Pathway In Human Keratinocytes Research Articles

Cannabis sativa (Hemp) seed-derived peptides WVYY and PSLPA modulate the Nrf2 signaling pathway in human keratinocytes.

Cannabis sativa (Hemp) seeds are used widely for cosmetic and therapeutic applications, and contain peptides with substantial therapeutic potential. Two key peptides, WVYY and PSLPA, extracted from hemp seed proteins were the focal points of this study. These peptides have emerged as pivotal contributors to the various biological effects of hemp seed extracts. Consistently, in the present study, the biological effects of WVYY and PSLPA were explored. We confirmed that both WVYY and PSLPA exert antioxidant and antibacterial effects and promote wound healing. We hypothesized the involvement of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in these observed effects, given that Nrf2 is reported to be a central player in the regulation of these observed effects. Molecular-level investigations unequivocally confirmed the role of the Nrf2 signaling pathway in the observed effects of WVYY and PSLPA, specifically their antioxidant effects. Our study highlights the therapeutic potential of hemp seed-derived peptides WVYY and PSLPA, particularly with respect to their antioxidant effects, and provides a nuanced understanding of their effects. Further, our findings can facilitate the investigation of targeted therapeutic applications and also underscore the broader significance of hemp extracts in biological contexts.

Persicaria senticosa extract mitigates ultraviolet B-induced photoaging by suppressing the mitogen-activated protein kinase/activator protein 1/matrix metalloproteinase 1 pathway in human keratinocytes and hairless mice

Ultraviolet (UV) irradiation has been identified as a key trigger for skin photoaging, characterized by the overproduction of matrix metalloproteinases (MMPs) and reactive oxygen species (ROS), along with the accelerated decomposition of extracellular matrix (ECM) proteins, ultimately contributing to the development of wrinkles. Persicaria senticosa (PS) extracts are recognized for their antioxidative properties and their importance in skin health. Nevertheless, there is a paucity of studies investigating the potential of PS in protecting the skin against photoaging. The present study aimed to assess the effectiveness of PS extracts in preventing photoaging and elucidating the molecular mechanisms involved in using immortalized human keratinocytes (HaCaT) and hairless mice. The major bioactive constituents of PS were identified as p-coumaric acid, isoquercitrin, quercetin-3-O-glucuronide, and quercetin. Aqueous extracts of PS exhibited the ability to mitigate UVB-induced cellular damage and diminished ROS generation in HaCaT cells. Moreover, treatment with PS effectively attenuated the upregulated expression of matrix metalloproteinase-1 (MMP-1) and collagen degradation induced by UVB exposure. The property of PS to counteract photoaging was related to its capacity to inhibit the UVB-induced phosphorylation of mitogen-activated protein kinase (MAPK) and suppress the subsequent activation of activator protein 1 (AP-1) signaling pathways. Moreover, in hairless mice exposed to UVB radiation, the application of PS significantly alleviated the development of skin wrinkles, diminished epidermal thickening, and mitigated collagen degradation. Notably, PS treatment resulted in the downregulation of the UVB-activated MAPK/AP-1/MMP-1 pathway in mouse skin tissues. These findings suggest that PS has the potential to serve as a therapeutic agent for treating photoaging, holding promises in both cosmeceutical and pharmaceutical applications.

Fermented black ginseng extract prevents UVB-induced inflammation by regulating the nc886-PKR pathway in human keratinocytes.

Continuous exposure of the skin to ultraviolet B (UVB) rays can cause inflammation and photodamage. In previous studies, we observed that the upregulation of nc886, a noncoding RNA (ncRNA), can alleviate UVB-induced inflammation through suppression of the protein kinase RNA (PKR) pathway. We aim to investigate the effect of fermented black ginseng extract (FBGE), which has been shown to increase the expression of nc886, on UVB-induced inflammation in keratinocytes. To confirm the cytotoxicity of FBGE, MTT assay was performed, and no significant cytotoxicity was found on human keratinocytes. The efficacies of FBGE were assessed through qPCR, Western blotting, and ELISA analysis which confirmed regulation of UVB-induced inflammation. The analysis results showed that FBGE inhibited the decrease in nc886 expression and the increase in the methylated nc886 caused by UVB. It also prevented the UVB-induced increase of metalloproteinase-9 (MMP-9), metalloproteinase-1 (MMP-1), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α). Additionally, FBGE suppressed the PKR-MAPK pathways activated by UVB. These results implicate that FBGE can alleviate UVB-induced inflammation through regulation of the nc886-PKR pathway.

