Human Neonatal Keratinocytes Research Articles

An integrative biology approach to understanding keratinocyte collective migration as stimulated by bioglass.

A critical phase of wound healing is the coordinated movement of keratinocytes. To this end, bioglasses show promise in speeding healing in hard tissues and skin wounds. Studies suggest that bioglass materials may promote wound healing by inducing positive cell responses in proliferation, growth factor production, expression of angiogenic factors, and migration. Precise details of how bioglass may stimulate migration are unclear, however, because the common assays for studying migration in wound healing focus on simplified outputs like rate of migration or total change in wound area. These outputs are limited in that they represent the average behavior of the collective, with no connection between the motion of the individual cells and the collective wound healing response. There is a need to apply more refined tools that identify how the motion of the individual cells changes in response to perturbations, such as by bioglass, and in turn affects motion of the cell collective. Here, we apply an integrative biology strategy that combines an in vitro wound healing assay using primary neonatal human keratinocytes with time lapse microscopy and quantitative image analysis. The resulting data set provides the cell velocity field, from which we define key metrics that describe cooperative migration phenotypes. Treatment with growth factors led to faster single-cell speeds compared to control, but the migration was not cooperative, with cells breaking away from their neighbors and migrating as individuals. Treatment with calcium or bioglass led to migration phenotypes that were highly collective, with greater coordination in space compared to control. We discuss the link between bioglass treatment and observed increases in free calcium ions that are hypothesized to promote these distinct coordinated behaviors in primary keratinocytes. These findings have been enabled by the unique descriptors developed through applying image analysis to interpret biological response in migration models. Insight Box/Paragraph Statement: Bioglasses are important materials for tissue engineering and have more recently shown promise in skin and wound healing by mechanisms tied to their unique ionic properties. The precise details, however, of how cell migration may be affected by bioglass are left unclear by traditional cell assay methods. The following describes the integration of migration assays of keratinocytes, cells critical for skin and wound healing, with the tools of time lapse microscopy and image analysis to generate a quantitative description of coordinated, tissue-like migration behavior, stimulated by bioglass, that would not have been accessible without the combination of these analytical tools.

TEAD1 Silencing Regulates Cell Proliferation and Resistance to 5-Fluorouracil in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a skin malignant tumor account for approximately one-third of all nonmelanoma skin cancers. Studies have shown that TEA domain transcription factor 1 (TEAD1) is discovered to be involved in the pathogenesis of some human cancers, but to our knowledge its role in cSCC has not been reported. Samples from 16 cSCC patients and 27 healthy individuals were obtained for immunohistochemical staining of TEAD1. The expressions of TEAD1 in SCL-1, HSC-1 cells compared with the primary neonatal human epithelial keratinocytes were detected by Western blot and RT-qPCR. Proliferation and cell cycle of TEAD1 knockdown in cSCC cell lines were examined by MTT and flow cytometry analysis. Annexin V/PI and JC-1 staining were used to determine the cell apoptosis. The expression of TEAD1 decreased significantly in cSCC compared to its expression in normal skin tissues and cell lines. Down-regulation of TEAD1 in cSCC cell lines promoted cell growth via regulation of the G2/M progression. Additionally, silence of TEAD1 also protected cells against 5-Fluorouracil-induced apoptosis and decreased the expression of apoptosis-related protein (p53). Our results suggested that TEAD1 expression is down-regulated and functioned as a tumor suppressor in cSCC and that it may serve as a biomarker or therapeutic target of cSCC.

Human Papillomavirus 16 E6 and E7 Oncoproteins Alter the Abundance of Proteins Associated with DNA Damage Response, Immune Signaling and Epidermal Differentiation.

The high-risk human papillomaviruses are oncogenic viruses associated with almost all cases of cervical carcinomas, and increasing numbers of anal, and oral cancers. Two oncogenic HPV proteins, E6 and E7, are capable of immortalizing keratinocytes and are required for HPV associated cell transformation. Currently, the influence of these oncoproteins on the global regulation of the host proteome is not well defined. Liquid chromatography coupled with quantitative tandem mass spectrometry using isobaric-tagged peptides was used to investigate the effects of the HPV16 oncoproteins E6 and E7 on protein levels in human neonatal keratinocytes (HEKn). Pathway and gene ontology enrichment analyses revealed that the cells expressing the HPV oncoproteins have elevated levels of proteins related to interferon response, inflammation and DNA damage response, while the proteins related to cell organization and epithelial development are downregulated. This study identifies dysregulated pathways and potential biomarkers associated with HPV oncoproteins in primary keratinocytes which may have therapeutic implications. Most notably, DNA damage response pathways, DNA replication, and interferon signaling pathways were affected in cells transduced with HPV16 E6 and E7 lentiviruses. Moreover, proteins associated with cell organization and differentiation were significantly downregulated in keratinocytes expressing HPV16 E6 + E7. High-risk HPV E6 and E7 oncoproteins are necessary for the HPV-associated transformation of keratinocytes. However their influence on the global dysregulation of keratinocyte proteome is not well documented. Here shotgun proteomics using TMT-labeling detected over 2500 significantly dysregulated proteins associated with E6 and E7 expression. Networks of proteins related to interferon response, inflammation and DNA damage repair pathways were altered.

