UVB Radiation Induces Apoptosis in Keratinocytes by Activating a Pathway Linked to “BLT2-Reactive Oxygen Species”

The Repair Enzyme Peptide Methionine-S-Sulfoxide Reductase Is Expressed in Human Epidermis and Upregulated by UVA Radiation

ΔNp63α Promotes Apoptosis of Human Epidermal Keratinocytes

Role of Phagocyte Oxidase in UVA-Induced Oxidative Stress and Apoptosis in Keratinocytes

Expression and Function of Neurotrophins and Their Receptors in Cultured Human Keratinocytes

Ca(2+) is an essential factor inducing keratinocyte differentiation due to the natural Ca(2+) gradient in the skin. However, the membrane mechanisms that mediate calcium entry and trigger keratinocyte differentiation had not previously been elucidated. In this study we demonstrate that Ca(2+)-induced differentiation up-regulates both mRNA and protein expression of a transient receptor potential highly Ca(2+)-selective channel, TRPV6. The latter mediates Ca(2+) uptake and accounts for the basal [Ca(2+)](i) in human keratinocytes. Our results show that TRPV6 is a prerequisite for keratinocyte entry into differentiation, because the silencing of TRPV6 in human primary keratinocytes led to the development of impaired differentiated phenotype triggered by Ca(2+). The expression of such differentiation markers as involucrin, transglutaminase-1, and cytokeratin-10 was significantly inhibited by small interfering RNA-TRPV6 as compared with differentiated control cells. TRPV6 silencing affected cell morphology and the development of intercellular contacts, as well as the ability of cells to stratify. 1,25-Dihydroxyvitamin D3, a cofactor of differentiation, dose-dependently increased TRPV6 mRNA and protein expression in human keratinocytes. This TRPV6 up-regulation led to a significant increase in Ca(2+) uptake in both undifferentiated and differentiated keratinocytes. We conclude that TRPV6 mediates, at least in part, the pro-differentiating effects of 1,25-dihydroxyvitamin D3 by increasing Ca(2+) entry, thereby promoting differentiation. Taken together, these data suggest that the TRPV6 channel is a key element in Ca(2+)/1,25-dihydroxyvitamin D3-induced differentiation of human keratinocytes.

LincRNA-p21 is a long noncoding RNA and a transcriptional target of p53 and HIF-1α. LincRNA-p21 regulates gene expression in cis and trans, mRNA translation, protein stability, the Warburg effect, and p53-dependent apoptosis and cell cycle arrest in doxorubicin-treated mouse embryo fibroblasts. p53 plays a key role in the response of skin keratinocytes to UVB-induced DNA damage by inducing cell cycle arrest and apoptosis. In skin cancer development, UVB-induced mutation of p53 allows keratinocytes upon successive UVB exposures to evade apoptosis and cell cycle arrest. We hypothesized that lincRNA-p21 has a key functional role in UVB-induced apoptosis and/or cell cycle arrest in keratinocytes and loss of lincRNA-p21 function results in the evasion of apoptosis and/or cell cycle arrest. We observed that lincRNA-p21 transcripts are highly inducible by UVB in mouse and human keratinocytes in culture and in mouse skin in vivo. LincRNA-p21 is regulated at the transcriptional level in response to UVB, and the UVB induction of lincRNA-p21 in keratinocytes and in vivo in mouse epidermis is primarily through a p53-dependent pathway. Knockdown of lincRNA-p21 blocked UVB-induced apoptosis in mouse and human keratinocytes, and lincRNA-p21 was responsible for the majority of UVB-induced and p53-mediated apoptosis in keratinocytes. Knockdown of lincRNA-p21 had no effect on cell proliferation in untreated or UVB-treated keratinocytes. An early event in skin cancer is the mutation of a single p53 allele. We observed that a mutant p53+/R172H allele expressed in mouse epidermis (K5Cre+/tg;LSLp53+/R172H) showed a significant dominant-negative inhibitory effect on UVB-induced lincRNA-p21 transcription and apoptosis in epidermis. We conclude lincRNA-p21 is highly inducible by UVB and has a key role in triggering UVB-induced apoptotic death. We propose that the mutation of a single p53 allele provides a pro-oncogenic function early in skin cancer development through a dominant inhibitory effect on UVB-induced lincRNA-p21 expression and the subsequent evasion of UVB-induced apoptosis.