Keratinocyte FABP5-VCP complex mediates recruitment of neutrophils in psoriasis

Abstract Psoriasis is a common chronic inflammatory skin disease. One of the hallmarks of psoriasis is the abundance of neutrophil infiltration in the skin lesions. However, the detailed molecular mechanisms of neutrophil chemotaxis and activation remains unclear. Here we demonstrated an upregulation of epidermal fatty acid binding protein (E-FABP, also known as FABP5) in skin tissue from both human psoriasis and imiquimod (IMQ)-induced psoriatic mouse model. Genetic deletion of FABP5 in global knockout mice and keratinocyte conditional (Krt6a-Cre) knockout mice, but not in myeloid cell conditional (Lysm-Cre) knockout mice, attenuated the skin erythema, thickness, and desquamation. Immunophenotypic analysis showed that FABP5 deficiency reduced the recruitment of Ly6G+ neutrophils in the dermis and ear of IMQ-treated mice. Mechanistically, activated keratinocytes in IMQ-treated mice produced chemokines and cytokines (e.g., CXCL8, IL-36γ) that triggered neutrophil chemotaxis and activation in a FABP5-dependent manner. Proteomic and IP analyses further identified that FABP5 interacted with a valosin-containing protein (VCP), a key player in NFκB signaling pathway in human keratinocytes. Silencing of FABP5 inhibited VCP-mediated IκBα phosphorylation and NFκB signaling activation. Collectively, these data suggest that FABP5 in keratinocytes interacts with VCP to activate NFκb signaling, thus playing an essential role in the neutrophil infiltration in psoriatic skin lesions. R01AI137324, #LINK#R01CA180986#ENDLINK#

Santamarine Isolated from Artemisia scoparia Inhibits UVB-induced Matrix Metalloproteinase Expression via Repression of MAPK/AP-1 Pathway in Human Keratinocytes

Background: Artemisia scoparia has a widespread distribution and can be found commonly in Eurasia. In traditional Korean medicine, A. scoparia leaves and flowers are used against urethral complications, phlogistic problems, and in the treatment of hepatitis.BRMethods and Results: In the present study, the protective effect of santamarine isolated from A. scoparia was evaluated in Ultraviolet B (UVB)-damaged keratinocytes. Results showed that santamarine suppressed the production of reactive oxygen species in a concentration-dependent manner. Treating cells with santamarine decreased the generation of Matrix Metalloproteinase (MMP)-1 compared to that in cells treated with UVB alone. Additionally, the mRNA levels of MMP-1 and MMP-3 were remarkably lower in cells treated with santamarine than those in cells with UVB irradiation. Furthermore, upregulated protein levels of MMP-1, MMP-2, MMP-3, and MMP-9 following UVB exposure were ameliorated by the introduction of santamarine. Investigation of the mechanisms underlying the photoprotective effect of santamarine showed enhanced inhibition of MAPK/AP-1 signaling following santamarine treatment. The presence of santamarine also recovered the UVB-induced decrease in collagen amount.BRConclusions: Overall, these results demonstrated that santamarine has a potential protective effect against UVB-induced skin photoaging in keratinocytes in vitro. The mechanism behind this effect was suggested to be via suppression of MAPK/AP-1 signaling.

Poria cocos Extract from Mushrooms Stimulates Aquaporin-3 via the PI3K/Akt/mTOR Signaling Pathway.

