Protective effect of inositol hexaphosphate against UVB damage in HaCaT cells and skin carcinogenesis in SKH1 hairless mice

UVB radiation damages keratinocytes, potentially inducing chronic skin damage, cutaneous malignancy, and suppression of the immune system. Naturally occurring agents have been considered for prevention and treatment of various kinds of cancer, including skin cancer. Inositol hexaphosphate (IP6), an antioxidant, is a naturally occurring polyphosphorylated carbohydrate that has shown a strong anticancer activity in several experimental models. We assessed the protective effects of IP6 against UVB irradiationinduced injury and photocarcinogenesis by using HaCaT cells (human immortalized keratinocytes) and SKH1 hairless mice. We found that IP6 counteracts the harmful effects of UVB irradiation and increases the viability and survival of UVB-exposed cells. Treatment with IP6 after UVB irradiation (30 mJ/cm(2)) arrested cells in the G(1) and G(2) M phases while decreasing the S phase of the cell cycle. Treatment with IP6 also decreased UVB-induced apoptosis and caspase 3 activation. Topical application of IP6 followed by exposure to UVB irradiation in SKH1 hairless mice decreased tumor incidence and multiplicity as compared with control mice. Our results suggest that IP6 protects HaCaT cells from UVB-induced apoptosis and mice from UVB-induced tumors.     

Positive Promoting Effects of Smilax China Flower Absolute on the Wound Healing/Skin Barrier Repair-Related Responses of HaCaT Human Skin Keratinocytes

Smilax china (SC) has pharmacological effects including anti-inflammatory activity, but its effects on skin wound healing and skin barrier function have not been investigated. Here, we investigated the effects of absolute extracted from SC flowers (SCF) on skin wound healing-linked responses and functional skin barrier proteins using human epidermal keratinocytes (HaCaT cells). SCF absolute contained 20 components and was non-toxic to HaCaT cells. The absolute increased the proliferation, migration, and sprout outgrowth of HaCaT cells, and enhanced the activations of serine/threonine-specific protein kinase and extracellular signal-regulated kinase1/2. In addition, it increased the syntheses of type I and IV collagens and the expressions of skin barrier proteins (filaggrin and loricrin). These results indicate SCF absolute may has positive effects on skin wound healing by accelerating keratinocyte migration and proliferation activities and collagen synthesis, and on skin barrier function by upregulating barrier proteins in keratinocytes. We suggest SCF absolute to be considered as a potential means of promoting skin wound and barrier repair.     

Apoptosis may underlie the pathology of zinc-deficient skin

The trace element zinc is essential for the survival and function of all cells. Zinc deficiency, whether nutritional or genetic, is fatal if left untreated. The effects of zinc deficiency are particularly obvious in the skin, seen as an erythematous rash, scaly plaques, and ulcers. Electron microscopy reveals degenerative changes within keratinocytes. Despite the well-documented association between zinc deficiency and skin pathology, it is not clear which cellular processes are most sensitive to zinc deficiency and could account for the typical pathological features. We used the cultured HaCaT keratinocyte line to obtain insight into the cellular effects of zinc deficiency, as these cells show many characteristics of normal skin keratinocytes. Zinc deficiency was induced by growing cells in the presence of the zinc chelator, TPEN, or by growth in zinc-deficient medium. Growth of cells in zinc-deficient medium resulted in a 44% reduction of intracellular zinc levels and a 75% reduction in the activity of the zinc-dependent enzyme, 5'-nucleotidase, relative to the control cells. Over a period of 7 days of exposure to zinc-deficient conditions, no changes in cell viability and growth, or in the cytoskeletal and cell adhesion systems, were found in HaCaT cells. At 7 days, however, induction of apoptosis was indicated by the presence of DNA fragmentation and expression of active caspase-3 in cells. These results demonstrate that apoptosis is the earliest detectable cellular change induced by zinc deficiency in HaCaT keratinocytes. Our observations account for many of the features of zinc deficiency, including the presence of degenerate nuclei, chromatin aggregates and abnormal organization of keratin, that may represent the later stages of apoptosis. In summary, a major causal role for apoptosis in the pathology of zinc deficiency in the skin is proposed. This role is consistent with the previously unexplained diverse range of degenerative cellular changes seen at the ultrastructural level in zinc-deficient keratinocytes.     