Homo Sapiens (Hsa)-microRNA (miR)-6727-5p Contributes to the Impact of High-Density Lipoproteins on Fibroblast Wound Healing In Vitro.

Chronic, non-healing wounds are a significant cause of global morbidity and mortality, and strategies to improve delayed wound closure represent an unmet clinical need. High-density lipoproteins (HDL) can enhance wound healing, but exploitation of this finding is challenging due to the complexity and instability of these heterogeneous lipoproteins. The responsiveness of primary human neonatal keratinocytes, and neonatal and human dermal fibroblasts (HDF) to HDL was confirmed by cholesterol efflux, but promotion of ‘scrape’ wound healing occurred only in primary human neonatal (HDFn) and adult fibroblasts (HDFa). Treatment of human fibroblasts with HDL induced multiple changes in the expression of small non-coding microRNA sequences, determined by microchip array, including hsa-miR-6727-5p. Intriguingly, levels of hsa-miR-6727-5p increased in HDFn, but decreased in HDFa, after exposure to HDL. Delivery of a hsa-miR-6727-5p mimic elicited repression of different target genes in HDFn (ZNF584) and HDFa (EDEM3, KRAS), and promoted wound closure in HDFn. By contrast, a hsa-miR-6727-5p inhibitor promoted wound closure in HDFa. We conclude that HDL treatment exerts distinct effects on the expression of hsa-miR-6727-5p in neonatal and adult fibroblasts, and that this is a sequence which plays differential roles in wound healing in these cell types, but cannot replicate the myriad effects of HDL.

Exploring the dermotoxicity of the mycotoxin deoxynivalenol: combined morphologic and proteomic profiling of human epidermal cells reveals alteration of lipid biosynthesis machinery and membrane structural integrity relevant for skin barrier function

Deoxynivalenol (vomitoxin, DON) is a secondary metabolite produced by Fusarium spp. fungi and it is one of the most prevalent mycotoxins worldwide. Crop infestation results not only in food and feed contamination, but also in direct dermal exposure, especially during harvest and food processing. To investigate the potential dermotoxicity of DON, epidermoid squamous cell carcinoma cells A431 were compared to primary human neonatal keratinocytes (HEKn) cells via proteome/phosphoproteome profiling. In A431 cells, 10 µM DON significantly down-regulated ribosomal proteins, as well as mitochondrial respiratory chain elements (OXPHOS regulation) and transport proteins (TOMM22; TOMM40; TOMM70A). Mitochondrial impairment was reflected in altered metabolic competence, apparently combined with interference of the lipid biosynthesis machinery. Functional effects on the cell membrane were confirmed by live cell imaging and membrane fluidity assays (0.1–10 µM DON). Moreover, a common denominator for both A431 and HEKn cells was a significant downregulation of the squalene synthase (FDFT1). In sum, proteome alterations could be traced back to the transcription factor Klf4, a crucial regulator of skin barrier function. Overall, these results describe decisive molecular events sustaining the capability of DON to impair skin barrier function. Proteome data generated in the study are fully accessible via ProteomeXchange with the accession numbers PXD011474 and PXD013613.

HiPSC-Derived Epidermal Keratinocytes from Ichthyosis Patients Show Altered Expression of Cornification Markers.

Inherited ichthyoses represent a large heterogeneous group of skin disorders characterised by impaired epidermal barrier function and disturbed cornification. Current knowledge about disease mechanisms has been uncovered mainly through the use of mouse models or human skin organotypic models. However, most mouse lines suffer from severe epidermal barrier defects causing neonatal death and human keratinocytes have very limited proliferation ability in vitro. Therefore, the development of disease models based on patient derived human induced pluripotent stem cells (hiPSCs) is highly relevant. For this purpose, we have generated hiPSCs from patients with congenital ichthyosis, either non-syndromic autosomal recessive congenital ichthyosis (ARCI) or the ichthyosis syndrome trichothiodystrophy (TTD). hiPSCs were successfully differentiated into basal keratinocyte-like cells (hiPSC-bKs), with high expression of epidermal keratins. In the presence of higher calcium concentrations, terminal differentiation of hiPSC-bKs was induced and markers KRT1 and IVL expressed. TTD1 hiPSC-bKs showed reduced expression of FLG, SPRR2B and lipoxygenase genes. ARCI hiPSC-bKs showed more severe defects, with downregulation of several cornification genes. The application of hiPSC technology to TTD1 and ARCI demonstrates the successful generation of in vitro models mimicking the disease phenotypes, proving a valuable system both for further molecular investigations and drug development for ichthyosis patients.