BackgroundPoria cocos (PC), a fungus, has been used for more than 2000 years as a food and medicine in China. PC and its components have various pharmacological effects on the skin, including immunomodulatory activities, barrier function improvement, and anti-tumor effects. However, the effect of PC in aquaporin-3 (AQP3) expression, which is essential for epidermal water permeability barrier maintenance, was not reported.MethodsThis study examined the mechanism through which the ethanol extract of the sclerotium of PC (EPC) promoted the expression of AQP3 in cultured human keratinocytes. Western blotting was used to investigate the expression of AQPs and the activation of phosphoinositide 3-kinase (PI3K)/Akt-related signaling molecules in HaCaT cells. Cells were treated with inhibitors of PI3K/Akt and mechanistic target of rapamycin (mTOR) prior to EPC treatment.ResultsEPC promoted the expression of AQP3 in HaCaT cells without affecting AQP1 and AQP2 expression. Phosphorylated Akt levels were increased by EPC treatment, and the inhibition of PI3K by LY2940002 resulted in a reduction in EPC-induced AQP3 expression. Furthermore, EPC stimulated the phosphorylation of p70S6K and AktSer473, which are downstream targets of mTORC1 and mTORC2, respectively. The mTOR complex inhibitors, rapamycin and Torin 1, partially reduced EPC-induced AQP3 expression.ConclusionThese results suggest that EPC increased expression of AQP3, which is important for skin moisturization, by activating the PI3K/Akt/mTOR signaling pathway in human keratinocytes.

Differential induction of TLR2/c-Jun and NF-κB signaling pathway in human keratinocytes infected by Trichophyton rubrum and Microsporum gypseum

Differential induction of TLR2/c-Jun and NF-κB signaling pathway in human keratinocytes infected by Trichophyton rubrum and Microsporum gypseum

Tetrandrine Enhances H2O2-Induced Apoptotic Cell Death Through Caspase-dependent Pathway in Human Keratinocytes.

Tetrandrine, a bis-benzylisoquinoline alkaloid, induces apoptosis of many types of human cancer cell. Hydrogen peroxide (H2O2) is a reactive oxygen species inducer; however, there are no reports to show whether pre-treatment of tetrandrine with H2O2 induces more cell apoptosis than H2O2 alone. Thus, the present study investigated the effects of tetrandrine on H2O2-induced cell apoptosis of human keratinocytes, HaCaT, in vitro. HaCaT cells were pre-treated with and without tetrandrine for 1 h, and then treated with H2O2 for examining cell morphological changes and cell viability using contrast-phase microscopy and propidium iodide (PI) exclusion assay, respectively. Cells were measured apoptotic cell death by using annexin V/PI double staining and further analyzed by flow cytometer. Cells were further assessed for DNA condensation using 2-(4-amidinophenyl)-6-indolecarbamidine staining. Western blotting was used to measure expression of apoptosis-associated proteins and confocal laser microscopy was used to measure the protein expression and nuclear translocation from the cytoplasm to nuclei. Pre-treatment of tetrandrine for 1 h and treatment with H2O2 enhanced H2O2-induced cell morphological changes and reduced cell viability, whilst increasing apoptotic cell death and DNA condensation. Furthermore, tetrandrine significantly increased expression of reactive oxygen species-associated proteins such as superoxide dismutase (Cu/Zn) and superoxide dismutase (Mn) but significantly reduced the level of catalase, which was also confirmed by confocal laser microscopy. It also increased expression of DNA repair-associated proteins ataxia telangiectasia mutated, ataxia-telangectasia and Rad3-related, phospho-P53, P53 and phosphorylated histone H2AX, and of pro-apoptotic proteins BCL2 apoptosis regulator-associated X-protein, caspase-3, caspase-8, caspase-9 and poly ADP ribose polymerase in HaCaT cells. These are the first and novel findings showing tetrandrine enhances H2O2-induced apoptotic cell death of HaCaT cells and may provide a potent approach for the treatment of proliferated malignant keratinocytes.

Prolidase Stimulates Proliferation and Migration through Activation of the PI3K/Akt/mTOR Signaling Pathway in Human Keratinocytes.

Recent reports have indicated prolidase (PEPD) as a ligand of the epidermal growth factor receptor (EGFR). Since this receptor is involved in the promotion of cell proliferation, growth, and migration, we aimed to investigate whether prolidase may participate in wound healing in vitro. All experiments were performed in prolidase-treated human keratinocytes assessing cell vitality, proliferation, and migration. The expression of downstream signaling proteins induced by EGFR, insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGF-β1), and β1-integrin receptors were evaluated by Western immunoblotting and immunocytochemical staining. To determine collagen biosynthesis and prolidase activity radiometric and colorimetric methods were used, respectively. Proline content was determined by applying the liquid chromatography coupled with mass spectrometry. We found that prolidase promoted the proliferation and migration of keratinocytes through stimulation of EGFR-downstream signaling pathways in which the PI3K/Akt/mTOR axis was involved. Moreover, PEPD upregulated the expression of β1-integrin and IGF-1 receptors and their downstream proteins. Proline concentration and collagen biosynthesis were increased in HaCaT cells under prolidase treatment. Since extracellular prolidase as a ligand of EGFR induced cell growth, migration, and collagen biosynthesis in keratinocytes, it may represent a potential therapeutic approach for the treatment of skin wounds.