Caffeine promotes ultraviolet B-induced apoptosis in human keratinocytes without complete DNA repair

In response to ultraviolet B damage, keratinocytes undergo apoptosis to eliminate damaged cells, thereby preventing tumorigenic transformation. Caffeine, the most widely consumed psychoactive substance, produces complex pharmacological actions; it has been shown to be chemopreventive in non-melamona skin cancer in mice through increasing apoptosis. Here we have investigated the molecular and cellular mechanisms in the pro-apoptotic effect of caffeine on UVB-irradiated human HaCaT keratinocytes. Pretreatment with caffeine increased UVB-induced apoptosis in HaCaT cells. Caffeine blocked UVB-induced Chk1 phosphorylation. In addition, similar to the effect of the PI3K inhibitor LY294002, caffeine also inhibited phosphorylation of AKT and up-regulation of COX-2, two critical oncogenic pathways in skin tumorigenesis. However, phosphorylation of EGFR or ERK was unaffected. Inhibiting ATR pathways by siRNA targeting ATR had little effect on UVB-induced apoptosis or AKT activation, indicating that the inhibitory effect of caffeine on apoptosis and the AKT pathway does not require the ATR pathway. Inhibiting AKT by caffeine blocked UVB-induced COX-2 up-regulation. Expression of constitutively active AKT that was not inhibited by caffeine was found to protect cells from caffeine-promoted apoptosis post-UVB irradiation, indicating that AKT is an essential inhibitory target for caffeine to promote apoptosis. Caffeine specifically sensitized cells with unrepaired DNA damage to UVB-induced apoptosis. These findings indicate that in HaCaT keratinocytes, inhibiting the AKT/COX-2 pathways through an ATR-independent pathway is a critical molecular mechanism by which caffeine promotes UVB-induced apoptosis of unrepaired keratinocytes for elimination.     

IL-4 suppresses UVB-induced apoptosis in skin

In this study, cutaneous role of IL-4 in UVB-induced apoptosis was investigated using transgenic mice with skin-specific expression of IL-4 (IL-4 Tg mice). The transgenic mice did not show any gross clinical abnormalities. However, epidermis was thickened and increased MHC class II positive cells were detected as well as enhanced expression of inflammatory cytokines such as IL-1 and TNF-alpha in skin. In addition, histological analysis revealed increased infiltration of lymphocytes, acanthosis, hyperkeratosis, and parakeratosis in skin of IL-4 Tg mice. The physiological effect of IL-4 overexpression in skin against environmental stimulus such as UVB was investigated by irradiating wild-type and IL-4 Tg mice with UVB followed by evaluation of apoptosis. The result demonstrated suppressed apoptosis in epidermis of IL-4 Tg mice compared with wild-type mice. To further assess anti-apoptotic function of IL-4 in keratinocytes, stable cell clones were made where IL-4 was constitutively overexpressed and examined for UVB-induced apoptosis. The results showed that apoptosis was remarkably decreased in IL-4 over-expressing cell clones compared with that in mock transfected cells. Collectively, data presented here shows that IL-4 has an inhibitory effect against UVB-induced apoptosis in keratinocytes, suggesting that IL-4 may be an important regulator in cutaneous immunity against UVB.     

