Apoptosis In Epidermal Keratinocytes Research Articles

P05-06 Protective role of peroxiredoxin V against UVB-induced apoptosis of epidermal keratinocytes

P05-06 Protective role of peroxiredoxin V against UVB-induced apoptosis of epidermal keratinocytes

The Role of Antiapoptotic Erythropoietin on Ultraviolet B-Induced Photodamaged Skin Through Inhibition of Sunburn Cells

Ultraviolet light causes photodamaged skin leading to photoaging skin. Ultraviolet-B (UV-B) causes epidermal keratinocyte apoptosis, namely sunburn cell, through apoptotic intrinsic pathway. Erythropoietin (EPO) has a role in cytoprotection in various tissues but its role to epidermal skin is not clear yet. This study was designed pretest-posttest control group design. Thirty two male mice Mus musculus, strain Balbc, were divided into control and treatment group, 16 mice each group. All mice were exposured by UV-B light 16mJ/cm2, distance 30 cm, duration 90 seconds, for 3 consecutive days. Four mice each group were randomly sacrificed as pretest data. The control mice were given aquadest subcutaneous injection 0.1 mL, and treatment mice were given EPO subcutaneous injection 0.1 mL (100 IU/kg BW). UVB were given everyday with same protocol above. Treatments were given 4 times, interval 3 days. All mice were sacrificed to examine sunburn cells. Data were performed as mean ± SD and analyzed by t-test using SPSS 17.0 with significant value p<0.05. Sunburn cells of pretest control were 25.00 ± 4.85% and treatment group were 24.83 ± 5.15%. Sunburn cells significantly decreased (p=0.002) in treatment group (31.5 ± 9.39%) than control (50.83 ± 6.70%). UV-B causes sunburn cells formation. EPO inhibits apoptosis through increasing NO production and eNOS expression, inhibiting caspase and proinflammatory cytokines, and increasing antiapoptotic protein. EPO has a role in skin apoptosis inhibition which is shown by decreased sunburn cells

Non-neuronal kappa-opioid receptor activation enhances epidermal keratinocyte proliferation, and modulates mast cell functions in human skin ex vivo.

Kappa-opioid receptor (KOR) activation reportedly elicits anti-inflammatory responses and can downregulate neuropeptide release from sensory nerve fibers. While this renders KOR agonists (KORAs) potentially interesting therapeutics in skin diseases associated with neurogenic inflammation, it remains poorly understood how KOR agonists impact on human skin and dermal mast cells (MCs) ex vivo, in the absence of functional innervation. The KORA 5a was administrated to the culture medium (200nmol/L and 1µmol/L) in human skin organ culture, thus mimicking a "systemic" mode of application. We show that KORA significantly increased epidermal thickness and upregulated the number and proliferation of epidermal keratinocytes. Unexpectedly, it also stimulated epidermal keratinocyte apoptosis in situ, compared with vehicle. Moreover, KORA significantly decreased the number of c-Kit-positive MCs, but did not significantly alter the number or degranulation of mature (tryptase- or toluidine blue-positive) MCs. These pilot observations render the tested KORA (5a) an interesting candidate for the management of inflammatory dermatoses in which MC-dependent neurogenic skin inflammation plays an important role (e.g. atopic dermatitis, psoriasis).

IFN-γ enhances cell-mediated cytotoxicity against keratinocytes via JAK2/STAT1 in lichen planus.

Lichen planus (LP) is a chronic debilitating inflammatory disease of unknown etiology affecting the skin, nails, and mucosa with no current FDA-approved treatments. It is histologically characterized by dense infiltration of T cells and epidermal keratinocyte apoptosis. Using global transcriptomic profiling of patient skin samples, we demonstrate that LP is characterized by a type II interferon (IFN) inflammatory response. The type II IFN, IFN-γ, is demonstrated to prime keratinocytes and increase their susceptibility to CD8+ T cell-mediated cytotoxic responses through MHC class I induction in a coculture model. We show that this process is dependent on Janus kinase 2 (JAK2) and signal transducer and activator of transcription 1 (STAT1), but not JAK1 or STAT2 signaling. Last, using drug prediction algorithms, we identify JAK inhibitors as promising therapeutic agents in LP and demonstrate that the JAK1/2 inhibitor baricitinib fully protects keratinocytes against cell-mediated cytotoxic responses in vitro. In summary, this work elucidates the role and mechanisms of IFN-γ in LP pathogenesis and provides evidence for the therapeutic use of JAK inhibitors to limit cell-mediated cytotoxicity in patients with LP.

