Following the publication of the paper “All-trans retinoic acid attenuates ultraviolet radiation-induced down-regulation of aquaporin-3 and water permeability in human keratinocytes”, J. Cell. Physiol., 215: 506–516. doi: 10.1002/jcp.21336, it has come to our attention that there were minor errors in the preparation of Figures 8B and Figure 9A. The corrected panels are now presented. The errors did not change the overall interpretation of the figures or the conclusion of the paper.

The Li‐Cor Odyssey Infrared Imaging System is pioneering the way for standard Western blot analysis by using direct infrared fluorescence detection. An advantage of the Li‐COR Odyssey Infrared Imaging System is that it provides a safer and cleaner method to detect proteins compared to traditional darkroom scans because each membrane does not have to be soaked in toxic detection reagents before the scan is initiated. However, one key problem with the Odyssey imaging system is that its standard stripping protocol fails to remove all of the antibodies from each membrane. In order to completely strip each membrane, some elements of the standard protocol had to be altered. We chose to manipulate the amount of time the membranes were exposed to the Newblot Stripping Buffer, the temperature at which the membranes were soaked in the buffer solution, and the concentration of the stripping buffer. We concluded that the optimal stripping procedure involved soaking the membranes in a 1:5 diluted buffer solution at 50°C for 30 minutes. Removing antibodies from a membrane is critical and our data suggests that these modifications to the original protocol offer a more effective stripping procedure.

While it is known that a cell in a hypotonic environment swells from an influx of water due to an osmotic imbalance, the pathways activated inside the cell are not as clearly observable or thoroughly investigated. To determine whether apoptotic and cell survival signaling pathways are activated, cultured human skin keratinocytes (HaCaT cell line) were exposed to increasing levels of hypotonicity from 0% dilution up to 50%. Western blotting analysis was used after 30 minutes of incubation. The results show that AMPK is activated when the dilution is increased, and the activity is peaked at 40% dilution and then dropped slightly at 50% dilution. Similarly, AKT is activated and the activity is peaked at 40% and 50% dilutions, suggesting that at this level of hypotonicity these pathways are activated for cell survival. The results also demonstrate that p38 and JNK are activated as the dilution is increased, and the activity is peaked at 40% dilution. Interestingly, the data from cell migration assay show that cell migration is increased gradually as dilution is increased, but dropped significantly at 50% dilution. Collectively, our data suggest that hypotonic stress affect both apoptotic and survival pathways and to certain degree enhance cell migration.

CpG‐DNA and its related synthetic CpG oligodeoxynucleotides (CpG‐ODNs) play an important role in immune cell survival and activation. Existing data have shown that AKT activation is involved in this process. However, the potential role of CpG‐ODN in protection against UV‐induced skin damage has thus far not been tested. In this study, we found that CpG‐ODN protects against UV‐induced cell death and apoptosis in both skin keratinocytes (HaCaT cells) and cultured dendritic cells (XS106, or DCs). In mechanism research, we found that CpG‐ODN pre‐treatment enhances UV induced activation of AKT (ser473, thr308 phosphorylation) as well as downstream signal TORC1 (S6K and 4E‐BP1 phosphorylation) in both HaCaT cells and DCs. AKT deficiency (by using AKT1/2 double knockout Mouse Embryonic Fibroblasts as well as LY 294002, a pharmacological inhibitors of AKT) or mTORC1 inhibition (by using rapamycin, a pharmacological inhibitor of mTORC1) largely neutralize the protective effects of CpG‐ODN, indicating that AKT and downstream signaling mTORC1 activation are necessary for CpG‐ODN induced protective effects against UV induced cell death. Our findings suggest that CpG‐ODN may be utilized to prevent from UV‐induced skin aging and provide a novel mechanism of protective effects against UV radiation.