Serum-derived exosomes accelerate scald wound healing in mice by optimizing cellular functions and promoting Akt phosphorylation

Wound exudate holds great clinical and research potential in wound repair via paracrine signaling. In essence, exudate is modified serum that contains a high concentration of exosomes. The aim of this study was to investigate the role of serum-derived exosomes in scald wound healing of NIH mice skin and to explore the underlying mechanisms. Hence, we constructed a deep second-degree scald model in NIH mice, testing the benefits of exosomes in the scald wound healing. The scratch wound assay, apoptosis assay and MTT assay were conducted to assess the effects of serum-derived exosomes on migration, apoptosis and proliferation of HaCaT cells and fibroblasts. Our results showed that serum-derived exosomes injected subcutaneously entered cells and effectively accelerated wound healing processes in mice. Additionally, serum-derived exosomes optimized functions of cells related to skin injury repair by stimulating fibroblast proliferation, promoting HaCaT cell migration, and suppressing apoptosis of HaCaT cells induced by heat stress. Further study revealed that serum-derived exosomes enhanced phosphorylation of the serine-threonine kinase Akt in scalded skin tissue. These results suggest a potential clinical use of serum-derived exosomes for treating skin scald.

     

Up-regulation of transient receptor potential canonical 1 (TRPC1) following sarco(endo)plasmic reticulum Ca2+ ATPase 2 gene silencing promotes cell survival: a potential role for TRPC1 in Darier's disease

The mechanism(s) involved in regulation of store operated calcium entry in Darier's disease (DD) is not known. We investigated the distribution and function of transient receptor potential canonical (TRPC) in epidermal skin cells. DD patients demonstrated up-regulation of TRPC1, but not TRPC3, in the squamous layers. Ca2+ influx was significantly higher in keratinocytes obtained from DD patients and showed enhanced proliferation compared with normal keratinocytes. Similar up-regulation of TRPC1 was also detected in epidermal layers of SERCA2+/- mice. HaCaT cells expressed TRPC1 in the plasma membrane. Expression of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA)2 small interfering RNA (siRNA) in HaCaT cells increased TRPC1 levels and thapsigargin-stimulated Ca2+ influx, which was blocked by store-operated calcium entry inhibitors. Thapsigargin-stimulated intracellular Ca2+ release was decreased in DD cells. DD keratinocytes exhibited increased cell survival upon thapsigargin treatment. Alternatively, overexpression of TRPC1 or SERCA2-siRNA in HaCaT cells demonstrated resistance to thapsigargin-induced apoptosis. These effects were dependent on external Ca2+ and activation of nuclear factor-kappaB. Isotretinoin reduced Ca2+ entry in HaCaT cells and decreased survival of HaCaT and DD keratinocytes. These findings put forward a novel consequence of compromised SERCA2 function in DD wherein up-regulation of TRPC1 augments cell proliferation and restrict apoptosis. We suggest that the anti-apoptotic effect of TRPC1 could potentially contribute to abnormal keratosis in DD.     

Cold atmospheric plasma ameliorates imiquimod-induced psoriasiform dermatitis in mice by mediating antiproliferative effects

Psoriasis is a chronic hyperproliferative skin disease characterised by excessive growth of keratinocytes. Indeed, inducing keratinocyte apoptosis is a key mechanism responsible for psoriatic plaques clearance following some important existing therapies, which display pro-oxidant activity. Cold atmospheric plasma (CAP), acting as a tuneable source of reactive oxygen and nitrogen species (RONS), can controllably transfer RONS to the cellular environment, deliver antiproliferative RONS concentrations and exert antiproliferative and proapoptotic effects. This study was undertaken to evaluate the therapeutic potential of CAP in psoriasis. We used cell models of psoriasis-like inflammation by adding lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNF-α) to HaCaT keratinocytes. Indirect plasma, plasma-activated medium (PAM), was administered to HaCaT cells. Atmospheric pressure plasma jet (APPJ) was applied directly to imiquimod (IMQ)-induced psoriasiform dermatitis in mice. The results showed that PAM induced an increase in intracellular ROS and caused keratinocyte apoptosis. Moreover, cells under inflammation showed lesser viability and larger apoptosis rate. With repeated administration of APPJ, psoriasiform lesions showed ameliorated morphological manifestation and reduced epidermal proliferation. Overall, this study supports that CAP holds good potential in psoriasis treatment.     