Elimination of KLK5 inhibits early skin tumorigenesis by reducing epidermal proteolysis and reinforcing epidermal microstructure

Epidermal desquamation involves a finely-tuned proteolytic cascade ensuring the regulated cleavage of desmosomes that releases old stratum corneum outermost layers. Although the roles of desmosomes in normal physiology are well-established, their putative involvement in cancer remains unexplored. The KLK5 protease is thought of having fundamental roles in epidermal proteolysis and homeostasis, and its aberrant activity has been linked to skin pathologies. We found that deletion of Klk5 results in significantly higher numbers of lengthier desmosomes and enhanced skin strength. Klk5−/− mice retained normal skin barrier function and are resistant to chemically-induced skin tumorigenesis. The resistance to tumorigenesis was not due to inhibition of inflammation, and on the contrary, absence of Klk5 increased the TPA-induced inflammatory skin response. We found that increased desmosomes and reduced proteolysis prevent oncogenic signaling by capturing β-catenin into the cytoplasm and facilitate epidermal keratinocyte apoptosis, thus, inhibiting tumor initiation. We highlight that the skin ultrastructure affects early neoplastic transformation by modulating intracellular signaling and suggest that tissue reinforcement provides a novel mode of tumor suppression.

Decreased expression of suprabasin induces aberrant differentiation and apoptosis of epidermal keratinocytes: Possible role for atopic dermatitis

BackgroundSuprabasin (SBSN), a secreted protein, is expressed in various epithelial tissues. The role of SBSN in epidermal differentiation and atopic dermatitis (AD) pathology remains largely unknown. ObjectiveTo evaluate the effects of SBSN on epidermal keratinocytes and its role in AD. MethodsWe examined the SBSN expression levels in the stratum corneum and the epidermis by proteome analysis and immunohistochemistry, respectively. The serum SBSN concentration was measured by ELISA. These values were compared between AD and healthy control. Morphological changes in the epidermis were investigated in SBSN-knockdown three-dimensional human living skin equivalent (LSE) model with or without IL-4/IL-13. ResultsEpidermal SBSN expression was decreased in AD lesional skin compared to healthy skin, as assessed by the stratum corneum proteome analysis and immunohistochemistry. The SBSN serum levels were significantly lower in AD patients than in normal subjects (P<0.05). The SBSN-deficient LSE exhibited compact stratum corneum, immature stratum granulosum, and increased keratinocyte apoptosis. Th2 cytokines, IL-4 and IL-13, did not affect SBSN expression in LSE. There were no differentiation-associated makers that were affected by the SBSN knockdown. SBSN deficiency-induced apoptosis of keratinocytes was exaggerated by IL-4/IL-13, and accordingly, the addition of recombinant SBSN induced significant keratinocyte proliferation (P<0.05). ConclusionOur data demonstrated that SBSN regulates normal epidermal barrier. Th2 cytokines unaffect SBSN expression in keratinocytes, but promote SBSN deficiency-induced apoptosis. It is suggested that SBSN has an anti-apoptotic activity, and its deficiency is involved in the pathogenesis of AD.

