Activation of Nrf2 in fibroblasts promotes a skin aging phenotype via an Nrf2-miRNA-collagen axis.

Aging is associated with progressive skin fragility and a tendency to tear, which can lead to severe clinical complications. The transcription factor NRF2 is a key regulator of the cellular antioxidant response, and pharmacological NRF2 activation is a promising strategy for the prevention of age-related diseases. Using a combination of molecular and cellular biology, histology, imaging and biomechanical studies we show, however, that constitutive genetic activation of Nrf2 in fibroblasts of mice suppresses collagen and elastin expression, resulting in reduced skin strength as seen in aged mice. Mechanistically, the "aging matrisome" results in part from direct Nrf2-mediated overexpression of a network of microRNAs that target mRNAs of major skin collagens and other matrix components. Bioinformatics and functional studies revealed high NRF2 activity in aged human fibroblasts in 3D skin equivalents and human skin biopsies, highlighting the translational relevance of the functional mouse data. Together, these results identify activated NRF2 as a promoter of age-related molecular and biomechanical skin features.

Adverse Effects of Vemurafenib on Skin Integrity: Hyperkeratosis and Skin Cancer Initiation Due to Altered MEK/ERK-Signaling and MMP Activity.

The BRAF inhibitor vemurafenib, approved for treating patients with BRAF V600E-mutant and unresectable or metastatic melanomas, rapidly induces cutaneous adverse events, including hyperkeratotic skin lesions and cutaneous squamous cell carcinomas (cSCC). To determine, how vemurafenib would provoke these adverse events, we utilized long-term in vitro skin equivalents (SEs) comprising epidermal keratinocytes and dermal fibroblasts in their physiological environment. We inserted keratinocytes with different genetic background [normal keratinocytes: NHEK, HaCaT (p53/mut), and HrasA5 (p53/mut+Hras/mut)] to analyze effects depending on the stage of carcinogenesis. We now show that vemurafenib activates MEK-ERK signaling in both, keratinocytes, and fibroblasts in vitro and in the in vivo-like SEs. As a consequence, vemurafenib does not provide a growth advantage but leads to a differentiation phenotype, causing accelerated differentiation and hyperkeratosis in the NHEK and normalized stratification and cornification in the transformed keratinocytes. Although all keratinocytes responded very similarly to vemurafenib in their expression profile, particularly with a significant induction of MMP1 and MMP3, only the HrasA5 cells revealed a vemurafenib-dependent pathophysiological shift to tumor progression, i.e., the initiation of invasive growth. This was shown by increased proteolytic activity allowing for penetration of the basement membrane and invasion into the disrupted underlying matrix. Blocking MMP activity, by the addition of ilomastat, prevented invasion with all corresponding degradative activities, thus substantiating that the RAS-RAF-MEK-ERK/MMP axis is the most important molecular basis for the rapid switch towards tumorigenic conversion of the HrasA5 keratinocytes upon vemurafenib treatment. Finally, cotreatment with vemurafenib and the MEK inhibitor cobimetinib prevented MEK-ERK hyperactivation and with that abolished both, the epidermal differentiation and the tumor invasion phenotype. This suggests that both cutaneous adverse events are under direct control of vemurafenib-dependent MEK-ERK hyperactivation and confirms the dependence on preexisting genetic alterations of the skin keratinocytes that determine the basis towards induction of tumorigenic progression.

Characterisation of the novel spontaneously immortalized and invasively growing human skin keratinocyte line HaSKpw.

We here present the spontaneously immortalised cell line, HaSKpw, as a novel model for the multistep process of skin carcinogenesis. HaSKpw cells were established from the epidermis of normal human adult skin that, without crisis, are now growing unrestricted and feeder-independent. At passage 22, clonal populations were established and clone7 (HaSKpwC7) was further compared to the also spontaneously immortalized HaCaT cells. As important differences, the HaSKpw cells express wild-type p53, remain pseudodiploid, and show a unique chromosomal profile with numerous complex aberrations involving chromosome 20. In addition, HaSKpw cells overexpress a pattern of genes and miRNAs such as KRT34, LOX, S100A9, miR21, and miR155; all pointing to a tumorigenic status. In concordance, HaSKpw cells exhibit reduced desmosomal contacts that provide them with increased motility and a highly migratory/invasive phenotype as demonstrated in scratch- and Boyden chamber assays. In 3D organotypic cultures, both HaCaT and HaSKpw cells form disorganized epithelia but only the HaSKpw cells show tumorcell-like invasive growth. Together, HaSKpwC7 and HaCaT cells represent two spontaneous (non-genetically engineered) "premalignant" keratinocyte lines from adult human skin that display different stages of the multistep process of skin carcinogenesis and thus represent unique models for analysing skin cancer development and progression.