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using invitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrinα6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Self-assembling 3D spheroid cultures of human neonatal keratinocytes have enhanced regenerative properties.

Relative to conventional two-dimensional (2-D) culture, three-dimensional (3-D) suspension culture of epithelial cells more closely mimics the in vivo cell microenvironment regarding cell architecture, cell to matrix interaction, and osmosis exchange. However, primary normal human keratinocytes (NHKc) rapidly undergo terminal differentiation and detachment-induced cell death (anoikis) upon disconnection from the basement membrane, thus greatly constraining their use in 3-D suspension culture models. Here, we examined the 3-D anchorage-free growth potential of NHKc isolated from neonatal skin explants of 59 different individuals. We found that 40% of all isolates naturally self-assembled into multicellular spheroids within 24 h in anchorage-free culture, while 60% did not. Placing a single spheroid back into 2-D monolayer culture yielded proliferating cells that expressed elevated levels of nuclear P63 and basal cytokeratin 14. These cells also displayed prolonged keratinocyte renewal and a gene expression profile corresponding to cellular heterogeneity, quiescence, and de-differentiation. Notably, spheroid-derived (SD) NHKc were enriched for a P63/K14 double-positive population that formed holoclonal colonies and reassembled into multicellular spheroids during 3-D suspension subculture. This study reveals marked phenotypic differences in neonatal keratinocyte suspension cultures isolated from different individuals and present a model system that can be readily employed to study epithelial cell behavior, along with a variety of dermatological diseases.

Self-Assembling 3D Spheroid Cultures of Human Neonatal Keratinocytes Have Enhanced Regenerative Properties

Relative to conventional two-dimensional (2-D) culture, three-dimensional (3-D) suspension culture of epithelial cells more closely mimics the in vivo cell microenvironment regarding cell architecture, cell to matrix interaction, and osmosis exchange. However, primary normal human keratinocytes (NHKc) rapidly undergo terminal differentiation and detachment-induced cell death (anoikis) upon disconnection from the basement membrane, thus greatly constraining their use in 3-D suspension culture models. Here, we examined the 3-D anchorage-free growth potential of NHKc isolated from neonatal skin explants of 59 different individuals. We found that 40% of all isolates naturally self-assembled into multicellular spheroids within 24 h in anchorage-free culture, while 60% did not. Placing a single spheroid back into 2-D monolayer culture yielded proliferating cells that expressed elevated levels of nuclear P63 and basal cytokeratin 14. These cells also displayed prolonged keratinocyte renewal and a gene expression profile corresponding to cellular heterogeneity, quiescence, and de-differentiation. Notably, spheroid-derived (SD) NHKc were enriched for a P63/K14 double-positive population that formed holoclonal colonies and reassembled into multicellular spheroids during 3-D suspension subculture. This study reveals marked phenotypic differences in neonatal keratinocyte suspension cultures isolated from different individuals. We present here a model system that can be readily employed to study epithelial cell behavior, along with a variety of dermatological diseases.

131 The effect of PM 2.5 from three rural USA areas on inflammatory markers in human keratinocytes

Poor air quality in large cities has shown to have a negative effect on human health and to induce skin inflammation and premature aging. Less attention has been paid to rural environments, thus the effects on skin of specific rural pollutants are largely unknown. In addition, the combined effect of rural PM and solar radiation is also of interest. Here we investigate the effects of fine particulate matter (PM2.5) collected from rural environments on interleukin (IL) and matrix metalloprotease (MMP) production in human neonatal keratinocytes in the absence or presence of solar simulated UVR (ssUVR).

Tyrosinase inhibitors with potent anti-senescence activity in human neonatal keratinocyte progenitors

Tyrosinase inhibitors with potent anti-senescence activity in human neonatal keratinocyte progenitors

Tyrosinase inhibitors with potent anti-senescence activity in human neonatal keratinocyte progenitors

Tyrosinase inhibitors with potent anti-senescence activity in human neonatal keratinocyte progenitors

Cytoprotective effect of a functional antipollutant blend through reducing B[a] P-induced intracellular oxidative stress and UVA exposure.