A novel approach for studying angiogenesis: A human skin equivalent with a capillary-like network

Angiogenesis results from an ordered set of events that can be modulated in vivo by a variety of angiogenesis-enhancing or inhibiting agents. We review in vitro angiogenesis models and the agents that enhance or inhibit angiogenesis. We also discuss a new in vitro angiogenesis model created within a skin equivalent. Briefly, endothelial cells were combined with the cutaneous cells of a standard skin equivalent and cultured in a chitosan cross-linked collagen-glycosaminoglycan scaffold of this endothelialized skin. This model enables the formation of capillary-like structures in a coculture environment containing newly synthesized extracellular matrix by fibroblasts and keratinocytes. Several morphological characteristics associated with the microvasculature in vivo were observed in the endothelialized skin equivalent such as histotypic organization of tubular structures, basement membrane deposition, and intercellular junction formation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Feasibility of repairing skin defects by VEGF165 gene-modified iPS-HFSCs seeded on a 3D printed scaffold containing astragalus polysaccharide

The preparation of biodegradable scaffolds loaded with cells and cytokine is a feature of tissue-engineered skin. IPSCs-based tissue-engineered skin treatment for wound repair is worth exploring. Healthy human skin fibroblasts were collected and reprogrammed into iPSCs. After gene modification and induction, CK19⁺/Integrinβ1⁺/CD200⁺ VEGF₁₆₅ gene-modified iPS-HFSCs^GFP were obtained and identified by a combination of immunofluorescence and RT-qPCR. Astragalus polysaccharide-containing 3D printed degradable scaffolds were prepared and co-cultured with VEGF₁₆₅ gene-modified iPS-HFSCs^GFP, and the biocompatibility and spatial structure of the tissue-engineered skin was analysed by cell counting kit-8 (CCK8) assay and scanning electron microscopy. Finally, the tissue-engineered skin was transplanted onto the dorsal trauma of nude mice, and the effect of tissue-engineered skin on the regenerative repair of total skin defects was evaluated by a combination of histology, immunohistochemistry, immunofluorescence, RT-qPCR, and in vivo three-dimensional reconstruction under two-photon microscopy. CK19⁺/Integrinβ1⁺/CD200⁺ VEGF₁₆₅ gene-modified iPS-HFSCs^GFP, close to the morphology and phenotype of human-derived hair follicle stem cells, were obtained. The surface of the prepared 3D printed degradable scaffold containing 200 μg/mL astragalus polysaccharide was enriched with honeycomb-like meshwork, which was more conducive to the proliferation of the resulting cells. After tissue-engineered skin transplantation, combined assays showed that it promoted early vascularization, collagen and hair follicle regeneration and accelerated wound repair. VEGF₁₆₅ gene-modified iPS-HFSCs^GFP compounded with 3D printed degradable scaffolds containing 200 μg/mL astragalus polysaccharide can directly and indirectly participate in vascular, collagen, and hair follicle regeneration in the skin, achieving more complete structural and functional skin regenerative repair.     

Downregulation of CD9 in Keratinocyte Contributes to Cell Migration via Upregulation of Matrix Metalloproteinase-9