Saturated and aromatic aldehydes originating from skin and cutaneous bacteria activate the Nrf2-keap1 pathway in human keratinocytes.

Skin homeostasis is constantly challenged by environmental factors, affecting its delicate redox balance. The skin is also home to a wide variety of bacterial species, including Staphylococci. The cutaneous redox state is governed by the Nrf2-keap1 pathway, which is responsible for the induction of phase II cytoprotective enzymes, thus sustaining a healthy oxidative state. As part of normal metabolism, both bacteria and cutaneous tissue emit copious amounts of volatile organic compounds (VOCs), one subgroup of which are aldehydes. α,β-unsaturated aldehydes are known activators of Nrf2-keap1 pathway by direct oxidation of the keap1 protein. However, we did not encounter reports of Nrf2 activation by saturated or aromatic aldehydes, neither bacteria nor skin-derived. We hypothesized that non-α,β-unsaturated aldehydes derived from skin or cutaneous bacteria may act as Nrf2-keap1 pathway activators and therefore afford protection against environmental insults. The saturated aldehydes nonanal and decanal (known skin metabolites) and the aromatic aldehyde benzaldehyde (known skin and Staphylococcus epidermidis metabolite) were shown to induce the Nrf2-keap1 pathway in human keratinocytes. We also identified a newly described aromatic aldehyde, 3-furaldehyde (3-FA), emitted from S. aureus and S. epidermidis cultures, which also activated the pathway. Moreover, Nrf2-keap1 induction led to a significant protection against UVB-induced apoptosis. The mechanism involved in this activation has been partially elucidated. This work emphasizes the importance of cutaneous bacteria, as well as healthy skin lipid peroxidation processes in the maintenance and regulation of the cellular antioxidant response, namely with regard to coping with environmental stressors.

Formation of SUMO3-conjugated chains of MAVS induced by poly(dA:dT), a ligand of RIG-I, enhances the aggregation of MAVS that drives the secretion of interferon-β in human keratinocytes

The retinoic-acid inducible gene (RIG)-I is a cytoplasmic pattern recognition receptor that senses single-stranded (ss) or double-stranded (ds) RNA. RIG-I also senses AT-rich dsDNA, poly(dA:dT), through the action of an RNA polymerase III-transcribed RNA intermediate. Upon the binding of an RNA ligand, RIG-I binds to the mitochondrial antiviral-signaling protein (MAVS) and induces the formation of filamentous aggregates of MAVS, leading to the formation of a signaling complex that drives Type I interferon (IFN) responses. In the current study, we investigated the issue of whether the SUMOylation of MAVS induced by poly(dA:dT) affects the aggregation of MAVS in the RIG-I/MAVS pathway in human keratinocytes. Our results show that the poly(dA:dT)-induced secretion of IFN-β was dependent on RIG-I and MAVS. The inhibition of SUMOylation by Ginkgolic acid or Ubc9 siRNA was found to inhibit the poly(dA:dT)-induced secretion of IFN-β, suggesting that the SUMOylation is required for the poly(dA:dT)-activated RIG-I/MAVS pathway, which drives the secretion of IFN-β. In addition, treatment with poly(dA:dT) enhanced the formation of polymeric chains of small-ubiquitin like modifiers (SUMO)3, but not SUMO1 and SUMO2, on MAVS. Our results also show that the conjugation of SUMO3 to MAVS induced by poly (dA:dT) enhanced the aggregation of MAVS. These collective results show that the formation of SUMO3-conjugated chains of MAVS induced by poly (dA:dT), a ligand of RIG-I, enhances the aggregation of MAVS which, in turn, drives the secretion of IFN-β in human keratinocytes.