761 The impact of 308-nm excimer laser on human keratinocytes of organ cultured human skin

The 308-nm excimer laser (EL) is a new ultraviolet B phototherapy instrument for various types of skin diseases including psoriasis, atopic dermatitis and vitiligo. Although the action of EL on these diseases is considered to be due to its anti-inflammatory properties at least by increasing the levels of regulatory T cells, the effects on keratinocytes have not been fully understood. Here, we investigated the effect of 308-nm EL on human keratinocytes by using human skin organ culture system. We irradiated human skin specimens by an EL device (Thera Beam UV308; USHIO, Tokyo, Japan) at various doses, including 300, 400 and 600 mJ/cm2 at day 0 and then organ cultured for 7 days. The effects of EL on the epidermis of organ cultured human skin were evaluated by HE histochemistry, Ki-67, TUNEL, β1 integrin, CK10, Involucrin and CK16 immunohistochemistry. As a result, organ cultured human skin irradiated at 600 mJ/cm2 at day 2 showed obvious damage of the epidermis. This was correlated to the significantly increased number of apoptotic cells detected by TUNEL immunofluorescence. However, obvious newly formed keratinized layer of epidermis was observed at 600 mJ/cm2 at day 5. Interestingly, the number of Ki-67 positive proliferating cells within the epidermis was significantly increased at 300, 400 and 600 mJ/cm2 after day 5. The expression of b1 integrin, which is one of epidermal stem cells (SC)s markers was significantly increased at 300 and 600 mJ/cm2 at day 5. In addition, the expression of differentiation-related molecules including CK10, CK16 and Involucrin was increased by EL at day 5. Here, we showed (1) EL irradiation not only induced apoptosis of epidermal keratinocytes but also stimulated their proliferation and differentiation, (2) EL also stimulated epidermal SCs. These results indicate that in addition to its well-known anti-inflammatory properties, EL may play a role as a stimulator for epidermal keratinocytes proliferation as well as SCs activity. This might be utilized for a novel treatment for wound healing.

IL-4 dysregulates microRNAs involved in inflammation, angiogenesis and apoptosis in epidermal keratinocytes.

IL-4 plays an important role in the pathogenesis of atopic dermatitis (AD) by dysregulating many key factors at the transcriptional level. In this study, a microRNA array technique and IL-4 transgenic mice were used to demonstrate that IL-4 dysregulates microRNAs involved in inflammation, angiogenesis, lymphangiogenesis and apoptosis. Of the 372 common microRNAs examined, 26 and one microRNAs were found to be up- and down-regulated, respectively. MicroRNA-101-5p, -122-5p, -142-3p, -204-5p, -335-3p, -376a-3p, -378a-5p, -639 and -9-5p are among the most significantly up-regulated microRNAs. MicroRNA-147a, the only one that was down- regulated in the present study, attenuates TLR-induced inflammatory responses. These dysregulated microRNAs may provide post-transcriptional regulation of key genes in AD.

SMAD7 in keratinocytes promotes skin carcinogenesis by activating ATM-dependent DNA repair and an EGFR-mediated cell proliferation pathway.

SMA- and MAD-related protein 7 (SMAD7) is a general inhibitor of transforming growth factor-β (TGF-β) signaling that acts through interaction and degradation of TGF-β receptors. SMAD7 has been demonstrated to be transcriptionally upregulated in chemical-induced skin tumors and TGF-β-treated normal keratinocytes. To evaluate the function of SMAD7 in skin carcinogenesis in vivo, Smad7 transgenic mice that specifically express either wild-type (WT) SMAD7 (TG-Smad7-WT) or mutant SMAD7 (TG-Smad7-MT) in keratinocytes, as well as Smad7 keratinocyte-specific knockout (Smad72f/2f-K14Cre) mice, were subjected to chemical-induced skin carcinogenesis. WT-SMAD7-expressing transgenic mice showed significantly greater papilloma formation than did non-TG control and Smad7-MT mice. The expression of WT-SMAD7 attenuated DNA damage-induced apoptosis in epidermal keratinocytes by stimulating the ATM-dependent DNA repair pathway. Nonetheless, overexpression of WT-SMAD7 caused a susceptibility to 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperproliferation through activation of epidermal growth factor (EGF) signaling. In agreement with the transgenic mouse data, keratinocyte-specific deletion of SMAD7 markedly suppressed the tumor formation by inhibiting ATM and epidermal growth factor receptor (EGFR) signaling. Moreover, specific inhibition of EGFR signaling attenuated the hyperproliferation and tumor formation in TG-Smad7-WT mice. Taken together, these data support a novel role for SMAD7 as a tumor promoter in skin carcinogenesis where SMAD7 stimulates the DNA repair pathway and EGFR signaling activation.