UV-type specific alteration of miRNA expression and its association with tumor progression and metastasis in SCC cell lines.

PURPOSE: UV exposure is the main risk factor for development of cutaneous squamous cell carcinoma (cSCC). While early detection greatly improves cSCC prognosis, locally advanced or metastatic cSCC has a severely impaired prognosis. Notably, the mechanisms of progression to metastatic cSCC are not well understood. We hypothesized that UV exposure of already transformed epithelial cSCC cells further induces changes which might be involved in the progression to metastatic cSCCs and that UV-inducible microRNAs (miRNAs) might play an important role. METHODS: Thus, we analyzed the impact of UV radiation of different quality (UVA, UVB, UVA + UVB) on the miRNA expression pattern in established cell lines generated from primary and metastatic cSCCs (Met-1, Met-4) using the NanoString nCounter platform. RESULTS: This analysis revealed that the expression pattern of miRNAs depends on both the cell line used per se and on the quality of UV radiation. Comparison of UV-induced miRNAs in cSCC cell lines established from a primary tumor (Met-1) and the respective (un-irradiated) metastasis (Met-4) suggest that miR-7-5p, miR-29a-3p and miR-183-5p are involved in a UV-driven pathway of progression to metastasis. This notion is supported by the fact that these three miRNAs build up a network of 81 potential target genes involved e.g. in UVA/UVB-induced MAPK signaling and regulation of the epithelial-mesenchymal transition. As an example, PTEN, a target of UV-upregulated miRNAs (miR-29a-3p, miR-183-5p), could be shown to be down-regulated in response to UV radiation. We further identified CNOT8, the transcription complex subunit 8 of the CCR4-NOT complex, a deadenylase removing the poly(A) tail from miRNA-destabilized mRNAs, in the center of this network, targeted by all three miRNAs. CONCLUSION: In summary, our results demonstrate that UV radiation induces an miRNA expression pattern in primary SCC cell line partly resembling those of metastatic cell line, thus suggesting that UV radiation impacts SCC progression beyond initiation.

EGFR/Ras-induced CCL20 production modulates the tumour microenvironment.

BACKGROUND: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment. METHODS: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo. RESULTS: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system. CONCLUSION: We propose that the chemokine axis CCL20-CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.

Methods in cell biology: Cell-derived matrices.

Three-dimensional (3D) in vitro skin and skin cancer models have become an invaluable tool in skin research. They go back to 1979, when Bell and colleagues reported on the establishment of a fibroblast-dependent collagen tissue (Bell, Ivarsson, & Merrill, 1979). On top of such tissue a stratified and differentiated epidermis could be established (Bell, Merrill, & Solomon, 1979). Hydrogel-based dermal equivalents have been generated ever since and upon co-culture with normal human skin keratinocytes, these constructs were then termed skin equivalents. Due to a number of deficiencies, the most important one being their restricted survival time, new developments helped to circumvent premature fibroblast activation and tissue destruction. By avoiding collagen for the dermal equivalent (DE), we proposed, a scaffold-based DE, allowing fibroblasts to reorganize the primary fibrin solution into an "authentic" dermal matrix (Boehnke et al., 2007; Stark et al., 2004, 2006). With this, our goal of a long-term skin equivalent-successful cultivation for several months-was achieved. Nevertheless, also this model presented limitations. One being its opaqueness made it difficult to image the intact tissue. Another draw-back was that tumor cells upon invasion used the scaffold as a guardrail leaving behind an unspecific invasion pattern. All this could be avoided by an approach, the fibroblast-derived matrix-based model, based on the work by Ahlfors and Billiar (2007) We here provide a protocol for this type of model, thereby providing the basis for future work in the field of skin research.

Molecular Profiling of Keratinocyte Skin Tumors Links Staphylococcus aureus Overabundance and Increased Human ß-Defensin-2 Expression to Growth Promotion of Squamous Cell Carcinoma.