770 CYP11A1-derived vitamin D3 hydroxyderivatives protect against UVB-induced skin inflammation through the modulation of NF-κB signaling pathway in human keratinocytes

770 CYP11A1-derived vitamin D3 hydroxyderivatives protect against UVB-induced skin inflammation through the modulation of NF-κB signaling pathway in human keratinocytes

Nc886, a non-coding RNA, inhibits UVB-induced MMP-9 and COX-2 expression via the PKR pathway in human keratinocytes

nc886, a long non-coding RNA (ncRNA) of 101 nucleotides in length, is known as a vault RNA or microRNA precursor. Despite the recent discovery that ncRNAs in the nucleus play a crucial role in regulating chromosomal transformation and transcription, only a few studies have focused on the function of ncRNAs in the cytoplasm, such as nc886. Several studies have investigated the function of nc886 as a suppressor of carcinogenesis and inflammation in different cancer cell types; however, its role in the skin has yet to be clearly elucidated. The two RNA binding sites for protein kinase RNA-activated (PKR) are located in the central region of the stable structure of nc886, which competes with other double-stranded RNA species. Successful binding results in decreased PKR activity. Among changes in skin cells induced by ultraviolet B (UVB) radiation, nc886 expression decreases, whereas PKR phosphorylation via mitogen-activated protein kinases (MAPKs) increases. Reduced nc886 expression leads to uncontrolled PKR activity and increases in the expression of inflammatory cytokines, matrix metalloproteinase-9 (MMP-9), type IV collagenase, and cyclooxygenase (COX-2), which ultimately accelerate inflammatory responses and skin aging. The present study investigated the regulatory mechanism associated with PKR activity and nc886–PKR binding in skin cell aging and inflammation. These results suggest a role for nc886 in controlling photoaging and inflammation in skin cells.

Amentoflavone-induced oxidative stress activates NF-E2-related factor 2 via the p38 MAP kinase-AKT pathway in human keratinocytes.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that responds to oxidative stress and xenobiotics. Multiple lines of evidence suggest that Nrf2 activation protects against aging, inflammation, and many diseases, including cancer. Nrf2 activators derived from natural sources have been widely studied. In this study, we investigated the effect of amentoflavone (AFN), a biflavonoid found in many plants, on Nrf2 signaling in human keratinocytes (HaCaT cells). AFN significantly increased ARE luciferase activity by Nrf2 accumulation in the nucleus. Subsequently, the levels of a Nrf2 target protein, NQO-1, were significantly increased by AFN in a dose- and time-dependent manner. To verify the mechanism of AFN-induced activation of Nrf2 signaling, we measured generation of reactive oxygen species (ROS). Interestingly AFN triggered mild ROS production. Additionally, AFN-induced Nrf2 activation was inhibited by N-acetyl cysteine. Therefore, we studied the effect of ROS-related signaling on Nrf2 by measuring the activation of AKT and members of the mitogen-activated protein kinase family, such as extracellular signal-regulated kinase (ERK1/2) and p38. The results showed that the pharmacological inhibitor of PI3K/AKT (LY294002) or p38 (SB 203580), but not ERK1/2 (U0126), abrogated AFN-induced Nrf2 activation. Subsequently, we found that silencing or inhibition of p38 resulted in decrease of AKT phosphorylation as well as inhibition of Nrf2 accumulation. Furthermore, we found that AFN stabilized Nrf2 by inhibiting its ubiquitination. Taken together, our results suggest that AFN contributes to Nrf2 activation through ROS-mediated activation of the p38-AKT pathway in HaCaT cells.

Rhododendron album Blume extract inhibits TNF-α/IFN-γ-induced chemokine production via blockade of NF-κB and JAK/STAT activation in human epidermal keratinocytes.