The role of Sprouty1 in the proliferation, differentiation and apoptosis of epidermal keratinocytes.

Sprouty (SPRY) 1 is one of the SPRY proteins that inhibits signalling from various growth factors pathways and has also been known as a tumour suppressor in various malignancies. However, no study elucidates the role of SPRY1 in the skin. Our study was conducted to determine the function of SPRY1 in human keratinocytes and the epidermis. In vitro primary cultured epidermal keratinocytes were used to investigate the proliferation, differentiation and apoptosis of these cells. We also established overexpression of SPRY1 in vitro and K14-SPRY1 transgenic mice. SPRY1 was mainly located in the cytoplasm of the epidermal keratinocytes from the granular epidermal layer of the skin and cultured cells. Overexpressed SPRY1 in keratinocytes resulted in up-regulation of P21, P27 and down-regulation of cyclin B1; decrease in MMP3 and integrin α6. SPRY1-overexpressed primary keratinocytes exhibited a lower proliferation and migration capability and higher rates of apoptosis. Epidermis of SPRY1-TG mice represented delayed wound healing. Proteomics analysis and GO enrichment showed DEPs of SPRY1 TG mice epidermis is significantly enriched in immune- and inflammatory-associated biological process. In summary, SPRY1 expression was inversely correlated with cell proliferation, migration and promote cell apoptosis of keratinocytes. SPRY1 maybe a negative feedback regulator in normal human epidermal keratinocytes and cutaneous inflammatory responses. Our study raised the possibility that enhancing expression of SPRY1 may have the potential to promote anti-inflammatory effects.

Protective Role of Mitochondrial Peroxiredoxin III against UVB-Induced Apoptosis of Epidermal Keratinocytes

UVB light induces generation of reactive oxygen species, ultimately leading to skin cell damage. Mitochondria are a major source of reactive oxygen species in UVB-irradiated skin cells, with increased levels of mitochondrial reactive oxygen species having been implicated in keratinocyte apoptosis. Peroxiredoxin III (PrxIII) is the most abundant and potent H2O2-removing enzyme in the mitochondria of most cell types. Here, the protective role of PrxIII against UVB-induced apoptosis of epidermal keratinocytes was investigated. Mitochondrial H2O2 levels were differentiated from other types of ROS using mitochondria-specific fluorescent H2O2 indicators. Upon UVB irradiation, PrxIII-knockdown HaCaT human keratinocytes and PrxIII-deficient (PrxIII-/-) mouse primary keratinocytes exhibited enhanced accumulation of mitochondrial H2O2 compared with PrxIII-expressing controls. Keratinocytes lacking PrxIII were subsequently sensitized to apoptosis through mitochondrial membrane potential loss, cardiolipin oxidation, cytochrome c release, and caspase activation. Increased UVB-induced epidermal tissue damage in PrxIII-/- mice was attributable to increased caspase-dependent keratinocyte apoptosis. Our findings show that mitochondrial H2O2 is a key mediator in UVB-induced apoptosis of keratinocytes and that PrxIII plays a critical role in protecting epidermal keratinocytes against UVB-induced apoptosis through eliminating mitochondrial H2O2. These findings support the concept that reinforcing mitochondrial PrxIII defenses may help prevent UVB-induced skin damage such as inflammation, sunburn, and photoaging.

Apoptosis as a Mechanism for Keratinocyte Death in Canine Toxic Epidermal Necrolysis.

In humans and dogs, toxic epidermal necrolysis (TEN) is a life-threatening dermatosis characterized by sudden epidermal death resulting in extensive skin detachment. There is little information on the pathogenesis of keratinocyte cell death in canine TEN. We studied the occurrence of apoptosis in skin lesions of dogs with TEN to determine if apoptosis contributes to the pathogenesis of this disease. Immunostaining with antibodies to activated caspase-3 and the terminal deoxynucleotidyl-transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling technique revealed positive apoptotic keratinocytes in basal and suprabasal epidermal compartments in 17 biopsy specimens collected from 3 dogs with TEN and 16 from 3 dogs with erythema multiforme (EM). There was no significant difference in the number of positively stained epidermal cells between TEN and EM. These results suggest that apoptosis of epidermal keratinocytes and lymphocytic satellitosis represent one of the early steps in the pathogenesis of canine TEN, as in the human disease counterpart.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN): could retinoids play a causative role?