Low Concentrations of Curcumin Induce Growth Arrest and Apoptosis in Skin Keratinocytes Only in Combination with UVA or Visible Light

Increased cellular ceramide accounts in part for UVB irradiation-induced apoptosis in cultured human keratinocytes with concurrent increased glucosylceramide but not sphingomyelin generation in these cells. Given that conversion of ceramide to non-apoptotic metabolites such as sphingomyelin and glucosylceramide protects cells from ceramide-induced apoptosis, we hypothesized that failed up-regulation of sphingomyelin generation contributes to ceramide accumulation following UVB irradiation. Because both sphingomyelin synthase and glucosylceramide synthase activities were significantly decreased in UVB-irradiated keratinocytes, we investigated whether alteration(s) in the function of ceramide transport protein (or CERT) required for sphingomyelin synthesis occur(s) in UVB-irradiated cells. Fluorescently labeled N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-ceramide) relocation to the Golgi was diminished after irradiation, consistent with decreased CERT function, whereas the CERT inhibitor N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (1R,3R isomer) (HPA-12) produced an equivalent effect. UVB irradiation also induced the rapid formation of a stable CERT homotrimer complex in keratinocytes as determined by Western immunoblot and mass spectrometry analyses, a finding replicated in HeLa, HEK293T, and HaCaT cells and in murine epidermis. Ceramide binding activity was decreased in recombinant CERT proteins containing the UVB-induced homotrimer. The middle region domain of the CERT protein was required for the homotrimer formation, whereas neither the pleckstrin homology (Golgi-binding) nor the START (ceramide-binding) domains were involved. Finally like UVB-treated keratinocytes, HPA-12 blockade of CERT function increased keratinocyte apoptosis, decreased sphingomyelin synthesis, and led to accumulation of ceramide. Thus, UVB-induced CERT homotrimer formation accounts, at least in part, for apoptosis and failed up-regulation of sphingomyelin synthesis following UVB irradiation, revealing that inactive CERT can attenuate a key metabolic protective mechanism against ceramide-induced apoptosis in keratinocytes.

Forskolin Protects Keratinocytes from UVB-Induced Apoptosis and Increases DNA Repair Independent of its Effects on Melanogenesis

Hypoxia is known to stimulate reactive oxygen species (ROS) generation. Because reduced glutathione (GSH) is compartmentalized in cytosol and mitochondria, we examined the specific role of mitochondrial GSH (mGSH) in the survival of hepatocytes during hypoxia (5% O2). 5% O2 stimulated ROS in HepG2 cells and cultured rat hepatocytes. Mitochondrial complex I and II inhibitors prevented this effect, whereas inhibition of nitric oxide synthesis with Nomega-nitro-L-arginine methyl ester hydrochloride or the peroxynitrite scavenger uric acid did not. Depletion of GSH stores in both cytosol and mitochondria enhanced the susceptibility of HepG2 cells or primary rat hepatocytes to 5% O2 exposure. However, this sensitization was abrogated by preventing mitochondrial ROS generation by complex I and II inhibition. Moreover, selective mGSH depletion by (R,S)-3-hydroxy-4-pentenoate that spared cytosol GSH levels sensitized rat hepatocytes to hypoxia because of enhanced ROS generation. GSH restoration by GSH ethyl ester or by blocking mitochondrial electron flow at complex I and II rescued (R,S)-3-hydroxy-4-pentenoate-treated hepatocytes to hypoxia-induced cell death. Thus, mGSH controls the survival of hepatocytes during hypoxia through the regulation of mitochondrial generation of oxidative stress.

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

The majority of prostate cancer-related deaths are associated with advanced and metastatic malignancies. Although anoikis resistance has been recognized as one of the hallmarks of metastatic prostate malignancies, the molecular events that cause anoikis resistance are poorly understood. In this study, we found that the detachment of PC-3 prostate cancer cells caused a time-dependent increase in the expression level of the leukotriene B4 receptor-2 (BLT2) and that BLT2 played a critical role in establishing anoikis resistance in these cells. Blocking BLT2 with the pharmacological inhibitor LY255283 or with RNAi knockdown clearly abolished anoikis resistance and resulted in severe apoptotic death. Additionally, we demonstrated that the activation of NADPH oxidase (NOX) and subsequent generation of reactive oxygen species (ROS) were downstream of BLT2 signaling and led to the activation of NF-κB, thus establishing anoikis resistance during cell detachment. Furthermore, we observed that the ectopic expression of BLT2 in normal prostate PWR-1E cells rendered the cells resistant to anoikis and apparently diminished apoptotic cell death following detachment. Taken together, our results suggest that BLT2-NOX-ROS-NF-κB cascade induction during detachment confers a novel mechanism of anoikis resistance in prostate cancer cells and potentially contributes to prostate cancer progression.