Rhododendron album Blume (RA) has traditionally been used as an herbal medicine and is considered to have anti‑inflammatory properties. It is a well‑known medicine for treatment of allergic or atopic diseases. In the present study, the biological effects of an RA methanol extract (RAME) on inflammation were investigated in tumor necrosis factor‑α (TNF‑α)/interferon‑γ (IFN‑γ)‑stimulated human keratinocytes. The present study aimed to investigate the potential mechanisms by which RAME inhibited TNF‑α/IFN‑γ‑induced expression of chemokines [thymus‑ and activation-regulated chemokine (TARC) and macrophage‑derived chemokine (MDC)] and cytokines [interleukin (IL)‑6 and IL‑8] through the nuclear factor‑κB (NF‑κB) pathway in human keratinocytes. The effects of RAME treatment on cell viability were investigated in TNF‑α/IFN‑γ‑stimulated HaCaT cells. The expression of TARC, MDC, IL‑6 and IL‑8 was assessed using reverse transcription‑quantitative polymerase chain reaction analysis or ELISA, and its effect on the inhibitory mitogen-activated protein kinase pathway was also studied using western blot analysis. TNF‑α/IFN‑γ induced the expression of IL‑6, IL‑8, TARC and MDC in a dose‑dependent manner through NF‑κB and Janus kinase/signal transducers and activators of transcription (JAK/STAT) activation. Notably, treatment with RAME significantly suppressed TNF-α/IFN-γ-induced expression of IL‑6, IL‑8, TARC, and MDC. In addition, RAME treatment inhibited the activation of NF‑κB and the JAK/STAT pathway in TNF‑α/IFN‑γ‑induced HaCaT cells. These results suggest that RAME decreases the production of chemokines and pro‑inflammatory cytokines by suppressing the NF‑κB and the JAK/STAT pathways. Consequently, RAME may potentially be used for treatment of atopic dermatitis.

3-Bromo-4,5-dihydroxybenzaldehyde Enhances the Level of Reduced Glutathione via the Nrf2-Mediated Pathway in Human Keratinocytes.

A natural bromophenol found in seaweeds, 3-bromo-4,5-dihydroxybenzaldehyde (BDB), has been shown to possess antioxidant effects. This study aimed to investigate the mechanism by which BDB protects skin cells subjected to oxidative stress. The effect of BDB on the protein and mRNA levels of glutathione-related enzymes and the cell survival of human keratinocytes (HaCaT cells) was investigated. BDB treatment increased the protein and mRNA levels of glutathione synthesizing enzymes and enhanced the production of reduced glutathione in HaCaT cells. Furthermore, BDB activated NF-E2-related factor 2 (Nrf2) and promoted its localization into the nucleus by phosphorylating its up-stream signaling proteins, extracellular signal–regulated kinase and protein kinase B. Thus, BDB increased the production of reduced glutathione and established cellular protection against oxidative stress via an Nrf2-mediated pathway.

Ultraviolet B radiation down-regulates ULK1 and ATG7 expression and impairs the autophagy response in human keratinocytes

Autophagy is a self-digestive pathway that helps to maintain cellular homeostasis, and many autophagy-related gene (ATG)s involved the regulation of the autophagy process. Ultraviolet light is a common stressor of skin, but it is unclear how autophagy is regulated after ultraviolet exposure in epidermal keratinocytes. Here, we found that the mRNAs of some key ATG genes such as ULK1, ATG5 and ATG7 exhibited significantly lower levels in the skin tissues of the face and chest with solar ultraviolet exposure, compared with perineal skin. Interestingly, UVB radiation down-regulated the expression of ULK1, ATG3 and ATG7, and it inhibited the autophagy flux via a mechanistic target of rapamycin (MTOR)-independent pathway in human keratinocytes. The inhibition of autophagy in UVB-treated keratinocytes cannot be restored by treatment with the MTOR-dependent autophagy inducer rapamycin. Importantly, UVB treatment perturbs the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II and LC3-II turnover in response to treatment with MTOR inhibitors (Torin 1 and pp242), as well as endoplasmic reticular stress (A23187 and tunicamycin), inositol pathway (L690,330) and autophagy inducers (resveratrol and STF62247). Our study demonstrates that UVB radiation down-regulates several key autophagy-related proteins and impairs the autophagy response in keratinocytes. This study demonstrates a linkage between autophagy and skin disorders associated with ultraviolet exposure.