The skin microbiota plays a prominent role in health and disease; however, its contribution to skin tumorigenesis is not well understood. We comparatively assessed the microbial community compositions from excision specimens of the main human non-melanoma skin cancers, actinic keratosis (AK), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). Keratinocyte skin tumors are characterized by significantly different microbial community compositions, wherein AK and SCC are more similar to each other than to BCC. Notably, in SCC, which represents the advanced tumor entity and frequently develops from AK, overabundance of Staphylococcus aureus, a known skin pathogen, was noted. Moreover, S. aureus overabundance was significantly associated with increased human ß-defensin-2 (hBD-2) expression in SCC. By challenging human SCC cell lines with S. aureus, a specific induction of hBD-2 expression and increased tumor cell growth was seen. Increased proliferation was also induced by directly challenging SCC cells with hBD-2. Together, our data indicate that a changed microbial community composition in SCC, specified by S. aureus overabundance, might promote tumor cell growth via modulation of hBD-2 expression.

Regulation and Role of GLI1 in Cutaneous Squamous Cell Carcinoma Pathogenesis.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin tumor in humans. Although current therapies are sufficient to clear the tumor in many cases, the overall risk of cSCC metastasis is still 5%. Alternative treatment options could help to overcome this situation. Here we focused on the role of the Hedgehog (HH) signaling pathway and its interplay with epidermal growth factor receptor (EGFR) signaling in cSCC. The analyses revealed that, despite lack of Sonic HH (SHH) expression, a subset of human cSCC can express GLI1, a marker for active HH signaling, within distinct tumor areas. In contrast, all tumors strongly express EGFR and the hair follicle stem cell marker SOX9 at the highly proliferative tumor-stroma interface, whereas central tumor regions with a more differentiated stratum spinosum cell type lack both EGFR and SOX9 expression. In vitro experiments indicate that activation of EGFR signaling in the human cSCC cell lines SCL-1, MET-1, and MET-4 leads to GLI1 inhibition via the MEK/ERK axis without affecting cellular proliferation. Of note, EGFR activation also inhibits cellular migration of SCL-1 and MET-4 cells. Because proliferation and migration of the cells is also not altered by a GLI1 knockdown, GLI1 is apparently not involved in processes of aggressiveness in established cSCC tumors. In contrast, our data rather suggest a negative correlation between Gli1 expression level and cSCC formation because skin of Ptch +/- mice with slightly elevated Gli1 expression levels is significantly less susceptible to chemically-induced cSCC formation compared to murine wildtype skin. Although not yet formally validated, these data open the possibility that GLI1 (and thus HH signaling) may antagonize cSCC initiation and is not involved in cSCC aggressiveness, at least in a subset of cSCC.

DNA methylation at an enhancer of the three prime repair exonuclease 2 gene (TREX2) is linked to gene expression and survival in laryngeal cancer.

BACKGROUND: Genetic aberrations in DNA repair genes are linked to cancer, but less is reported about epigenetic regulation of DNA repair and functional consequences. We investigated the intragenic methylation loss at the three prime repair exonuclease 2 (TREX2) locus in laryngeal (n = 256) and colorectal cancer cases (n = 95) and in pan-cancer data from The Cancer Genome Atlas (TCGA). RESULTS: Significant methylation loss at an intragenic site of TREX2 was a frequent trait in both patient cohorts (p = 0.016 and < 0.001, respectively) and in 15 out of 22 TCGA studies. Methylation loss correlated with immunohistochemically staining for TREX2 (p < 0.0001) in laryngeal tumors and improved overall survival of laryngeal cancer patients (p = 0.045). Chromatin immunoprecipitation, demethylation experiments, and reporter gene assays revealed that the region of methylation loss can function as a CCAAT/enhancer binding protein alpha (CEBPA)-responsive enhancer element regulating TREX2 expression. CONCLUSIONS: The data highlight a regulatory role of TREX2 DNA methylation for gene expression which might affect incidence and survival of laryngeal cancer. Altered TREX2 protein levels in tumors may affect drug-induced DNA damage repair and provide new tailored therapies.

T-lymphocyte profiles differ between keratoacanthomas and invasive squamous cell carcinomas of the human skin.