Tetraspanin CD9 has been implicated in various cellular and physiological processes, including cell migration. In our previous study, we found that wound repair is delayed in CD9-null mice, suggesting that CD9 is critical for cutaneous wound healing. However, many cell types, including immune cells, endothelial cells, keratinocytes and fibroblasts undergo marked changes in gene expression and phenotype, leading to cell proliferation, migration and differentiation during wound repair, whether CD9 regulates kerationcytes migration directly remains unclear. In this study, we showed that the expression of CD9 was downregulated in migrating keratinocytes during wound repair in vivo and in vitro. Recombinant adenovirus vector for CD9 silencing or overexpressing was constructed and used to infect HaCaT cells. Using cell scratch wound assay and cell migration assay, we have also demonstrated that downregulation of CD9 promoted keratinocyte migration in vitro, whereas CD9 overexpression inhibited cell migration. Moreover, CD9 inversely regulated the activity and expression of MMP-9 in keratinocytes, which was involved in CD9-regulated keratinocyte migration. Importantly, CD9 silencing-activated JNK signaling was accompanied by the upregulation of MMP-9 activity and expression. Coincidentally, we found that SP600125, a JNK pathway inhibitor, decreased the activity and expression of MMP-9 of CD9-silenced HaCaT cells. Thus, our results suggest that CD9 is downregulated in migrating keratinocytes in vivo and in vitro, and a low level of CD9 promotes keratinocyte migration in vitro, in which the regulation of MMP-9 through the JNK pathway plays an important role.     

Detection of poly(ADP-ribose) by immunocytochemistry: a sensitive new method for the early identification of UVB- and H2O2-induced apoptosis in keratinocytes

Apoptosis is an active form of cell death that is initiated by a variety of stimuli, including reactive oxygen species (ROS) and ultraviolet (UV) radiation. Poly (ADP-ribose) (PAR) is formed upon activation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), and therefore was suggested as a new marker of apoptosis. Since DNA of epidermal cells represents a well-known chromophore for UVB irradiation, and UVB is known to generate H2O2 in keratinocytes, we hypothesized that PAR is a very sensitive marker of UVB- and H2O2-induced apoptosis in keratinocytes. In order to test this hypothesis, human immortalized keratinocytes (HaCaT) were UVB-irradiated or treated with H2O2, and subsequently apoptosis was identified by comparing conventional parameters such as morphological analysis, DNA laddering, and TUNEL assay, with PAR formation. Both, UVB and H2O2 treatment induced PAR formation in HaCaT cells in a dose-dependent manner, and its formation was detected as early as 4 h after irradiation, and at lower UVB doses (10 mJ/cm2) than observed by DNA laddering and the TUNEL assay. In conclusion, the detection of PAR formation is a very sensitive and early method for the identification of apoptotic cells in UVB-induced apoptosis of human keratinocytes.     

microRNA-106b derived from endothelial cell–secreted extracellular vesicles prevents skin wound healing by inhibiting JMJD3 and RIPK3

Intriguingly, microRNAs (miRs) transferred as cargo in extracellular vesicles (EVs) can modulate wound healing through their regulation of fibroblast functions. In this study, we investigated the effects of miR-106b transfer via EVs derived from human umbilical vein endothelial cells (HUVECs) on skin wound healing. Dual-luciferase reporter gene assay identified that miR-106b could target and inhibit JMJD3. RT-qPCR analysis showed EVs isolated from HUVECs had enriched expression of miR-106b. LL29 fibroblast cells and HaCaT keratinocytes were co-cultured with HUVEC-derived EVs, in which miR-106b had been up-regulated or down-regulated by its mimic or inhibitor. The co-culture with HUVEC-derived EVs increased miR-106b expression, and reduced the viability and adhesion of LL29 and HaCaT cells, whereas the inhibition of miR-106b in HUVEC-derived EVs enhanced the viability and adhesion of LL29 and HaCaT cells through up-regulation of JMJD3. Next, we showed that JMJD3 overexpression enhanced LL29 and HaCaT cell viability and adhesion through elevating RIPK3, which induced the phosphorylation of AKT during the wound-healing process. We next developed a mouse skin wound model to investigate the actions of miR-106b in vivo after 14 days. The delivery of miR-106b via HUVEC-derived EVs delayed wound healing through suppression of collagen I content and angiogenesis, but had no effects on pro-inflammatory cytokines. In conclusion, miR-106b from HUVEC-derived EVs inhibits JMJD3 and RIPK3, leading to the inhibition of skin wound healing, thus constituting a new therapeutic target.     