Calcium hypochlorite compositions are primarily used for disinfectant and general sanitation purposes in swimming pools. It has not been shown that calcium hypochlorite induces apoptosis in human skin cells. Therefore, we undertook this study to investigate whether and how calcium hypochlorite induces skin cell apoptosis using human skin keratinocytes cell line HaCaT cells. After treatment of HaCaT cells with different doses of calcium hypochlorite (Pulsar® Plus Briquettes), cell proliferation was measured using an MTT Dye assay. Detection of specific proteins from apoptosis pathways were analyzed using Western blot at different time intervals. Increased calcium hypochlorite concentrations lead to increased apoptosis in cultured HaCaT cells. It is also shown that prolonged exposure to calcium hypochlorite result in an increased rate of apoptosis. Although it is known that calcium hypochlorite is an effective disinfectant in swimming pools, little is known about its effects on human skin cells. Our results thus suggest that prolonged exposure at specific concentrations of calcium hypochlorite lead to varying rates of apoptosis in human keratinocytes.

AMP-activated protein kinase or AMPK is an evolutionarily conserved sensor of cellular energy status, activated by a variety of cellular stresses that deplete ATP. However, the possible involvement of AMPK in UV- and H2O2-induced oxidative stresses that lead to skin aging or skin cancer has not been fully studied. We demonstrated for the first time that UV and H2O2 induce AMPK activation (Thr172 phosphorylation) in cultured human skin keratinocytes. UV and H2O2 also phosphorylate LKB1, an upstream signal of AMPK, in an epidermal growth factor receptor-dependent manner. Using compound C, a specific inhibitor of AMPK and AMPK-specific small interfering RNA knockdown as well as AMPK activator, we found that AMPK serves as a positive regulator for p38 and p53 (Ser15) phosphorylation induced by UV radiation and H2O2 treatment. We also observed that AMPK serves as a negative feedback signal against UV-induced mTOR (mammalian target of rapamycin) activation in a TSC2-dependent manner. Inhibiting mTOR and positively regulating p53 and p38 might contribute to the pro-apoptotic effect of AMPK on UV- or H2O2-treated cells. Furthermore, activation of AMPK also phosphorylates acetyl-CoA carboxylase or ACC, the pivotal enzyme of fatty acid synthesis, and PFK2, the key protein of glycolysis in UV-radiated cells. Collectively, we conclude that AMPK contributes to UV- and H2O2-induced apoptosis via multiple mechanisms in human skin keratinocytes and AMPK plays important roles in UV-induced signal transduction ultimately leading to skin photoaging and even skin cancer.

Aquaporins (AQPs) are a family of 13 small ( approximately 30 kDa/monomer), hydrophobic, integral membrane proteins. AQPs are expressed in various epithelial and endothelial cells involved in fluid transport. Here, we demonstrated for the first time that AQP1 is expressed in cultured human retinal pigment epithelial (RPE) cells (ARPE-19 cell line). Ultraviolet radiation (UVB) and H2O2, two major factors causing RPE cell damage, induced AQP1 downregulation which was mediated by MEK/ERK activation. UV and H2O2 as well as AQP1-specific siRNA knockdown impaired water permeability of ARPE-19 cells. Notably, pretreatment with all-trans retinoic acid attenuated UV- and H2O2-induced AQP1 downregulation and water permeability impairment. Considering that water permeability is involved in multiple functions of RPE cells such as cellular junction formation, fluid or protein exchange and barrier formation, our data elucidated a novel mechanism through which UV radiation and oxidative stress induce eye cell damage. Our results further support the notion that all-trans retinoic acid might be useful for protection against UV or oxidative stress-induced eye cell damage.

mTOR acts as an important integrator of several upstream signals, including growth factors, nutrients, energy levels, and stresses. We found in this study that UVB radiation induces mTOR activation in human skin keratinocytes and both cultured mouse dendritic cells and mouse monocyte derived dendritic cells. Using various specific inhibitors and gene manipulation methods, we observed that UVB‐induced mTOR activation is dependent on EGFR‐mediated AKT activation. We also found that TSC2/mTOR, downstream of AKT, is involved in UVB‐induced S6K activation. Inhibition of EGFR, AKT and mTOR enhances UVB‐induced apoptosis. Collectively, our data suggest that EGFR mediated mTOR activation, together with its upstream signals, serve as a novel survival signal against UVB‐induced cell apoptosis.