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are overlapping manifestations on a spectrum of acute drug-induced conditions associated with severe blistering, skin peeling, and multi-organ damage. TEN is an eruption resembling severe scalding, with ≥30% skin detachment. SJS is a mild form of TEN, characterized histologically by epidermal keratinocyte apoptosis with dermo-epidermal separation and extensive small blisters with <10% body surface skin detachment. The syndrome can be induced by numerous medications and typically occurs 1–4 weeks after the initiation of therapy. Granulysin is found in the lesions of patients with SJS/TEN and plays a significant pathogenic role in the condition, but the overall mechanisms linking medications, granulysin, and disease manifestations remain obscure. This paper reviews evidence suggesting that the different medications implicated in SJS/TEN have the common property of interacting and synergizing with endogenous retinoids (vitamin A and its congeners), in many instances causing the latter to accumulate in and damage the liver, the main storage organ for vitamin A. It is hypothesized that liver damage leads to the spillage of toxic retinoid compounds into the circulation, resulting in an endogenous form of hypervitaminosis A and cytotoxicity with widespread apoptosis, mediated by granulysin and recognized as SJS/TEN. Subject to testing, the model suggests that symptom worsening could be arrested at onset by lowering the concentration of circulating retinoids and/or granulysin via phlebotomy or plasmapheresis or by pharmacological measures to limit their expression.

Effluent syntaxin3 from dying cells affords protection against apoptosis in epidermal keratinocytes

Ultra-violet B (UVB)-induced oxidative stress crucially perturbs the epidermal homeostasis, and the skin is endowed with protective mechanisms to take action against such damage. Here, we show the possible involvement of t-SNARE protein syntaxin3, a membrane fusion mediator of cytoplasmic vesicles, and which is released from dying keratinocytes, to play a role in this response. UVB irradiation, which generates reactive oxidative stress in cells, was shown to lead to the keratinocyte cell death accompanied by a release of cytoplasmic syntaxin3. We found that such extracellularly sourced syntaxin3 completely blocked the processing of a crucial effector for apoptotic cell death, caspase-3, and thus facilitated the survival of keratinocytes damaged by oxidative stress. These results demonstrate the latent prosurvival function of syntaxin3 and underline the importance of intracellular molecular elements for the maintenance of homeostasis in epidermal keratinocytes.

Identification of an Interstitial 18p11.32-p11.31 Duplication Including the EMILIN2 Gene in a Family with Porokeratosis of Mibelli

Porokeratosis is a rare disease of epidermal keratinization characterized by the histopathological feature of the cornoid lamella, a column of tightly fitted parakeratocytic cells, whose etiology is still unclear. Porokeratosis of Mibelli is a subtype of porokeratosis presenting a single plaque or a small number of plaques of variable size located unilaterally on limbs. It frequently appears in childhood and occurs with a higher incidence in males. Cytogenetic analyses were performed in all members of the family on lesioned and uninvolved skin. An array-CGH analysis was also performed utilizing the Human Genome CGH Microarray Kit G3 400 with 5.3 KB overall median probe spacing. Gene expression was performed on skin fibroblasts. In this study, we describe a Caucasian healthy 4-year-old child and his father showing features of porokeratosis of Mibelli. Array-CGH analysis revealed an interstitial 429.5 Kb duplication of chromosome 18p11.32-p11.3 containing four genes, namely: SMCHD1, EMILIN2, LPIN2, and MYOM1 both in patient and his father. EMILIN2 resulted overexpressed on skin fibroblasts. Also other members of this family, without evident signs of porokeratosis, carried the same duplication. Among these genes, we focused our attention on elastin microfibril interfacer 2 (EMILIN2) gene. Apoptosis plays a fundamental role in maintaining epidermal homeostasis, balancing keratinocytes proliferation, and forming the stratum corneum. EMILIN2 is known to trigger the apoptosis of different cell lines negatively affecting cell survival. It is expressed in the skin. We could speculate that the duplication and overexpression of EMILIN2 cause an abnormal apoptosis of epidermal keratinocytes and alter the process of keratinization, even if other epigenetic and genetic factors could also be involved. Our results could contribute to a better understanding of the pathogenesis of porokeratosis of Mibelli.