Pterostilbene protects against UVB-induced photo-damage through a phosphatidylinositol-3-kinase-dependent Nrf2/ARE pathway in human keratinocytes

ABSTRACTObjective: Ultraviolet B (UVB) irradiation is the initial etiological factor for various skin disorders, including erythema, sunburn, photoaging, and photocarcinogenesis. Pterostilbene (Pter) displayed remarkable antioxidant, anti-inflammatory, and anticarcinogenic activities. This study aimed to investigate the effective mechanism of Pter against UVB-induced photodamage in immortalized human keratinocytes.Methods: Human keratinocytes were pretreated with Pter (5 and 10 μM) for 24 h prior to UVB irradiation (300 mJ/cm2). Harvested cells were analyzed by MTT, DCFH-DA, comet, western blotting, luciferase promoter, small interference RNA transfection, and quantitative real-time polymerase chain reaction assay.Results: Pter significantly attenuated UVB-induced cell death and reactive oxygen species (ROS) generation, and effectively increased nuclear translocation of NF-E2-related factor-2 (Nrf2), expression of Nrf2-dependent antioxidant enzymes, and DNA repair activity. Moreover, the protective effects of Pter were abolished by small interference RNA-mediated Nrf2 silencing. Furthermore, Pter was also found to induce the phosphorylation of Nrf2 and the known phosphatidylinositol-3-kinase (PI3K) phosphorylated kinase, Akt. The specific inhibitor of PI3K, LY294002, successfully abrogated Pter-induced Nrf2 phosphorylation, activation of Nrf2-antioxidant response element pathway, ROS scavenging ability, and DNA repair activity.Conclusion: The present study indicated that Pter effectively protected against UVB-induced photodamage by increasing endogenous defense mechanisms, scavenging UVB-induced ROS, and aiding in damaged DNA repair through a PI3K-dependent activation of Nrf2/ARE pathway.

Arsenic Induces p62 Expression to Form a Positive Feedback Loop with Nrf2 in Human Epidermal Keratinocytes: Implications for Preventing Arsenic-Induced Skin Cancer.

Exposure to inorganic arsenic in contaminated drinking water poses an environmental public health threat for hundreds of millions of people in the US and around the world. Arsenic is a known carcinogen for skin cancer. However, the mechanism by which arsenic induces skin cancer remains poorly understood. Here, we have shown that arsenic induces p62 expression in an autophagy-independent manner in human HaCaT keratinocytes. In mouse skin, chronic arsenic exposure through drinking water increases p62 protein levels in the epidermis. Nrf2 is required for basal and arsenic-induced p62 up-regulation. p62 knockdown reduces arsenic-induced Nrf2 activity, and induces sustained p21 up-regulation. p62 induction is associated with increased proliferation in mouse epidermis. p62 knockdown had little effect on arsenic-induced apoptosis, while it decreased cell proliferation following arsenic treatment. Our findings indicate that arsenic induces p62 expression to regulate the Nrf2 pathway in human keratinocytes and suggest that targeting p62 may help prevent arsenic-induced skin cancer.

Extracellular Vesicles from Adipose-Derived Mesenchymal Stem/Stromal Cells Accelerate Migration and Activate AKT Pathway in Human Keratinocytes and Fibroblasts Independently of miR-205 Activity.

Mesenchymal stem/stromal cells (MSCs) are promising tools in cell therapy. They secrete extracellular vesicles (EVs) that carry different classes of molecules that can promote skin repair, but the mechanisms are poorly understood. Skin wound healing is a complex process that requires the activity of several signaling pathways and cell types, including keratinocytes and fibroblasts. In this study, we explored whether adipose tissue MSC-derived EVs could accelerate migration and proliferation of keratinocytes and fibroblasts, activate the AKT pathway, and promote wound healing in vivo. Furthermore, we evaluated if EV effects are miR-205 dependent. We found that MSC EVs had an average diameter of 135 nm. Keratinocytes and fibroblasts exposed to EVs exhibited higher levels of proliferation, migration, and AKT activation. Topical administration of EVs accelerated skin wound closure. Knockdown of miR-205 decreased AKT phosphorylation in fibroblasts and keratinocytes, whereas migration was decreased only in keratinocytes. Moreover, knockdown of miR-205 failed to inhibit AKT phosphorylation in fibroblasts and keratinocytes exposed to EVs. About the mechanism of EV effects, we found that incubation with EVs prevented inhibition of AKT activation by miR-205 knockdown, suggesting that EVs activate AKT independently of miR-205. In conclusion, we demonstrated that EVs are a promising tool for wound healing.