AMP‐activated protein kinase or AMPK is an evolutionarily conserved sensor of cellular energy status, activated by a variety of cellular stresses that deplete ATP as well as other stresses. However, the possible involvement of AMPK in UV‐ and H2O2 ‐induced skin aging is not fully studied. We show for the first time that UV and H2O2 induce AMPK activation in cultured human skin keratinocytes (HaCaT cells). UV and H2O2 also induce LKB1, the possible upstream signal of AMPK, in an EGFR dependent manner. Using Compound C, a specific inhibitor of AMPK and AMPK specific siRNA knockdown as well as AMPK activator AICAR, we found that AMPK serves as a positive regulator for p38 and p53 (ser 15) activation induced by UV radiation or H2O2 treatment. We also observed that AMPK activation pathway serves a negative feedback signal pathway against UV‐induced mTOR activation in a TSC2 dependent manner. Inhibiting mTOR and activating p53 and p38 may mediate AMPK's pro‐apoptotic effect in UV or H2O2 treated cells. Furthermore, activation AMPK also phosphorylates acetyl‐CoA carboxylase or ACC, the pivotal enzyme of fatty acid synthesis, and PFK2, the key protein of glycolysis in UV‐radiated cells which may explain reduced adipogenesis in UV or H2O2 treated 3T3‐L1 adipocyte and skin keratinocytes. Collectively, our results demonstrate that UV and H2O2 induce EGFR/LKB1/AMPK signal pathway activation in cultured skin keratinocytes, which plays important role on UV or H2O2‐induced signal transduction and skin aging.

Over-expression of EGFR, as in most cases of ovarian cancer, is associated with advanced-stage disease and poor prognosis. Activation of EGFR signaling pathway is involved in increased cell proliferation, angiogenesis, metastasis and decreased apoptosis. Tyrosine kinase activity is essential for signal transduction and receptor down-regulation. However, we found in this study that tyrosine kinase activity is not necessary in ligand-induced EGFR down-regulation in ovarian cancer cell line CaOV3 cells. EGFR tyrosine kinase inhibitors, such as PD153035, AG1478, as well as non-specific tyrosine kinase inhibitor PP2 cannot reverse EGF-induced down-regulation of EGFR. These findings thus permit us to develop the following exciting but unconventional strategy to sensitize cancer cells, namely, by priming ovarian cancer cells with EGF and EGFR inhibitor PD153035, before chemotherapy. This priming procedure down-regulates EGFR without induction of mitogenic signals such as ERK and PI3K/AKT. EGF plus EGFR inhibitor-primed ovarian cancer cells display increased sensitivity to taxol-induced cell death, resistant to EGF-induced cell migration and cell proliferation as well as ERK and PI3K/AKT activation. Further studies showed that PD153035, which does not reverse ligand-induced EGFR down-regulation, blocks EGF-induced EGFR activation as well as EGFR’s binding to c-cbl and Grb2. Taken together, we contend that priming with EGFR inhibitors plus EGF inhibits cell signaling pathways leading to cell proliferation and survival, while down-regulating EGFR. This priming approach sensitizes ovarian cancer cells and would ultimately result in better chemotherapeutical outcome.

Vitiligo is an acquired and progressive disorder manifested by the selective destruction of melanocytes in the skin. An extremely high level of hydrogen peroxide (H2O2) in plasma as well as in lesional skin has been reported in vitiligo patients. High H2O2 level has been suggested to be responsible for the disappearance of melanocytes in vitiligo. JNK and p38 MAPK are strongly induced by oxidative stress and related to neuron loss in neurodegenerative disorders. Minocycline, an antibiotic possessing antioxidant activity, is capable of attenuating oxidative stress-induced neurotoxicity. To investigate whether minocycline rescues melanocytes from H2O2-induced apoptosis, cultured mouse melanocytes (B10BR) were treated with H2O2 in the presence or absence of minocycline. Our data showed that H2O2 decreases cell viability in a concentration-dependent manner which is attenuated by minocycline. Also, H2O2 treatment activates JNK and p38 MAPK, and executive caspase 3 in B10BR cells. Minocycline significantly inhibits H2O2-induced activation of JNK, p38 MAPK and caspase 3. Collectively, we concluded that minocycline protects melanocytes against H2O2-induced apoptosis in vitro. Its protective effect is associated with the inhibition of JNK and p38 MAPK. Our findings suggest that minocycline, a clinically well-tolerated, safe antibiotic, may be used to prevent melanocyte loss in the early stage of vitiligo.