Influence of an anti-photosensitivity mixture on acute photodamage-induced cell apoptosis in BALB/c

Objective To investigate the action mechanism of an anti-photosensitivity mixture on skin photodamage. Methods Twenty-eight BLAB/c mice were divided into 4 groups, i.e., normal control group,treatment group, negative and positive control groups; the last three groups were irradiated with a single dose of UVB at 300 mJ/cm2 after 7-day pretreatment with sodium chloride physiological solution, anti-photosensitivity mixture, and hydroxychloroquine, respectively. Twenty-four hours after the irradiation, mice were killed and skin tissue samples were obtained at the irradiated sites. Terminal deoxynucleotidyl transferase （TdT）-mediated dUTP nick-end labeling （TUNEL） and immunohistochemical staining were carried out to detect cell apoptosis,Fas and Caspase-3 protein expressions respectively. Results An increase was observed in the expression level of Fas and Caspase-3 and in the apoptotic index in keratinocytes from UV-irradiated mice compared with unirradiated control mice （all P ＜ 0.01 ）. In comparison with sodium chloride physiological solution, the antiphotosensitivity mixture suppressed the UV irradiation-induced increase in the expression intensity of Fas and Caspase-3 and apoptotic index in keratinocytes （P ＜ 0.05 or 0.01 ）. Conclusions The anti-photosensitivity mixture could alleviate UV-induced inflammatory damage to and apoptosis in epidermal keratinocytes, likely by regulating cell apoptosis and Caspase-3 pathway.

Relationship of apoptosis in epidermal keratinocytes with efficacy of epidermal grafting in patients with vitiligo.

Objective To investigate the relationship between apoptosis in epidermal keratinocytes and efficacy of epidermal grafting. Methods Epidermal specimens were obtained from donor sites and depigmented area of 44 patients with vitiligo receiving epidermal grafting. The apoptosis in keratinocytes was determined by terminal deoxynucleotidyl transferase end-labeling (TUNEL) assay, and the expressions of caspase 3, 8, 9 as well as bcl-2 and P53 were evaluated by immunohistochemistry. Results As TUNEL assay showed, the number of apoptotic keratinocytes in epidermis from depigmented area differed significantly from that from donor sites.The expressions of caspase 3, 8 and 9 were mainly located in the membrane and cytoplasm of keratinocytes,and positive keratinocytes were predominately distributed in the middle and lower layer of the epidermis. Of the 44 patients, 19, 15 and 16 were positive for the expressions of caspase 3, 8 and 9 in the depigmented epidermis,respectively, and 9, 5 and 4 for those in the donor epidermis, respectively. P53 was expressed in neither donor epidermis nor depigmented epidermis, while Bcl-2 was weakly positive in donor epidermis and negative in depigmented epidermis. The number of apoptotic keratinocytes was higher in donor epidermis from patients failing to respond to the transplantation than in that from patients successfully treated by transplantation (15.83 ± 2.69 vs.9.24 ± 1.80, t = 10.96, P < 0.01 ). Conclusions There is an obvious apoptosis in keratinocytes from depigmented epidermis of patients with vitiligo, together with an increase in the expression of caspase 3, 8 and 9. The apoptosis in keratinocytes may be related to the efficacy of epidermal transplantation. Key words: Vitiligo; Keratinocytes; Apoptosis; Transplantation

Hydrolytic Pathway Protects against Ceramide-Induced Apoptosis in Keratinocytes Exposed to UVB