Benzo[a]pyrene (B[a]P) is a ubiquitous environmental pollutant that reacts with skin and induces intracellular oxidative stress through reactive oxygen species (ROS) accumulation. The antipollution properties of natural extracts, especially including antioxidants, for inhibiting ROS in cells are gaining importance, in addition to the anticancer effects attributed to them. In this study, a commercial functional antipollutant blend of plant extracts consisting of ellagic acid standardized Punica granatum peel extract, Sambucus nigra fruit extract, Prunus cerasus seed extract, and hydrolyzed wheat protein with high antioxidant properties and UV damage-protective properties attributed to each one was investigated. The cytoprotective effect of this functional antipollutant blend was determined by ROS assay through reducing the level of intracellular ROS induced by B[a]P as an oxidative stress factor in human neonatal keratinocytes and fibroblast cells. In addition, the cytoprotective effect of the functional antipollutant blend after UVA exposure was also determined. It is shown that the oxidative damage induced by B[a]P and UVA, which are the most abundant factors of chemical and physical pollution, would be prevented by the functional antipollutant blend. Thus, it can be concluded that this antipollutant functional blend may offer a promising ingredient for the cosmetic industry's skincare products.

998 EMT-like phenotype in normal keratinocytes driven by TLR3 activation

Toll-Like-Receptor 3 (TLR3), a pattern recognition receptor that recognizes dsRNA, activation results in NFkB or interferon-regulatory factor 3 (IRF3) mediated gene transcription of inflammatory cytokines (e.g. IL-6) and type I and III interferons (e.g. IFN-λ). TLR3 also contributes to induced-pluripotent stem cell (IPS) programming, wound healing and skin regeneration through Wnt, SHH, and epithelial-mesenchymal transition (EMT) pathways. Our hypothesis is that TLR3 signaling within normal keratinocytes contributes to non-melanoma skin cancer (NMSC) through activation of EMT. In vitro, normal neonatal human embryonic keratinocytes (NHEKs) treated with a TLR3 agonist, polyinosinic:polycytidylic acid (poly IC), exhibit sustained EMT morphology changes that correlate with elevated levels of the intermediate filament Vimentin (∼2 fold), and transcription factors, ZEB1 (∼3-fold), SNAI1 (∼2-fold), and TWIST1 (∼2-fold), all key EMT markers. Poly IC treatment significantly increased levels of IL-6 (∼8-fold) and IFN-λ1 (∼200-fold) in normal keratinocytes. However, treatment of NHEKs with recombinant IL-6 or IFN-λ1 proteins do not induce an EMT-like morphology or increase in EMT gene expression. UVB radiation, the major player in NMSC development, also activates TLR3 signaling in keratinocytes. In vitro, NHEKs exposed to UVB (10mJ/cm2) exhibit similar EMT-like morphology and gene expression changes as poly IC treated cells. Chemical inhibition of TLR3 (CU CPT 4a) prior to poly IC or UVB exposure prevents EMT gene expression. We performed RNA-sequencing to investigate common pathways activated by both Poly IC and UVB to uncover additional downstream players responsible for driving the TLR3 induced EMT-phenotype. Taken together, TLR3 activation and the resulting EMT-like phenotype is a result of multiple players, and not likely driven by a single downstream mediator of TLR3 signaling.

The Role of Serotonin during Skin Healing in Post-Thermal Injury.

Post-burn trauma significantly raises tissue serotonin concentration at the initial stages of injury, which leads us to investigate its possible role in post burn wound healing. Therefore, we planned this study to examine the role of serotonin in wound healing through in vitro and in vivo models of burn injuries. Results from in vitro analysis revealed that serotonin decreased apoptosis and increased cell survival significantly in human fibroblasts and neonatal keratinocytes. Cellular proliferation also increased significantly in both cell types. Moreover, serotonin stimulation significantly accelerated the cell migration, resulting in narrowing of the scratch zone in human neonatal keratinocytes and fibroblasts cultures. Whereas, fluoxetine (a selective serotonin reuptake inhibitor) and ketanserin (serotonin receptor 2A inhibitor) reversed these effects. Scald burn mice model (20% total body surface area) showed that endogenous serotonin improved wound healing process in control group, whereas fluoxetine and ketanserin treatments (disruptors of endogenous serotonin stimulation), resulted in poor reepithelization, bigger wound size and high alpha smooth muscle actin (α-SMA) count. All of these signs refer a prolonged differentiation state, which ultimately exhibits poor wound healing outcomes. Collectively, data showed that the endogenous serotonin pathway contributes to regulating the skin wound healing process. Hence, the results of this study signify the importance of serotonin as a potential therapeutic candidate for enhancing skin healing in burn patients.

