme NAD+ availability improved glandular cell proliferation and alleviated the dry eye disease phenotype. Both women and men express 3ß-HSD in the meibomian gland. Enhancing local steroidogenesis may help combat age-associated meibomian gland dysfunction.

EGFR-mediated epidermal stem cell motility drives skin regeneration through COL17A1 proteolysis.

Skin regenerative capacity declines with age, but the underlying mechanisms are largely unknown. Here we demonstrate a functional link between epidermal growth factor receptor (EGFR) signaling and type XVII collagen (COL17A1) proteolysis on age-associated alteration of keratinocyte stem cell dynamics in skin regeneration. Live-imaging and computer simulation experiments predicted that human keratinocyte stem cell motility is coupled with self-renewal and epidermal regeneration. Receptor tyrosine kinase array identified the age-associated decline of EGFR signaling in mouse skin wound healing. Culture experiments proved that EGFR activation drives human keratinocyte stem cell motility with increase of COL17A1 by inhibiting its proteolysis through the secretion of tissue inhibitor of metalloproteinases 1 (TIMP1). Intriguingly, COL17A1 directly regulated keratinocyte stem cell motility and collective cell migration by coordinating actin and keratin filament networks. We conclude that EGFR-COL17A1 axis-mediated keratinocyte stem cell motility drives epidermal regeneration, which provides a novel therapeutic approach for age-associated impaired skin regeneration.

Dynamic stem cell selection safeguards the genomic integrity of the epidermis.

Maintaining genomic integrity and stability is crucial for life; yet, no tissue-driven mechanism that robustly safeguards the epithelial genome has been discovered. Epidermal stem cells (EpiSCs) continuously replenish the stratified layers of keratinocytes that protect organisms against various environmental stresses. To study the dynamics of DNA-damaged cells in tissues, we devised an in vivo fate tracing system for EpiSCs with DNA double-strand breaks (DSBs) and demonstrated that those cells exit from their niches. The clearance of EpiSCs with DSBs is caused by selective differentiation and delamination through the DNA damage response (DDR)-p53-Notch/p21 axis, with the downregulation of ITGB1. Moreover, concomitant enhancement of symmetric cell divisions of surrounding stem cells indicates that the selective elimination of cells with DSBs is coupled with the augmented clonal expansion of intact stem cells. These data collectively demonstrate that tissue autonomy through the dynamic coupling of cell-autonomous and non-cell-autonomous mechanisms coordinately maintains the genomic quality of the epidermis.

Stem cell spreading dynamics intrinsically differentiate acral melanomas from nevi.

Early differential diagnosis between malignant and benign tumors and their underlying intrinsic differences are the most critical issues for life-threatening cancers. To study whether human acral melanomas, deadly cancers that occur on non-hair-bearing skin, have distinct origins that underlie their invasive capability, we develop fate-tracing technologies of melanocyte stem cells in sweat glands (glandular McSCs) and in melanoma models in mice and compare the cellular dynamics with human melanoma. Herein, we report that glandular McSCs self-renew to expand their migratory progeny in response to genotoxic stress and trauma to generate invasive melanomas in mice that mimic human acral melanomas. The analysis of melanocytic lesions in human volar skin reveals that genetically unstable McSCs expand in sweat glands and in the surrounding epidermis in melanomas but not in nevi. The detection of such cell spreading dynamics provides an innovative method for an early differential diagnosis of acral melanomas from nevi.

Distinct types of stem cell divisions determine organ regeneration and aging in hair follicles.

Hair follicles, mammalian mini-organs that grow hair, miniaturize during aging, leading to hair thinning and loss. Here we report that hair follicle stem cells (HFSCs) lose their regenerative capabilities during aging owing to the adoption of an atypical cell division program. Cell fate tracing and cell division axis analyses revealed that while HFSCs in young mice undergo typical symmetric and asymmetric cell divisions to regenerate hair follicles, upon aging or stress, they adopt an atypical 'stress-responsive' type of asymmetric cell division. This type of division is accompanied by the destabilization of hemidesmosomal protein COL17A1 and cell polarity protein aPKCλ and generates terminally differentiating epidermal cells instead of regenerating the hair follicle niche. With the repetition of these atypical divisions, HFSCs detach from the basal membrane causing their exhaustion, elimination and organ aging. The experimentally induced stabilization of COL17A1 rescued organ homeostasis through aPKCλ stabilization. These results demonstrate that distinct stem cell division programs may govern tissue and organ aging.