OBJECTIVE:Aquaporin (AQP) water channels are expressed in high-grade tumor cells of different tissue origins. Based on the involvement of AQPs in angiogenesis and cell migration as well as our previous studies which show that AQP3 is involved in human skin fibroblasts cell migration, in this study, we investigated whether AQP3 is expressed in cultured human ovarian cancer cell line CaOV3 cells, and whether AQP3 expression in these cells enhances cell migration and metastatic potential.METHODS:Cultured CaOV3 cells were treated with EGF and/or various reagents and subjected to cell migration assay by phagokinetic track mobility assay or biochemical analysis for expression or activation of proteins by SDS-PAGE/Western blot analysis.RESULTS:In this study, we demonstrate that AQP3 is expressed in CaOV3 cells. EGF induces CaOV3 migration and up-regulates AQP3 expression. EGF-induced cell migration is inhibited by specific AQP3 siRNA knockdown or AQP3 water transport inhibitor CuSO4 and NiCl2. We also find that curcumin, a well known anti-ovarian cancer drug, down-regulates AQP3 expression and reduces cell migration in CaOV3, and the effects of curcumin are mediated, at least in part, by its inhibitory effects on EGFR and downstream AKT/ERK activation.CONCLUSIONS:Collectively, our results provide evidence for AQP3-facilitated ovarian cancer cell migration, suggesting a novel function for AQP3 expression in high-grade tumors. The results that curcumin inhibits EGF-induced up-regulation of AQP3 and cell migration, provide a new explanation for the anticancer potential of curcumin.

Ultraviolet radiation (UV) induces apoptosis and functional maturation in skin dendritic cells (DCs). However, the molecular mechanisms through which UV activates DCs have not been thoroughly investigated. In this study, we examined the mechanisms of activation and apoptosis of DCs after UV irradiation by focusing on epidermal growth factor receptor (EGFR). Our previous studies have demonstrated that in addition to cognate ligands, EGFR is also activated by UVB irradiation in cultured human skin keratinocytes in vitro and in human skin in vivo. We found for the first time in this study that UV also induces EGFR activation in cultured mouse skin DCs (XS 106 cell line) as well as mouse monocyte-derived dendritic cells (MoDCs). Pharmacological inhibition of EGFR tyrosine kinase significantly inhibits UV-induced ERK, p38, and JNK MAP kinases, and their effectors, transcription factors c-Fos and c-Jun. Inhibition of EGFR also suppresses UV-induced activation of PI3K/AKT/mTOR/S6K and NF-kappaB signal transduction pathways. Our data demonstrated that UV induces LKB1/AMPK pathway, also dependent on EGFR trans-activation. We further observed that MAPK, LKB1/AMPK, PI3K/AKT/mTOR/S6K as well as NF-kappaB activation are impaired in EGFR-/- cells compared to wide type MEF cells after UV radiation. Taken together, we conclude that UV induces multiple signaling pathways mediated by EGFR trans-activation leading to possible maturation, apoptosis and survival, and EGFR activation protects against UV-induced apoptosis in cultured mouse dendritic cells.

AMP-activated protein kinase (AMPK), an evolutionarily conserved serine/threonine protein kinase, serves as an energy sensor in all eukaryotic cells. Recent findings suggest that AMPK activation strongly suppresses cell proliferation and induces cell apoptosis in a variety of cancer cells. Our study demonstrated that chemopreventive agent curcumin strongly activates AMPK in a p38-dependent manner in CaOV3 ovarian cancer cells. Pretreatment of cells with compound C (AMPK inhibitor) and SB203580 (p38 inhibitor) attenuates curcumin-induced cell death. We also observed that curcumin induces p53 phosphorylation (Ser 15) and both compound C and SB203580 pretreatment inhibit p53 phosphorylation. Collectively, our data suggest that AMPK is a new molecular target of curcumin and AMPK activation partially contributes to the cytotoxic effect of curcumin in ovarian cancer cells.

