Zhigang Bi

EGFR-mediated expression of aquaporin-3 is involved in human skin fibroblast migration

AQP3 (aquaporin-3), known as an integral membrane channel in epidermal keratinocytes, facilitates water and glycerol movement into and out of the skin. Here, we demonstrate that AQP3 is also expressed in cultured human skin fibroblasts, which under normal wound healing processes migrate from surrounding tissues to close the wound. EGF (epidermal growth factor), which induced fibroblast migration, also induced AQP3 expression in a time- and dose-dependent manner. CuSO4 and NiCl2, previously known as AQP3 water transport inhibitors, as well as two other bivalent heavy metals Mn2+ and Co2+, inhibited EGF-induced cell migration in human skin fibroblasts. AQP3 knockdown by small interfering RNA inhibited EGF-induced AQP3 expression and cell migration. Furthermore, an EGFR (EGF receptor) kinase inhibitor, PD153035, blocked EGF-induced AQP3 expression and cell migration. MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK inhibitor U0126 and PI3K (phosphoinositide 3-kinase) inhibitor LY294002 also inhibited EGF-induced AQP3 expression and cell migration. Collectively, our findings show for the first time that AQP3 is expressed in human skin fibroblasts and that EGF induces AQP3 expression via EGFR, PI3K and ERK signal transduction pathways. We have provided evidence for a novel role of AQP3 in human skin fibroblast cell migration, which occurs during normal wound healing.

Curcumin attenuates EGF-induced AQP3 up-regulation and cell migration in human ovarian cancer cells

ObjectiveAquaporin (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.MethodsCultured 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.ResultsIn 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.ConclusionsCollectively, 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.

All-trans retinoic acid attenuates ultraviolet radiation-induced down-regulation of aquaporin-3 and water permeability in human keratinocytes

One of the major characteristics of human skin photoaging induced by ultraviolet (UV) radiation is the dehydration of the skin. Water movement across plasma membrane occurs via diffusion through lipid bilayer and via aquaporins (AQPs). We find that UV induces aquaporin‐3 (AQP3) down‐regulation in human skin keratinocytes. MEK/ERK inhibitors PD98059 and U0126 inhibit UV‐induced down‐regulation of AQP3. Antioxidant N‐acetyl‐L‐cysteine or NAC blocks UV‐induced MEK/ERK activation and down‐regulation of AQP3. All‐trans retinoic acid or atRA, while alone inducing AQP3 expression, attenuates UV‐induced down‐regulation of AQP3 and water permeability. Using special inhibitors, we find that activation of EGFR and inhibition on ERK activation are involved in atRAs protective effects against UV‐induced AQP3 down‐regulation. Using specific AQP3s water transport inhibitors and siRNA knockdown, we observe that AQP3 is involved in cell migration and in vitro wound healing. UV‐induced AQP3 down‐regulation results in reduced water permeability, decreased cell migration, and delayed wound healing, which are attenuated by atRA pretreatment. We conclude that atRA protects against UV‐induced down‐regulation AQP3 and decrease in water permeability, reduction in cell migration and delayed in vitro wound healing via trans‐activation of EGFR and inhibition on ROS‐mediated MEK/ERK pathway. This novel finding provides evidence to support possible involvement of AQP3 in UV induced skin dehydration. J. Cell. Physiol. 215: 506–516, 2008.

Deguelin Induces Both Apoptosis and Autophagy in Cultured Head and Neck Squamous Cell Carcinoma Cells

Head and neck squamous cell carcinoma (HNSCC) represents more than 5% of all cancers diagnosed annually in United States and around the world. Despite advances in the management of patients with this disease, the survival has not been significantly improved, and the search for potential alternative therapies is encouraging. Here we demonstrate that deguelin administration causes a significant HNSCC cell death. Deguelin induces both cell apoptosis and autophagy by modulating multiple signaling pathways in cultured HNSCC cells. Deguelin inhibits Akt signaling, and down-regulates survivin and cyclin-dependent kinase 4 (Cdk4) expressions, by disrupting their association with heat shock protein-90 (Hsp-90). Deguelin induces ceramide production through de novo synthase pathway to promote HNSCC cell death. Importantly, increased ceramide level activates AMP-activated protein kinase (AMPK), which then directly phosphorylates Ulk1 and eventually leads to cell autophagy. We found that a low dose of deguelin sensitized HNSCC cells to 5-FU. Finally, using a nude mice Hep-2 xenograft model, we also showed a significant anti-tumor ability of deguelin in vivo. Together, we suggest that deguelin may represent a novel and effective chemo-agent against HNSCC.

Perifosine sensitizes UVB-induced apoptosis in skin cells: new implication of skin cancer prevention?

We demonstrate here that a relative low dose of perifosine significantly enhanced UVB-induced apoptosis in skin cells (keratinocytes and fibroblasts), associated with a significant increase of reactive oxygen species (ROS) and ceramide production as well as multiple perturbations of diverse cell signaling pathways, shifting to a significant pro-apoptosis outcomes. Perifosine inhibited UVB-induced pro-survival Akt/mammalian target of rapamycin (mTOR) and ERK activation, while facilitating pro-apoptotic AMP-activated protein kinas (AMPK), c-Jun-NH(2)-kinase (JNK), and p53 activation; these signaling changes together promoted a striking increase in skin cell apoptosis and a significantly reduced amount of DNA damages. Our results suggest that perifosine may represent a novel skin cancer prevention strategy.

