Spermidine-induced recovery of human dermal structure and barrier function by skin microbiome.

An unbalanced microbial ecosystem on the human skin is closely related to skin diseases and has been associated with inflammation and immune responses. However, little is known about the role of the skin microbiome on skin aging. Here, we report that the Streptococcus species improved the skin structure and barrier function, thereby contributing to anti-aging. Metagenomic analyses showed the abundance of Streptococcus in younger individuals or those having more elastic skin. Particularly, we isolated Streptococcus pneumoniae, Streptococcus infantis, and Streptococcus thermophilus from face of young individuals. Treatment with secretions of S. pneumoniae and S. infantis induced the expression of genes associated with the formation of skin structure and the skin barrier function in human skin cells. The application of culture supernatant including Streptococcal secretions on human skin showed marked improvements on skin phenotypes such as elasticity, hydration, and desquamation. Gene Ontology analysis revealed overlaps in spermidine biosynthetic and glycogen biosynthetic processes. Streptococcus-secreted spermidine contributed to the recovery of skin structure and barrier function through the upregulation of collagen and lipid synthesis in aged cells. Overall, our data suggest the role of skin microbiome into anti-aging and clinical applications.

Synthesis of Kisspeptin-Mimicking Fragments and Investigation of their Skin Anti-Aging Effects.

In recent years, a number of active materials have been developed to provide anti-aging benefits for skin and, among them, peptides have been considered the most promising candidate due to their remarkable and long-lasting anti-wrinkle activity. Recent studies have begun to elucidate the relationship between the secretion of emotion-related hormones and skin aging. Kisspeptin, a neuropeptide encoded by the KISS1 gene, has gained attention in reproductive endocrinology since it stimulates the reproductive axis in the hypothalamus; however, the effects of Kisspeptin on skin have not been studied yet. In this study, we synthesized Kisspeptin-10 and Kisspeptin-E, which are biologically active fragments, to mimic the action of Kisspeptin. Next, we demonstrated the anti-aging effects of the Kisspeptin-mimicking fragments using UV-induced skin aging models, such as UV-induced human dermal fibroblasts (Hs68) and human skin explants. Kisspeptin-E suppressed UV-induced 11 beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) stimulation leading to a regulation of skin aging related genes, including type I procollagen, matrix metalloproteinases-1 (MMP-1), interleukin-6 (IL-6), and IL-8, and rescued the skin integrity. Taken together, these results suggest that Kisspeptin-E could be useful to improve UV-induced skin aging by modulating expression of stress related genes, such as 11ß-HSD1.

Caviar Extract and Its Constituent DHA Inhibits UVB-Irradiated Skin Aging by Inducing Adiponectin Production.

In this study, caviar (sturgeon eggs) was used to elucidate its roles in adiponectin production and skin anti-aging. Recently, caviar has been largely used not only as a nutritional food, but also in cosmetic products. In particular, it has been reported that docosahexaenoic acid (DHA), as one of the main phospholipid components of caviar extract, induces intracellular lipid accumulation and the expression of adiponectin in adipocytes. Although adipocytes are well known to be associated with the skin dermis by secreting various factors (e.g., adiponectin), the effects of caviar extract and DHA on the skin are not well studied. Here, we demonstrate the effects of caviar extract and DHA on adipocyte differentiation and adiponectin production, resulting in a preventive role in UV-irradiated skin aging. Caviar extract and DHA enhanced adipocyte differentiation and promoted the synthesis of transcription factors controlling adipocyte differentiation and adiponectin. In addition, the mRNA expression levels of matrix metalloproteinase-1 (MMP-1) were decreased in UVB-irradiated Hs68 fibroblasts that were cultured in conditioned medium from caviar extract or DHA-treated differentiated adipocytes. Taken together, these results indicate that caviar extract and DHA induce adipocyte differentiation and adiponectin production, thereby inhibiting UVB-induced premature skin aging via the suppression of MMP-1 production.

2'-Fucosyllactose Attenuates Particulate Matter-Induced Inflammation via Inhibition of Hypoxia-Inducible Factor in Keratinocytes.

