Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components.

Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5-genes essential for autophagosome formation-was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.

Culture insert device with perfusable microchannels enhancesin vitroskin model development and barrier function assessment.

The ever-stricter regulations on animal experiments in the field of cosmetic testing have prompted a surge in skin-related research with a special focus on recapitulation of thein vivoskin structurein vitro. In vitrohuman skin models are seen as an important tool for skin research, which in recent years attracted a lot of attention and effort, with researchers moving from the simplest 2-layered models (dermis with epidermis) to models that incorporate other vital skin structures such as hypodermis, vascular structures, and skin appendages. In this study, we designed a microfluidic device with a reverse flange-shaped anchor that allows culturing of anin vitroskin model in a conventional 6-well plate and assessing its barrier function without transferring the skin model to another device or using additional contraptions. Perfusion of the skin model through vascular-like channels improved the morphogenesis of the epidermis compared with skin models cultured under static conditions. This also allowed us to assess the percutaneous penetration of the tested caffeine permeation and vascular absorption, which is one of the key metrics for systemic drug exposure evaluation.

Chemical- and Drug-Induced Allergic, Inflammatory, and Autoimmune Diseases Via Haptenation.

Haptens are small molecules that only elicit an immune response when bound to proteins. Haptens initially bind to self-proteins and activate innate immune responses by complex mechanisms via inflammatory cytokines and damage-associated molecular patterns and the subsequent upregulation of costimulatory signals such as cluster of differentiation 86 (CD86) on dendritic cells. Subsequent interactions between CD86 and CD28 on T cells are critically important for properly activating naive T cells and inducing interleukin 2 production, leading to the establishment of adaptive immunity via effector and memory T cells. Accumulating evidence revealed the involvement of haptens in the development of various autoimmune-like diseases such as allergic, inflammatory, and autoimmune diseases including allergic contact dermatitis, atopy, asthma, food allergy, inflammatory bowel diseases, hemolytic anemia, liver injury, leukoderma, and even antitumor immunity. Therefore, the development of in vitro testing alternatives to evaluate in advance whether a substance might lead to the development of these diseases is highly desirable. This review summarizes and discusses recent advances in chemical- and drug-induced allergic, inflammatory, and autoimmune diseases via haptenation and the possible molecular underlying mechanisms, as well as in vitro testing alternatives to evaluate in advance whether a substance might cause the development of these diseases.

Upregulation of CD86 and IL-12 by rhododendrol in THP-1 cells cocultured with melanocytes through ROS and ATP.

BACKGROUND: The tyrosinase inhibitor rhododendrol (RD), used as a skin whitening agent, reportedly has the potential to induce leukoderma. OBJECTIVE: Although an immune response toward melanocytes was demonstrated to be involved in leukoderma, the molecular mechanism is not fully understood. METHODS: We hypothesized that if RD is a pro-hapten and tyrosinase-oxidized RD metabolites are melanocyte-specific sensitizers, the sensitizing process could be reproduced by the human cell line activation test (h-CLAT) cocultured with melanocytes (h-CLATw/M) composed of human DC THP-1 cells and melanoma SK-MEL-37 cells. Cell surface expression, ROS generation and ATP release, mRNA expression, and the effects of several inhibitors were examined. RESULTS: When RD was added to the h-CLATw/M, the expression of cell-surface CD86 and IL-12 mRNA was greatly enhanced in THP-1 cells compared with those in the h-CLAT. The rapid death of melanoma cells was induced, with ROS generation and ATP release subsequently being greatly enhanced, resulting in the cooperative upregulation of CD86 and IL-12. Consistent with those observations, an ROS inhibitor, ATP receptor P2X7 antagonist, or PERK inhibitor antagonized the upregulation. CD86 upregulation was similarly observed with another leukoderma-inducible tyrosinase inhibitor, raspberry ketone, but not with the leukoderma noninducible skin-whitening agents ascorbic acid and tranexamic acid. CONCLUSION: RD is a pro-hapten sensitizer dependent on tyrosinase that induces ROS generation and ATP release from melanocytes for CD86 and IL-12 upregulation in DCs, possibly leading to the generation of tyrosinase-specific cytotoxic T lymphocytes. The coculture system h-CLATw/M may be useful for predicting the sensitizing potential to induce leukoderma.

The Rpd3/HDAC complex is present at the URS1 cis-element with hyperacetylated histone H3.

In eukaryotes, the hypoacetylated state of histone N-terminal lysines at many gene-promoters, which is created by histone deacetylases (HDACs), is changed to the hyperacetylated state by the function of histone acetyltransferases (HATs) upon transcription activation. Although much insight has been obtained to date as to how modification of the histone tail regulates gene expression, little is known about how the transition between the unmodified and modified states takes place. In Saccharomyces cerevisiae, the HDAC complex containing Rpd3 (Rpd3L) represses the transcription of several sets of genes through the URS1 cis-element. We found that the histone H3 acetylation level at the URS1 of seven genes (INO1, CAT2, ACS1, YAT1, RIM4, CRC1, and SIP4) was elevated in the presence of Rpd3/HDAC in growth in acetate-containing medium (YPA), suggesting that a mechanism that regulates HDAC activity is present in this organism. The biological significance of this phenomenon is discussed below.

The long non-coding RNA FLJ46906 binds to the transcription factors NF-κB and AP-1 and regulates expression of aging-associated genes.

Several features differentiate aged cells from young cells, many of which are due to changes in gene expression during the aging process. The mechanisms of altered gene expression in aging cells remain incompletely understood, and we hypothesized that long non-coding (lnc) RNAs mediate at least some of these changes. We screened for alterations in lncRNA expression with aging in skin fibroblasts and identified the lncRNA FLJ46906 to be consistently upregulated with aging in-vivo and in-vitro. The function of this lncRNA has not been known. Here we show that FLJ46906 regulates several aging-associated genes, including IL1B, IL6, CXCL8, TGFB1, and ELN. We suggest that these effects are mediated through NF-κB and AP-1, because these aging-associated genes are regulated by NF-κB and AP-1, and because we found that FLJ46906 directly binds to these two transcription factors. This data supports a role of the lncRNA FLJ46906 in the aging process.