Although ceramides (Cers) are key constituents of the epidermal permeability barrier, they also function as apoptogenic signals for UVB irradiation-induced apoptosis in epidermal keratinocytes. As epidermis is continuously exposed to UV irradiation, we hypothesized that Cer hydrolysis protects keratinocytes from UVB-induced apoptosis by attenuating Cer levels. Both low-dose UVB (L-UVB) (< 35 mJ cm(-2)) and high-dose UVB (H-UVB) (> or = 45 mJ cm(-2)) irradiation inhibited DNA synthesis in cultured human keratinocytes, but apoptosis occurred only after H-UVB. Whereas Cer production increased after both L- and H-UVB, it normalized only in L-UVB-exposed keratinocytes, but remained elevated after H-UVB. Both acidic ceramidase (aCDase) and neutral ceramidase (nCDase) activities declined after L- and H-UVB, but returned to normal only in L-UVB cells, with decreased CDase activities or mRNA or protein levels being sustained in H-UVB cells. Inhibition of CDase using either a CDase inhibitor, N-oleoylethanolamine, or small interfering RNA (siRNA) (either to a- and/or n-CDase(s)) sensitized keratinocytes to L-UVB-induced apoptosis in parallel with further Cer accumulation. Blockade of sphingosine kinase 1 (SPHK1) (but not SPHK2) by siRNA also increased apoptosis in L-UVB keratinocytes, revealing that conversion of sphingosine to sphingosine-1-phosphate (S1P) further protects keratinocytes from UVB-induced cell death. Thus, Cer → sphingosine → S1Pmetabolic conversion protects against UVB-induced, Cer-mediated apoptosis in keratinocytes, but excessive UVB overwhelms this mechanism, thereby leading to keratinocyte apoptosis.

T-lymphocyte-induced, fas-mediated apoptosis is associated with early keratinocyte differentiation

The development of eczematous lesions is thought to be due in part to a breakdown in skin barrier function as a result of T lymphocytes (T cells) invading the skin causing epidermal keratinocyte apoptosis. In this study, we investigated the interaction of T cells and keratinocytes on apoptosis and terminal differentiation using an in vitro co-culture system. Experiments were performed using the HaCaT keratinocyte cell line or normal human epidermal keratinocytes. Activated human peripheral blood-derived T cells were found to induce Fas-dependent keratinocyte apoptosis by up to sixfold. Increased Fas was associated with increased IFN-gamma. The T-cell apoptotic signal was found to target preferentially keratinocytes in the very early stages of terminal differentiation, such as those with low levels of alpha 6-integrin expression, and result in subsequent increased caspase 3 activity. This observation was accompanied by a marked increase in keratinocyte ICAM-1 expression and its ligand LFA-1 on T cells. Our data suggest that T cells may initiate the onset of keratinocyte terminal differentiation making them more susceptible to Fas-dependent cell death signals delivered by the T cells.

AP-2αInhibits c-MYC Induced Oxidative Stress and Apoptosis in HaCaT Human Keratinocytes

AP-2α and c-MYC are important transcription factors involved in multiple cellular processes. They each display the paradoxical capacities to stimulate both cell proliferation and apoptosis under different conditions. In the present study we found that over expression of c-MYC was associated with accumulation of reactive oxygen species (ROS) and apoptosis in human keratinocytes, both of which were significantly inhibited by co-expression of AP-2. The effects of AP-2 on c-MYC were active at several levels. First, AP-2 and c-MYC were confirmed to interact at the protein level as previously described. In addition, forced expression of AP-2 significantly decreased steady state levels of c-MYC mRNA and protein. These findings suggested that AP-2 may have a direct effect on the c-myc gene. Chromatin immunoprecipitation assays demonstrated that AP-2 proteins bound to a cluster of AP-2 binding sites located within a 2 kb upstream regulatory region of c-myc These results suggest that the negative regulation of AP-2 on c-MYC activity was achieved through binding of AP-2 protein to the c-myc gene. The effects of AP-2 on c-MYC induced ROS accumulation and apoptosis in epidermal keratinocytes are likely to play an important role in cell growth, differentiation and carcinogenesis of the skin.