2'-Fucosyllactose (2FL) is the most abundant component of the oligosaccharide content in human milk. It has been reported that 2FL has the ability to protect against infectious disease caused by bacterial pathogens. In this study, we investigated the protective effects of 2FL on particulate matter (PM)10-induced pro-inflammatory cytokines in HaCaT keratinocytes. 2FL reduced PM10-induced excess expression of interleukin (IL)-6, IL-8, IL-1α and IL-1ß in HaCaT keratinocytes. In addition, PM10 also increased hypoxia-inducible factor (HIF)-1α protein levels; however, 2FL inhibited the accumulation of HIF-1α protein and the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/Akt stimulated by PM10. Furthermore, 2FL improved PM10-induced the decrease in epidermal thickness and integrity of the cornified layer in the reconstructed human epidermal skin model (RHE). In our results, 2FL inhibited PM10-induced pro-inflammatory mediators by regulating the HIF-1α/PI3K/Akt pathway and protected the skin epidermis against PM10 irritation. Taken together, these results suggest that 2FL can be used as a primary ingredient in cosmeceutical products to alleviate skin irritation and inflammation caused by urban air pollution.

FoxO3 coordinates metabolic pathways to maintain redox balance in neural stem cells.

Forkhead Box O (FoxO) transcription factors act in adult stem cells to preserve their regenerative potential. Previously, we reported that FoxO maintains the long-term proliferative capacity of neural stem/progenitor cells (NPCs), and that this occurs, in part, through the maintenance of redox homeostasis. Herein, we demonstrate that among the FoxO3-regulated genes in NPCs are a host of enzymes in central carbon metabolism that act to combat reactive oxygen species (ROS) by directing the flow of glucose and glutamine carbon into defined metabolic pathways. Characterization of the metabolic circuit observed upon loss of FoxO3 revealed a drop in glutaminolysis and filling of the tricarboxylic acid (TCA) cycle. Additionally, we found that glucose uptake, glucose metabolism and oxidative pentose phosphate pathway activity were similarly repressed in the absence of FoxO3. Finally, we demonstrate that impaired glucose and glutamine metabolism compromises the proliferative potential of NPCs and that this is exacerbated following FoxO3 loss. Collectively, our findings show that a FoxO3-dependent metabolic programme supports redox balance and the neurogenic potential of NPCs.

FoxOs in neural stem cell fate decision.

Neural stem cells (NSCs) persist over the lifespan of mammals to give rise to committed progenitors and their differentiated cells in order to maintain the brain homeostasis. To this end, NSCs must be able to self-renew and otherwise maintain their quiescence. Suppression of aberrant proliferation or undesired differentiation is crucial to preclude either malignant growth or precocious depletion of NSCs. The PI3K-Akt-FoxO signaling pathway plays a central role in the regulation of multiple stem cells including one in the mammalian brain. In particular, members of FoxO family transcription factors are highly expressed in these stem cells. As an important downstream effector of growth, differentiation, and stress stimuli, mammalian FoxO transcription factor family controls cellular proliferation, oxidative stress response, homeostasis, and eventual maintenance of long-term repopulating potential. The review will focus on the current understanding of FoxO function in NSCs as well as discuss their biological activities that contribute to determining neural stem cell fate.

MAP17 is associated with the T-helper cell cytokine-induced down-regulation of filaggrin transcription in human keratinocytes.

In the meta-analysis of public microarray databases for different skin diseases, we revealed seven commonly up-regulated genes, DSG3, KRT6, MAP17, PLSCR1, RPM2, SOD2 and SPRR2B. We postulated that the genes selected from the meta-analysis may be potentially associated with the abnormal keratinocyte differentiation. To demonstrate this postulation, we alternatively evaluated whether the genes of interest in the meta-analysis can be regulated by T-helper (Th) cell cytokines in normal human epidermal keratinocytes (NHEK). We found that MAP17 was significantly up-regulated in response to interferon-gamma, interleukin 4 (IL-4), IL-6, IL-17A or IL-22 in NHEK. Interestingly, MAP17 was originally reported to interact with PDZK1; in turn, the PDZK1 gene is localized within the atopic dermatitis-linked region on human chromosome 1q21. In an attempt to evaluate whether MAP17 regulates the expression of cornified envelope-associated genes at the 1q21 locus, such as filaggrin, loricrin and involucrin, we found that the over-expression of MAP17 in HaCaT keratinocytes significantly decreased the expression of filaggrin. Taken together, the Th cell cytokine-induced up-regulation of MAP17 expression may be linked to the down-regulation of filaggrin in NHEK, which may be associated with the abnormal epidermal differentiation observed in the dermatological diseases.