BACKGROUND: T-lymphocytes are involved in tumor progression and regression. Actinic keratoses (AK) are atypical proliferations of keratinocytes of the skin. Some AK progress into invasive cutaneous squamous cell carcinomas (cSCC). Keratoacanthomas (KA) are either classified as a cSCC subtype or a benign tumor with histologic resemblance to well-differentiated cSCC as it is supposed to regress spontaneously. In contrast, cSCC represent malignant tumors that may metastasize. OBJECTIVES: To compare the T-lymphocyte profiles of AK, KA and cSCC in relation to PD-L1 expression. METHODS: Tissue micro-arrays of 103 cases of AK, 43 cases of KA and 106 cases of cSCC were stained by immunohistochemistry for E-cadherin, CD3, CD4, CD8, FOXp3, and the receptor-ligand pair PD-1/PD-L1. Immunohistological scores were computationally determined to assess PD-L1 expression as well as the expression profiles of T-lymphocytes. RESULTS: AK had lower numbers of CD3+ and PD-1+ cells compared to KA and lower numbers of CD3+, CD8+ and PD-1+ cells in comparison with cSCC. KA showed significantly higher numbers of CD4+ and FOXp3+ cells as well as lower numbers of CD8+ cells in comparison with invasive cSCC. cSCC expressed significantly more PD-L1 in comparison with AK and KA. Among cSCC PD-L1 expression was higher in moderately and poorly-differentiated cSCC than in well-differentiated cSCC. Increased PD-L1 expression also correlated with increased numbers of CD4+, CD8+ and FOXp3+ cells in cSCC. CONCLUSIONS: Tumor-associated T-lymphocyte infiltrates showed significant differences between AK, KA and invasive cSCC. PD-L1 expression correlated with invasion of T-cell infiltrates in invasive cSCC.

An RNAi Screen Reveals an Essential Role for HIPK4 in Human Skin Epithelial Differentiation from iPSCs.

Molecular mechanisms responsible for the development of human skin epithelial cells are incompletely understood. As a consequence, the efficiency to establish a pure skin epithelial cell population from human induced pluripotent stem cells (hiPSCs) remains poor. Using an approach including RNAi and high-throughput imaging of early epithelial cells, we identified candidate kinases involved in their differentiation from hiPSCs. Among these, we found HIPK4 to be an important inhibitor of this process. Indeed, its silencing increased the amount of generated skin epithelial precursors at an early time point, increased the amount of generated keratinocytes at a later time point, and improved growth and differentiation of organotypic cultures, allowing for the formation of a denser basal layer and stratification with the expression of several keratins. Our data bring substantial input regarding regulation of human skin epithelial differentiation and for improving differentiation protocols from pluripotent stem cells.

ß-2-himachalen-6-ol protects against skin cancer development in vitro and in vivo.

BACKGROUND: Previous studies in our laboratory showed that Daucus carota oil extract (DCOE) possesses remarkable in-vitro anticancer activity and antitumour promoting effect against DMBA/TPA skin carcinogenesis in mice. Chemical analysis of DCOE led to the isolation of the ß-2-himachalen-6-ol (HC), major sesquiterpene with a potent anticancer activity against various colon, breast, brain and skin cancer cells. This study investigated the anticancer activity of HC against invasive epidermal squamous cell carcinoma cells and evaluated its effect in a DMBA/TPA skin carcinogenesis Balb/c murine model. METHODS: HaCaT-ras II-4 epidermal squamous cells were treated with HC (1, 5, 10, 25 and 50 µg/ml), and cell viability was evaluated with WST 1 assay kit. Cell cycle analysis was carried out by flow cytometry, and pro/anti-apoptotic proteins were measured using Western blot. The effect of topical and intraperitoneal (IP) treatment with HC in mice was assessed using the DMBA/TPA skin carcinogenesis model. Cisplatin (2.5 mg/kg; IP) was used as a positive control. Papilloma incidence, yield and volume were monitored, and isolated papillomas were assessed for their pro/anti-apoptotic proteins and morphology. RESULTS: ß-2-himachalen-6-ol showed a dose-dependent decrease in cell survival with an IC50 and IC90 of 8 and 30 µg/ml, respectively. Flow cytometry analysis revealed that treatment with 10 µg/ml HC significantly increased the number of cells undergoing late apoptosis (28%), while 25 µg/ml caused a larger cell shift towards late apoptosis (46.6%) and necrosis (39%). A significant decrease in protein levels of p53 and Bcl-2 and a significant increase in p21 and Bax were observed. Also, there was a significant decrease in p-Erk and p-Akt protein levels. The treatment of mice (IP and topical) with HC caused a significant decrease in papilloma yield, incidence and volume. Similar effects were observed with cisplatin treatment, but HC-treated groups exhibited twofold to threefold increase in survival rates. Similar patterns in the pro- and anti-apoptotic proteins were observed in mice treated with HC, except for a significant increase in p53 protein. CONCLUSIONS: In conclusion, HC treatment induced cell cycle arrest (low dose) and promoted apoptosis partly via inhibition of the MAPK/ERK and PI3K/AKT pathways with no significant toxicity to laboratory mice.