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP‐labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full‐thickness cutaneous wound site in streptozotocin‐induced diabetic mice. Wounds treated with MSC‐ADM demonstrated an increased percentage of wound closure. The treatment of MSC‐ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col‐I) fibers synthesis via second harmonic generation imaging. The synthesis of Col‐I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP‐labeled MSCs during wound healing was simultaneously traced via two‐photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo‐multiplier tube.      

Regulation of eNOS in normal and diabetes-impaired skin repair: implications for tissue regeneration.

An important role of inducible nitric oxide (NO) synthase for epithelial action during skin repair has been well established. Although a delayed healing of skin wounds has been recently described for eNOS-deficient mice, a participation of endothelial-type NO synthase (eNOS) in skin repair largely remains unclear. In this study we determined the expression pattern of eNOS during wound healing in healthy and in diabetic mice. Remarkably, normal repair in healthy animals was characterized by a moderate induction of eNOS at the mRNA and protein level, whereas diabetes-impaired healing was associated with a clearly reduced eNOS protein expression. Immunohistochemistry revealed the endothelial lining of blood vessels within the granulation tissue, and also keratinocytes of the wound margins, the developing neo-epithelium, and the hair follicles to express eNOS protein. Keratinocyte-derived expression of eNOS could be confirmed at the mRNA level in vitro for human primary keratinocytes and the keratinocyte cell line HaCaT. Furthermore, eNOS enzymatic activity most likely contributes to epithelial regeneration, as eNOS-deficient (eNOS -/-) animals exhibited reduced wound margin epithelia associated with reduced keratinocyte proliferation.     

Docosahexaenoic acid reverts resistance to UV-induced apoptosis in human keratinocytes: involvement of COX-2 and HuR

The dramatic increase in the incidence of nonmelanoma skin cancer over the last decades has been related to the augmented exposure to ultraviolet (UV) radiation (UVR). It is known that apoptosis is induced as a protective mechanism after the acute irradiation of keratinocytes, whereas apoptotic resistance and carcinogenesis may follow the chronic exposure to UVR. We found that not all the human keratinocytes lines studied underwent apoptosis following acute exposure to UVR (10-60 mJ/cm²). Whereas UVR induced apoptosis in the HaCaT cells, NCTC 2544 and nr-HaCaT cells showed apoptosis resistance. The cytokeratin pattern of the apoptosis-resistant cells indicated that they possessed a degree of differentiation lower than that of HaCaT cells. They also showed an enhanced expression of cyclooxygenase-2 (COX-2), an early marker of carcinogenesis in various tissues, including skin. n-3 polyunsaturated fatty acids have drawn increasing interest as nutritional factors with the potential to reduce UVR carcinogenesis, and since they are apoptosis inducers and COX-2 inhibitors in cancer cells, we investigated the ability of n-3 polyunsaturated fatty acids to influence the resistance to UVR-induced apoptosis in keratinocytes. We observed that docosahexaenoic acid (DHA) reverted the resistance of nr-HaCaT cells to UVR-induced apoptosis, increasing the Bax/Bcl-2 ratio and caspase-3 activity, and reduced COX-2 levels by inhibiting the expression of the human antigen R (HuR), a known COX-2 mRNA stabilizer in keratinocytes. The transfection of nr-HaCaT cells with HuR siRNA mimicked the proapoptotic effect of DHA. Overall, our findings further support the role of DHA as a suitable anticarcinogenic factor against nonmelanoma skin cancers.     