Mutant ASXL1 induces age-related expansion of phenotypic hematopoietic stem cells through activation of Akt/mTOR pathway.

Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.

Evaluation of the proliferative potential of skin keratinocytes and fibroblasts isolated from critical limb ischemia patients.

Impaired wound healing in critical limb ischemia (CLI) results from multiple factors that affect many cell types and their behavior. Epidermal keratinocytes and dermal fibroblasts play crucial roles in wound healing. However, it remains unclear whether these cell types irreversibly convert into a non-proliferative phenotype and are involved in impaired wound healing in CLI. Here, we demonstrate that skin keratinocytes and fibroblasts isolated from CLI patients maintain their proliferative potentials. Epidermal keratinocytes and dermal fibroblasts were isolated from the surrounding skin of foot wounds in CLI patients with diabetic nephropathy on hemodialysis, and their growth potentials were evaluated. It was found that keratinocytes from lower limbs and trunk of patients can give rise to proliferative growing colonies and can be serially passaged. Fibroblasts can also form colonies with a proliferative phenotype. These results indicate that skin keratinocytes and fibroblasts maintain their proliferative capacity even in diabetic and ischemic microenvironments and can be reactivated under appropriate conditions. This study provides strong evidence that the improvement of the cellular microenvironments is a promising therapeutic approach for CLI and these cells can also be used for potential sources of skin reconstruction.

NUAK2 localization in normal skin and its expression in a variety of skin tumors with YAP.

BACKGROUND: NUAK2 is a critical gene that participates in the carcinogenesis of various types of cancers including melanomas. However, the expression patterns of NUAK2 in normal skin and in various types of skin tumors have not been fully elucidated to date. OBJECTIVES: To elucidate the distribution and localization of NUAK2 expression in normal skin, and characterize the expression patterns of NUAK2 and YAP in various types of skin tumors. METHODS: In this study, we characterized the expression of NUAK2 in tissues by developing a novel NUAK2-specific monoclonal antibody and using that to determine NUAK2 expression patterns in normal skin and in 155 cases of various types of skin tumors, including extramammary Paget's disease (EMPD), squamous cell carcinoma (SCC), Bowen's disease (BD), actinic keratosis (AK), basal cell carcinoma (BCC) and angiosarcoma (AS). Further, we analyzed the expression patterns of YAP and p-Akt in those tumors. RESULTS: Our analyses revealed that NUAK2 is expressed at high frequencies in EMPD, SCC, BD, AK, BCC and AS. The expression of p-Akt was positively correlated with tumor size in EMPD (P = 0.001). Importantly, the expression of NUAK2 was significantly correlated with YAP in SCC (P = 0.012) and in BD (P = 0.009). CONCLUSIONS: Our results suggest that the YAP-NUAK2 axis has critical importance in the tumorigenesis of SCC and BD, and that therapeutic modalities targeting the YAP-NUAK2 axis may be an effective approach against skin tumors including SCC and BD.

Epistatic connections between microphthalmia-associated transcription factor and endothelin signaling in Waardenburg syndrome and other pigmentary disorders.

Waardenburg syndrome (WS) is an inherited sensorineural deafness condition in humans caused by melanocyte deficiencies in the inner ear and forelock. Mutation of microphthalmia-associated transcription factor (MITF) is known to produce WS type IIA whereas mutations of either endothelin (EDN) or its receptor endothelin receptor B (EDNRB) produce WS type IV. However, a link between MITF haploinsufficiency and EDN signaling has not yet been established. Here we demonstrate mechanistic connections between EDN and MITF and their functional importance in melanocytes. Addition of EDN to cultured human melanocytes stimulated the phosphorylation of MITF in an EDNRB-dependent manner, which was completely abolished by mitogen-activated protein kinase kinase inhibition. The expression of melanocyte-specific MITF mRNA transcripts was markedly augmented after incubation with EDN1 and was followed by increased expression of MITF protein. Up-regulated expression of MITF was found to be mediated via both the mitogen-activated protein kinase-p90 ribosomal S6 kinase-cAMP response element-binding protein (CREB) and cAMP-protein kinase A-CREB pathways. In addition, EDNRB expression itself was seen to be dependent on MITF. The functional importance of these connections is illustrated by the ability of EDN to stimulate expression of melanocytic pigmentation and proliferation markers in an MITF-dependent fashion. Collectively these data provide mechanistic and epistatic links between MITF and EDN/EDNRB, critical melanocytic survival factors and WS genes.