Age, gender and UV-exposition related effects on gene expression in in vivo aged short term cultivated human dermal fibroblasts.

Ageing, the progressive functional decline of virtually all tissues, affects numerous living organisms. Main phenotypic alterations of human skin during the ageing process include reduced skin thickness and elasticity which are related to extracellular matrix proteins. Dermal fibroblasts, the main source of extracellular fibrillar proteins, exhibit complex alterations during in vivo ageing and any of these are likely to be accompanied or caused by changes in gene expression. We investigated gene expression of short term cultivated in vivo aged human dermal fibroblasts using RNA-seq. Therefore, fibroblast samples derived from unaffected skin were obtained from 30 human donors. The donors were grouped by gender and age (Young: 19 to 25 years, Middle: 36 to 45 years, Old: 60 to 66 years). Two samples were taken from each donor, one from a sun-exposed and one from a sun-unexposed site. In our data, no consistently changed gene expression associated with donor age can be asserted. Instead, highly correlated expression of a small number of genes associated with transforming growth factor beta signalling was observed. Also, known gene expression alterations of in vivo aged dermal fibroblasts seem to be non-detectable in cultured fibroblasts.

Wild carrot pentane-based fractions suppress proliferation of human HaCaT keratinocytes and protect against chemically-induced skin cancer.

BACKGROUND: Previous studies in our laboratory showed that the Lebanese Daucus carota ssp. carota (wild carrot) oil extract possesses in vitro and in vivo anticancer activities. The present study aims to examine the cytotoxic effect of Daucus carota oil fractions on human epidermal keratinocytes and evaluate the chemopreventive activity of the pentane diethyl ether fraction on DMBA/TPA induced skin carcinogenesis in mice. METHODS: Wild carrot oil extract was chromatographed to yield four fractions (F1, 100% pentane; F2, 50:50 pentane:diethyl ether; F3, 100% diethyl ether; F4 93:7 chloroform:methanol). The cytotoxic effect of fractions (10, 25, 50 and 100 µg/mL) was tested on human epidermal keratinocytes (non-tumorigenic HaCaT cells and tumorigenic HaCaT-ras variants) using WST a ssay. Cell cycle phase distribution of tumorigenic HaCaT-ras variants was determined by flow cytometry post-treatment with F2 fraction. Apoptosis related proteins were also assessed using western blot. The antitumor activity of F2 fraction was also evaluated using a DMBA/TPA induced skin carcinoma in Balb/c mice. RESULTS: All fractions exhibited significant cytotoxicity, with HaCaT cells being 2.4-3 times less sensitive than HaCaT-ras A5 (benign tumorigenic), and HaCaT-ras II4 (malignant) cells. GC-MS analysis revealed the presence of a major compound (around 60%) in the pentane/diethylether fraction (F2), identified as 2-himachalen-6-ol. Treatment of HaCaT-ras A5 and HaCaT-ras II4 cells with F2 fraction resulted in the accumulation of cells in the sub-G1 apoptotic phase and decreased the population of cells in the S and G2/M phases. Additionally, F2 fraction treatment caused an up-regulation of the expression of pro-apoptotic (Bax) and down-regulation of the expression of anti-apoptotic (Bcl2) proteins. A decrease in the phosphorylation of AKT and ERK was also observed. Intraperitoneal treatment with F2 fraction (50 or 200 mg/kg) in the DMBA/TPA skin carcinogenesis mouse model showed a significant inhibition of papilloma incidence (mice with papilloma), yield (number of papilloma/mouse) and volume (tumor relative size) at weeks 15, 18 and 21. CONCLUSION: The present data reveal that F2 fraction has a remarkable antitumor activity against DMBA/TPA-induced skin carcinogenesis, an effect that may be mediated through inhibition of the MAPK/ERK and PI3K/AKT pathways.