mTOR (mammalian target of rapamycin) serves as a signal integrator of several upstream signals, including growth factors, nutrients, energy levels, and stresses. We demonstrate in this study that UVB radiation induces mTOR activation in human skin keratinocytes (HaCaT cells) and both cultured mouse dendritic cells (XS 106 cell line) and mouse monocyte derived dendritic cells (MoDC). Using specific inhibitors and gene manipulation methods, we observed that UVB induced mTOR activation is dependent on EGFR‐mediated AKT activation. We also found that TSC2/mTOR, downstream of AKT, is involved in UVB‐induced S6K activation. UVB radiation also induces activation of AMPK (AMP‐activated protein kinase) and p38 as well as its upstream signal LKB1. Our data suggests that LKB1/AMPK/p38 serve as a negative feedback pathway against UVB‐induced mTOR activation. We conclude that mTOR activation, together with its upstream signals, serves as a novel survival signal against UVB‐induced cell death.

SIRT1 is a member of a highly conserved gene family (sirtuins) encoding nicotinamide adenine dinucleotide (NAD)+‐dependent deacetylases, originally found to deacetylate histones leading to increased DNA stability and prolonged survival in yeast and higher organisms, including mammals. SIRT1 has been found to function as a deacetylase for numerous protein targets involved in various cellular pathways, including stress responses, apoptosis and axonal degeneration. However, the role of SIRT1 in ultraviolet (UV) signalling pathways remains unknown. Using cell culture and Western blot analysis in this study we found that SIRT1 is expressed in cultured human skin keratinocytes. Both UV radiation and H2O2, two major inducers of skin cell damage, down‐regulate SIRT1 in a time‐ and dose‐dependent manner. We observed that reactive oxygen species‐mediated JNK activation is involved in this SIRT1 down‐regulation. SIRT1 activator, resveratrol, which has been considered as an important antioxidant, protects against UV‐ and H2O2‐induced cell death, whereas SIRT inhibitors such as sirtinol and nicotinamide enhance cell death. Activation of SIRT1 negatively regulates UV‐ and H2O2‐induced p53 acetylation, because nicotinamide and sirtinol as well as SIRT1 siRNA enhance UV‐ and H2O2‐induced p53 acetylation, whereas SIRT1 activator resveratrol inhibits it. We also found that SIRT1 is involved in UV‐induced AMP‐activated protein kinase (AMPK) and downstream acetyl‐CoA carboxylase (ACC), phosphofructose kinase‐2 (PFK‐2) phosphorylation. Collectively, our data provide new insights into understanding of the molecular mechanisms of UV‐induced skin aging, suggesting that SIRT1 activators such as resveratrol could serve as new anti‐skin aging agents.

The most common adverse effects that are related to all-trans retinoic acid (atRA) treatment are irritation and dryness of the skin. atRA therapy is reported to impair barrier function as achieved by trans-epidermal water loss (TEWL). Treatment with nicotinamide prior to initiation of atRA therapy provides additional barrier protection and thus reduces susceptibility of retinoic acid. Our previous studies showed that atRA upregulates aquaporin 3 (AQP3) in cultured human skin keratinocytes and fibroblasts. Others have demonstrated that in atopic dermatitis, overexpression of AQP3 is linked to elevated TEWL and that nicotinamide treatment reduces skin TEWL. In this study, we observed that while atRA upregulates AQP3 expression in cultured human skin keratinocytes (HaCaT cells), nicotinamide attenuates the effect of atRA in a concentration-dependent manner. atRA treatment induces EGFR and ERK activation. PD153035, an EGFR inhibitor, and U0126, an ERK inhibitor, inhibit atRA-induced upregulation of AQP3. Nicotinamide also inhibits atRA-induced activation of EGFR/ERK signal transduction and decreases water permeability by downregulating AQP3 expression. Collectively, our results indicate that the effect of atRA on AQP3 expression is at least partly mediated by EGFR/ERK signaling in cultured human skin keratinocytes. Nicotinamide attenuates atRA-induced AQP3 expression through inhibition of EGFR/ERK signal transduction and eventually decreases water permeability and water loss. Our study provides insights into the molecular mechanism through which nicotinamide reverses the side effects of dryness in human skin after treatment with atRA.