Ultraviolet Light Induces Apoptosis via Direct Activation of CD95 (Fas/APO-1) Independently of Its Ligand CD95L

Induction of apoptosis in keratinocytes by UV light is a critical event in photocarcinogenesis. Although p53 is of importance in this process, evidence exists that other pathways play a role as well. Therefore, we studied whether the apoptosis-related surface molecule CD95 (Fas/APO-1) is involved. The human keratinocyte cell line HaCaT expresses CD95 and undergoes apoptosis after treatment with UV light or with the ligand of CD95 (CD95L). Incubation with a neutralizing CD95 antibody completely prevented CD95L-induced apoptosis but not UV-induced apoptosis, initially suggesting that the CD95 pathway may not be involved. However, the protease CPP32, a downstream molecule of the CD95 pathway, was activated in UV-exposed HaCaT cells, and UV-induced apoptosis was blocked by the ICE protease inhibitor zVAD, implying that at least similar downstream events are involved in CD95- and UV-induced apoptosis. Activation of CD95 results in recruitment of the Fas-associated protein with death domain (FADD) that activates ICE proteases. Immunoprecipitation of UV-exposed HaCaT cells revealed that UV light also induces recruitment of FADD to CD95. Since neutralizing anti-CD95 antibodies failed to prevent UV-induced apoptosis, this suggested that UV light directly activates CD95 independently of the ligand CD95L. Confocal laser scanning microscopy showed that UV light induced clustering of CD95 in the same fashion as CD95L. Prevention of UV-induced CD95 clustering by irradiating cells at 10°C was associated with a significantly reduced death rate. Together, these data indicate that UV light directly stimulates CD95 and thereby activates the CD95 pathway to induce apoptosis independently of the natural ligand CD95L. These findings further support the concept that UV light can affect targets at the plasma membrane, thereby even inducing apoptosis.     

YAC tripeptide of epidermal growth factor promotes the proliferation of HaCaT keratinocytes through activation of EGFR

Epidermal growth factor (EGF) is known to play key roles in skin regeneration and wound-healing. Here, we demonstrate that Pep2-YAC, a tripeptide covering residues 29-31 in the B loop of EGF, promotes the proliferation of HaCaT keratinocytes with activity comparable to EGF. The treatment of HaCaT cells with Pep2-YAC induced phosphorylation, internalization, and degradation of EGFR and organization of signaling complexes, which consist of Grb2, Gab1, SHP2, and PI3K. In addition, it sti mulated the phosphorylation of ERK1/2 at Thr 202/Tyr 204 and of Akt1 at Ser 473 and the nuclear translocation of EGFR, STAT3, c-Jun, and c-Fos. These results suggest that Pep2-YAC may be useful as a therapeutic agent for skin regeneration and wound-healing as an EGFR agonist. [BMB Reports 2014; 47(10): 581-586]     

Differential activity of UV-DDB in mouse keratinocytes and fibroblasts: impact on DNA repair and UV-induced skin cancer

UV-damaged DNA-binding protein (UV-DDB) is essential for global genome nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers (CPD) and accelerates repair of 6-4 photoproducts (6-4PP). The high UV-induced skin cancer susceptibility of mice compared to man has been attributed to low expression of the UV-DDB subunit DDB2 in mouse skin cells. However, DDB2 knockout mice exhibit enhanced UVB skin carcinogenesis indicating that DDB2 protects mice against UV-induced skin cancer. To resolve these apparent contradictory findings, we systematically investigated the NER capacity of mouse fibroblasts and keratinocytes. Compared to fibroblasts, keratinocytes exhibited an increased level of UV-DDB activity, contained significantly higher levels of other NER proteins (i.e. XPC and XPB) and displayed efficient repair of CPD. At low UVB dosages, the difference in skin cancer susceptibility between DDB2 KO and wild type mice was even much more pronounced than previously reported with high dose UVB exposures. Hence, our observations show that mouse keratinocytes express sufficient levels of UV-DDB for efficient repair of photolesions and efficient protection against UV-induced skin cancer at physiological relevant UV exposure.