Aryl Hydrocarbon Receptor in Keratinocytes Is Essential for Murine Skin Barrier Integrity.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in adaptive cell functions, and it is highly active in the epidermis. AhR ligands can accelerate keratinocyte differentiation, but the precise role of AhR in the skin barrier is unknown. Our study showed that transepidermal water loss, a parameter of skin barrier integrity, is high in AhR-deficient mice. Experiments with conditionally AhR-deficient mouse lines identified keratinocytes as the primary cell population responsible for high transepidermal water loss. Electron microscopy showed weaker intercellular connectivity in the epidermis of keratinocytes in AhR-knockout mice, and gene expression analysis identified many barrier-associated genes as AhR targets. Moreover, AhR-deficient mice had higher interindividual differences in their microbiome. Interestingly, removing AhR ligands from the diet of wild-type mice mimicked AhR deficiency with respect to the impaired barrier; conversely, re-addition of the plant-derived ligand indole-3-carbinol rescued the barrier deficiency even in aged mice. Our results suggest that functional AhR expression is critical for skin barrier integrity and that AhR represents a molecular target for the development of therapeutic approaches for skin barrier diseases, including by dietary intervention.

Loss of tumorigenic potential upon transdifferentiation from keratinocytic into melanocytic lineage.

Lineage-specific transcription factors determine the cell fate during development. Direct conversion of several cell types into other lineages has been achieved by the overexpression of specific transcription factors. Even cancer cells have been demonstrated to be amenable to transdifferentiation. Here, we identified a distinct set of transcription factors, which are sufficient to transform cells of the keratinocytic lineage to melanocyte-like cells. Melanocyte marker expression was induced and melanosome formation was observed in non-tumorigenic keratinocytes (HaCaT) and tumorigenic squamous cell carcinoma (MET-4) cells. Moreover, reduced proliferation, cell metabolism, invasion and migration were measured in vitro in transdifferentiated MT-MET-4 cells. A loss of tumorigenic potential of squamous cell carcinoma cells could be due to the upregulation of the melanocyte differentiation associated gene IL-24. Our data show that cells from the keratinocytic lineage can be transdifferented into the melanocytic lineage and provide a proof of principle for a potential new therapeutic strategy.

Mitotic Diversity in Homeostatic Human Interfollicular Epidermis.

Despite decades of skin research, regulation of proliferation and homeostasis in human epidermis is still insufficiently understood. To address the role of mitoses in tissue regulation, we utilized human long-term skin equivalents and systematically assessed mitoses during early epidermal development and long-term epidermal regeneration. We now demonstrate four different orientations: (1) horizontal, i.e., parallel to the basement membrane (BM) and suggestive of symmetric divisions; (2) oblique with an angle of 45°-70°; or (3) perpendicular, suggestive of asymmetric division. In addition, we demonstrate a fourth substantial fraction of suprabasal mitoses, many of which are committed to differentiation (Keratin K10-positive). As verified also for normal human skin, this spatial mitotic organization is part of the regulatory program of human epidermal tissue homeostasis. As a potential marker for asymmetric division, we investigated for Numb and found that it was evenly spread in almost all undifferentiated keratinocytes, but indeed asymmetrically distributed in some mitoses and particularly frequent under differentiation-repressing low-calcium conditions. Numb deletion (stable knockdown by CRISPR/Cas9), however, did not affect proliferation, neither in a three-day follow up study by life cell imaging nor during a 14-day culture period, suggesting that Numb is not essential for the general control of keratinocyte division.

Body mass index and leukocyte telomere length dynamics among older adults: Results from the ESTHER cohort.

OBJECTIVE: Telomere length (TL) has been proposed as a biomarker of ageing, which might be used to identify individuals at higher risk of age-related diseases. Obesity is a well-known risk factor for several diseases. This study aims to analyse the associations of BMI with TL and the rate of TL change in older adults. METHODS: Leukocyte TL (LTL) was measured by quantitative PCR in blood samples of 3600 older adults aged 50-75 years obtained at the baseline examination of a population-based cohort study in Germany. For longitudinal analyses, measurements were repeated in blood samples obtained at 8-year follow-up from 1000 participants. Multivariate linear regression models were used to estimate associations of BMI with LTL and changes in LTL over time. RESULTS: LTL was inversely associated with age (r = -0.090, p < 0.0001). BMI and LTL associations varied according to age (p for interaction = 0.021). BMI was significantly inversely associated with LTL in those younger than 60 years (-6 basepairs per 1 kg/m(2) difference in BMI). In particular, weight gain during adulthood was inversely associated with LTL in a dose-response manner in this age group, with those having gained ≥ 30 kg having significantly shorter telomeres (-209 basepairs) than those who maintained their weight. No clear patterns were observed between any of BMI-related variables and the rate of LTL change. CONCLUSIONS: Our cross-sectional analysis supports suggestions that weight gain during adulthood and obesity may contribute to shorter telomere length below 60 years of age, but this relationship could not be shown longitudinally.