NFATc1 mediates HDAC-dependent transcriptional repression of osteocalcin expression during osteoblast differentiation.

We previously reported that the in vivo and in vitro suppression of Nuclear Factor of Activated T Cells (NFAT) signaling increases osteoblast differentiation and bone formation. To investigate the mechanism by which NFATc1 regulates osteoblast differentiation, we established an osteoblast cell line that overexpresses a constitutively active NFATc1 (ca-NFATc1). The activation of NFATc1 significantly inhibits osteoblast differentiation and function, demonstrated by inhibition of alkaline phosphatase activity and mineralization as well as a decrease in gene expression of early and late markers of osteoblast differentiation such as osterix and osteocalcin, respectively. By focusing on the specific role of NFATc1 during late differentiation, we discovered that the inhibition of osteocalcin gene expression by NFATc1 was associated with a repression of the osteocalcin promoter activity, and a decrease in TCF/LEF transactivation. Also, overexpression of NFATc1 completely blocked the decrease in total histone deacetylase (HDAC) activity during osteoblast differentiation and prevented the hyperacetylation of histones H3 and H4. Mechanistically, we show by Chromatin Immunoprecipitation (ChIP) assay that the overexpression of NFATc1 sustains the binding of HDAC3 on the proximal region of the osteocalcin promoter, resulting in complete hypoacetylation of histones H3 and H4 when compared to GFP-expressing osteoblasts. In contrast, the inhibition of NFATc1 nuclear translocation either by cyclosporin or by using primary mouse osteoblasts with deleted calcineurin b1 prevents HDAC3 from associating with the proximal regulatory site of the osteocalcin promoter. These preliminary results suggest that NFATc1 acts as a transcriptional co-repressor of osteocalcin promoter, possibly in an HDAC-dependent manner.

Conditional disruption of calcineurin B1 in osteoblasts increases bone formation and reduces bone resorption.

We recently reported that the pharmacological inhibition of calcineurin (Cn) by low concentrations of cyclosporin A increases osteoblast differentiation in vitro and bone mass in vivo. To determine whether Cn exerts direct actions in osteoblasts, we generated mice lacking Cnb1 (Cn regulatory subunit) in osteoblasts (DeltaCnb1(OB)) using Cre-mediated recombination methods. Transgenic mice expressing Cre recombinase, driven by the human osteocalcin promoter, were crossed with homozygous mice that express loxP-flanked Cnb1 (Cnb1(f/f)). Microcomputed tomography analysis of tibiae at 3 months showed that DeltaCnb1(OB) mice had dramatic increases in bone mass compared with controls. Histomorphometric analyses showed significant increases in mineral apposition rate (67%), bone volume (32%), trabecular thickness (29%), and osteoblast numbers (68%) as well as a 40% decrease in osteoclast numbers as compared with the values from control mice. To delete Cnb1 in vitro, primary calvarial osteoblasts, harvested from Cnb1(f/f) mice, were infected with adenovirus expressing the Cre recombinase. Cre-expressing osteoblasts had a complete inhibition of Cnb1 protein levels but differentiated and mineralized more rapidly than control, green fluorescent protein-expressing cells. Deletion of Cnb1 increased expression of osteoprotegerin and decreased expression of RANKL. Co-culturing Cnb1-deficient osteoblasts with wild type osteoclasts demonstrated that osteoblasts lacking Cnb1 failed to support osteoclast differentiation in vitro. Taken together, our findings demonstrate that the inhibition of Cnb1 in osteoblasts increases bone mass by directly increasing osteoblast differentiation and indirectly decreasing osteoclastogenesis.

Cyclosporin A elicits dose-dependent biphasic effects on osteoblast differentiation and bone formation.