Excess exposure of skin to ultraviolet B (UVB) results in the appearance of so-called sunburn cells. Although it has been demonstrated that sunburn cells represent apoptotic keratinocytes, the molecular mechanisms for UVB-induced apoptosis in keratinocytes have not been fully elucidated. The cytokine, TNF-alpha, has been shown to induce apoptosis in a variety of cell types. Since UVB induces keratinocytes to release TNF-alpha, we hypothesized that TNF-alpha is involved in UVB-induced apoptosis in keratinocytes. In order to confirm this hypothesis and to further delineate which type of TNF receptor signaling mediates the apoptosis pathway, we performed both in vivo and in vitro experiments using gene-targeted knockout mice lacking either the TNF p55 receptor or the TNF p75 receptor. In the in vivo study, wild-type and mutant mice were exposed to UVB, and apoptotic keratinocytes were detected by examining DNA fragmentation using in situ nick-end labeling. For the in vitro experiments, keratinocytes derived from the wild-type and mutant mice were irradiated with UVB, and the degree of apoptosis was determined by flow cytometry, nick-end labeling of DNA, and a DNA ladder assay. Both in vivo and in vitro studies demonstrated that the deletion of TNF receptor p55 could suppress UVB-induced apoptosis in keratinocytes. Our observations support the notion that TNF-alpha is involved in UVB-induced keratinocyte apoptosis, and demonstrate that p55 receptor signaling plays a pivotal role in this event.

The greatest environmental risk factor for developing non-melanoma skin cancer (NMSC) is ultraviolet B (UVB) radiation. Both complexes of the mammalian target of rapamycin (mTOR) signaling pathway, the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-resistant mTOR complex 2 (mTORC2), are stimulated in response to UVB. Cell survival in UVB-induced keratinocyte carcinogenesis has been shown to be mTORC2-depedent. The tumor suppressor FOXO3a, a member of the Forkhead box family of proteins, is an important transcription factor involved in regulating cell survival. FOXO3a activity is attenuated as a result of AKT activation, which is downstream of mTORC2. UVB has been shown to activate AKT through the mTORC2 pathway; however, the role of FOXO3a in UVB-induced apoptosis in keratinocytes has yet to be studied. To investigate the role of mTORC2 and FOXO3a in UVB-induced apoptosis, we utilized both human keratinocytes (HaCaT cells) as well as inducible Rictor-deficient mouse embryonic fibroblasts (iRictKO cells) following treatment with 4-hydroxy-tamoxifen (4OHT). We also employed the use of inhibitors and shRNA targeting Rictor or mSIN1. The Rictor and mSIN1 proteins are key structural components unique to mTORC2 and their removal dramatically lowers mTORC2 activity. Finally, we compared the effects of shRNA targeting FOXO3a with the expression of a constitutively active mutant FOXO3a. For all the experiments described, cells were exposed to an apoptotic dose of UVB and allowed to incubate for defined experimentally established intervals. Our results show that there is an mTORC2-dependent regulation of FOXO3a expression, localization, and activity upon UVB-irradiation. Through western analysis we demonstrate that UVB exposure promotes the phosphorylation of FOXO3a by AKT in cells with intact mTORC2 signaling, which is repressed when mTORC2 is inhibited. This phosphorylation has been shown in other systems to reduce FOXO3a activity by causing a cytoplasmic localization of the protein. In agreement with this, we observed a rapid cytoplasmic localization of FOXO3a and decreased FOXO3a promoter activation in response to UVB, which is rescued following disruption of mTORC2 signaling. Additionally, we find that FOXO3a protein expression is reduced upon UVB-irradiation when mTORC2-signaling is reduced. These data support previous findings that suggest the mTORC2-driven FOXO3a cytoplasmic sequestration protects FOXO3a from dephosphorylation and degradation. Taken together, these studies provide strong evidence that mTORC2 plays a critical role in regulating FOXO3a expression, localization and activity following UVB-irradiation. In addition, our results support the hypothesis that the mTORC2-dependent UVB-induced apoptosis is due at least in part to FOXO3a. Supported by ES19242 (LMS) Citation Format: Robert P. Feehan, Lisa M. Shantz. The role of the mTORC2-dependent regulation of FOXO3a in UVB-induced apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 27. doi:10.1158/1538-7445.AM2015-27

Ultraviolet-B-Induced Apoptosis of Keratinocytes: Evidence for Partial Involvement of Tumor Necrosis Factor-α in the Formation of Sunburn Cells