Differentiation of Keratinocytes Modulates Skin HPA Analog.

It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus-pituitary-adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA). Expression of CRF, UCN1-3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R, and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies. Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from days 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium. Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. J. Cell. Physiol. 232: 154-166, 2017. © 2016 Wiley Periodicals, Inc.

Crosstalk between adrenergic and toll-like receptors in human mesenchymal stem cells and keratinocytes: a recipe for impaired wound healing.

Previous studies demonstrate that skin wounds generate epinephrine (EPI) that can activate local adrenergic receptors (ARs), impairing healing. Bacterially derived activators of Toll-like receptors (TLRs) within the wound initiate inflammatory responses and can also impair healing. In this study, we examined the hypothesis that these two pathways crosstalk to one another, using EPI and macrophage-activating lipopeptide-2 (MALP2) to activate ARs and TLR2, respectively, in human bone marrow-derived mesenchymal stem cells (BM-MSCs) and neonatal keratinocytes (NHKs). BM-MSCs exposed to EPI significantly (p < .05) increased TLR2 message (sevenfold BM-MSCs), TLR2 protein (twofold), and myeloid differentiation factor 88 (MyD88) (fourfold). Conversely, activation of TLR2 by MALP2 in these cells increased β2-AR message (twofold in BM-MSCs, 2.7-fold in NHKs), β2-AR protein (2.5-fold), phosphorylation of β-AR-activated kinase (p-BARK, twofold), and induced release of EPI from both cell types (twofold). Treating cells with EPI and MALP2 together, as would be encountered in a wound, increased β2-AR and p-BARK protein expression (sixfold), impaired cell migration (BM-MSCs- 21%↓ and NHKs- 60%↓, p < .002), and resulted in a 10-fold (BM-MSCs) and 51-fold (NHKs) increase in release of IL-6 (p < .001) responses that were remarkably reduced by pretreatment with β2-AR antagonists. In vivo, EPI-stressed animals exhibited impaired healing, with elevated levels of TLR2, MyD88, and IL-6 in the wounds (p < .05) relative to nonstressed controls. Thus, our data describe a recipe for decreasing cell migration and exacerbating inflammation via novel crosstalk between the adrenergic and Toll-like receptor pathways in BM-MSCs and NHKs.

Promise of Human Induced Pluripotent Stem Cells in Skin Regeneration and Investigation

Induced pluripotent stem cell (iPSC) technology has initiated a new era in biomedical science. The skin has been realized as an ideal platform for iPSC applications; unlike other organs, the skin is easily accessible, highly proliferative, and reconstitutable. Currently, skin equivalents can be generated from iPSCs not only from healthy individuals but also from patients with genodermatoses, providing novel platforms for dissecting disease pathophysiology and establishing cell-based therapy. With their developmental plasticity, iPSCs may also enable the regeneration of skin appendages. The iPSC technology may provide novel remedies for intractable disorders, once key issues particularly, safety concerns, are cleared.

Polyandric Acid A, a Clerodane Diterpenoid from the Australian Medicinal Plant Dodonaea polyandra, Attenuates Pro-inflammatory Cytokine Secretion in Vitro and in Vivo

Dodonaea polyandra is a medicinal plant used traditionally by the Kuuku I'yu (Northern Kaanju) indigenous people of Cape York Peninsula, Australia. The most potent of the diterpenoids previously identified from this plant, polyandric acid A (1), has been examined for inhibition of pro-inflammatory cytokine production and other inflammatory mediators using well-established acute and chronic mouse ear edema models and in vitro cellular models. Topical application of 1 significantly inhibited interleukin-1β production in mouse ear tissue in an acute model. In a chronic skin inflammation model, a marked reduction in ear thickness, associated with significant reduction in myeloperoxidase accumulation, was observed. Treatment of primary neonatal human keratinocytes with 1 followed by activation with phorbol ester/ionomycin showed a significant reduction in IL-6 secretion. The present study provides evidence that the anti-inflammatory properties of 1 are due to inhibition of pro-inflammatory cytokines associated with skin inflammation and may be useful in applications for skin inflammatory conditions including psoriasis and dermatitis.

Cycloastragenol Is a Potent Telomerase Activator in Neuronal Cells: Implications for Depression Management

Cycloastragenol (CAG) is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB) activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression-like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression. © 2014 S. Karger AG, Basel