Cyclosporin A (CsA) is thought to prevent immune reactions after organ transplantation by inhibiting calcineurin (Cn) and its substrate, the Nuclear Factor of Activated T Cells (NFAT). A dichotomy exists in describing the effects of CsA on bone formation. The concept that the suppression of Cn/NFAT signaling by CsA inhibits bone formation is not entirely supported by many clinical reports and laboratory animal studies. Gender, dosage and basal inflammatory activity have all been suggested as explanations for these seemingly contradictory reports. Here we examine the effects of varying concentrations of CsA on bone formation and osteoblast differentiation and elucidate the role of NFATc1 in this response. We show that low concentrations of CsA (<1 microM in vitro and 35.5 nM in vivo) are anabolic as they increase bone formation, osteoblast differentiation, and bone mass, while high concentrations (>1 microM in vitro and in vivo) elicit an opposite and catabolic response. The overexpression of constitutively active NFATc1 inhibits osteoblast differentiation, and treatment with low concentrations of CsA does not ameliorate this inhibition. Treating osteoblasts with low concentrations of CsA (<1 microM) increases fra-2 gene expression and protein levels in a dose-dependent manner as well as AP-1 DNA-binding activity. Finally, NFATc1 silencing with siRNA increases Fra-2 expression, whereas NFATc1 overexpression inhibits Fra-2 expression. Therefore, NFATc1 negatively regulates osteoblast differentiation, and its specific inhibition may represent a viable anabolic therapy for osteoporosis.

NFATc1: a novel anabolic therapeutic target for osteoporosis.

Bone loss and osteoporosis are major public health problems in the elderly. With increasing life expectancy in the United States, the number of people that will develop age-related bone loss and osteoporosis is expected to rise to over 61 million by 2020. Osteoblast differentiation is a crucial aspect of bone formation and remodeling, a process severely compromised in osteoporosis. Almost all the FDA-approved treatments for building healthier bones, excluding parathyroid hormone (PTH), do not address the decrease in osteoblast differentiation seen in osteoporosis and rather are designed to target osteoclasts and bone resorption. The purpose of this study is to examine the effects of NFAT inhibition on osteoblast differentiation and to elucidate the mechanism of its action. Here we demonstrate that the inhibition of calcineurin (Cn) by using cyclosporine A (CsA) increases osteoblast differentiation, both in vivo and in vitro. Furthermore, the specific inhibition of NFATc1 by siRNA increased Fra-2 expression in osteoblasts. Taken together, our results point the way to a novel mechanism to aid in the development of anabolic treatment for osteoporosis.

Fas binding to calmodulin regulates apoptosis in osteoclasts.

Promotion of osteoclast apoptosis is one therapeutic approach to osteoporosis. Calmodulin, the major intracellular Ca(2+) receptor, modulates both osteoclastogenesis and bone resorption. The calmodulin antagonist, trifluoperazine, rescues bone loss in ovariectomized mice (Zhang, L., Feng, X., and McDonald, J. M. (2003) Endocrinology 144, 4536-4543). We show here that a 3-h treatment of mouse osteoclasts with either of the calmodulin antagonists, tamoxifen or trifluoperazine, induces osteoclast apoptosis dose-dependently. Tamoxifen, 10 microm, and trifluoperazine, 10 microm, induce 7.3 +/- 1.8-fold and 5.3 +/- 0.9-fold increases in osteoclast apoptosis, respectively. In Jurkat cells, calmodulin binds to Fas, the death receptor, and this binding is regulated during Fas-mediated apoptosis (Ahn, E. Y., Lim, S. T., Cook, W. J., and McDonald, J. M. (2004) J. Biol. Chem. 279, 5661-5666). In osteoclasts, calmodulin also binds Fas. When osteoclasts are treated with 10 microm trifluoperazine, the binding between Fas and calmodulin is dramatically decreased at 15 min and gradually recovers by 60 min. A point mutation of the Fas death domain in the Lpr(-cg) mouse renders Fas inactive. Using glutathione S-transferase fusion proteins, the human Fas cytoplasmic domain is shown to bind calmodulin, whereas a point mutation (V254N) comparable with the Lpr(-cg) mutation in mice has markedly reduced calmodulin binding. Osteoclasts derived from Lpr(-cg) mice have diminished calmodulin/Fas binding and are more sensitive to calmodulin antagonist-induced apoptosis than those from wild-type mice. Both tamoxifen- and trifluoperazine-induced apoptosis are increased 1.6 +/- 0.2-fold in Lpr(-cg)-derived osteoclasts compared with osteoclasts derived from wild-type mice. In summary, calmodulin antagonists induce apoptosis in osteoclasts by a mechanism involving interference with calmodulin binding to Fas. The effects of calmodulin/Fas binding on calmodulin antagonist-induced apoptosis may open a new avenue for therapy for osteoporosis.