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway.

UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaT cell line demonstrated that UVB radiation and hydrogen peroxide (H(2)O(2)) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H(2)O(2)-induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H(2)O(2)-induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-Ds critical role in UVB/oxidative stress-induced skin cell death.

Protective effect of astragaloside IV against matrix metalloproteinase-1 expression in ultraviolet-irradiated human dermal fibroblasts.

Ultraviolet (UV) irradiation induces skin photoaging associated with up-regulated matrix metalloproteinase (MMP) expression. Inhibition of MMP expression is suggested to alleviate photoaging induced by UV irradiation. Astragaloside IV (As-IV), one of the main active ingredients of Astragalus membranaceus (Fisch) Bge, has been reported to have various biological activities. However, its anti-photoaging effect has not been examined to date. In the present study, we observed the effect of As-IV on matrix metalloproteinase-1 (MMP-1) expression in UV-irradiated human dermal fibroblasts (HDFs). We found that treatment with As-IV significantly decreased UV-induced MMP-1 expression at the messenger RNA and protein levels. In addition, western blotting analysis revealed that As-IV concentration-dependently suppressed UV-induced phosphorylation of extracellular-regulated protein kinase, Jun-N-terminal kinase and p38 mitogen-activated protein kinase (MAPK). Furthermore, treatment with As-IV markedly inhibited UV-induced nuclear factor kappaB (NF-κB) activity. These results suggest that As-IV down-regulates UV-induced MMP-1 expression, perhaps through suppression of MAPK and NF-κB activation in HDFs. As-IV is thus a potential agent for the management of skin photoaging.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-SIN1 association mediates ultraviolet B (UVB)-induced Akt Ser-473 phosphorylation and skin cell survival

BackgroundThe exposure of skin keratinocytes to Ultraviolet (UV) irradiation leads to Akt phosphorylation at Ser-473, which is important for the carcinogenic effects of excessive sun exposure. The present study investigated the underlying mechanism of Akt Ser-473 phosphorylation by UVB radiation.ResultsWe found that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and mammalian target of rapamycin (mTOR) complex 2 (mTORC2) were both required for UVB-induced Akt Ser-473 phosphorylation in keratinocytes. Inhibition of DNA-PKcs activity via its inhibitor NU7026, a dominant-negative kinase-dead mutation, RNA interference (RNAi) or gene depletion led to the attenuation of UVB-induced Akt Ser-473 phosphorylation. Meanwhile, siRNA silencing or gene depletion of SIN1, a key component of mTORC2, abolished Akt Ser-473 phosphorylation by UVB. Significantly, we discovered that DNA-PKcs was associated with SIN1 in cytosol upon UVB radiation, and this complexation appeared required for Akt Ser-473 phosphorylation. Meanwhile, this DNA-PKcs-SIN1 complexation by UVB was dependent on epidermal growth factor receptor (EGFR) activation, and was disrupted by an EGFR inhibitor (AG1478) or by EGFR depletion. UVB-induced complexation between DNA-PKcs and mTORC2 components was also abolished by NU7026 and DNA-PKcs mutation. Finally, we found that both DNA-PKcs and SIN1 were associated with apoptosis resistance of UVB radiation, and inhibition of them by NU7026 or genetic depletion significantly enhanced UVB-induced cell death and apoptosis.ConclusionTaken together, these results strongly suggest that DNA-PKcs-mTORC2 association is required for UVB-induced Akt Ser-473 phosphorylation and cell survival, and might be important for tumor cell transformation.

Quercetin Attenuates UV- and H2O2-induced Decrease of Collagen Type I in Cultured Human Lens Epithelial Cells

Ultraviolet (UV) radiation is related to cataract formation. The dynamics of matrix proteins play crucial roles in cell proliferation, cell migration, and the remodeling of lens capsule and, possibly, cataract formation. However, the change of dynamics of matrix proteins, such as collagens, in lens cells in response to UV radiation has not been investigated. Using cultured human lens epithelial cells, we, for the first time, demonstrate that UV radiation induces a decrease of collagen type I in a time- and dose-dependent manner. Hydrogen peroxide (H(2)O(2)) also induces a collagen type I decrease in a similar pattern. We observed that UV and H(2)O(2) induce JNK and its downstream component, c-Jun, activation in both a time- and dose-dependent manner. The pharmacologic inhibitor of JNK or JNKi inhibits UV-induced JNK and c-Jun activation and attenuates a UV-induced decrease of collagen type I. Quercetin, a well known antioxidant, also protects against a UV- and H(2)O(2)-induced decrease of collagen type I in a dose-dependent manner. Quercetin inhibits UV- and H(2)O(2)-induced JNK and c-Jun activation. Collectively, we conclude that quercetin attenuates both a UV- and H(2)O(2)-induced decrease of collagen type I via the inhibiting of JNK/c-Jun activity. Understanding the cellular-signaling pathways involved in the UV- and H(2)O(2)-induced decrease of collagen type I may reveal potential therapeutic targets for the UV-induced cataract.

Gremlin inhibits UV-induced skin cell damages via activating VEGFR2-Nrf2 signaling.

Ultra Violet (UV) radiation induces reactive oxygen species (ROS) production, DNA oxidation and single strand breaks (SSBs), which will eventually lead to skin cell damages or even skin cancer. Here, we tested the potential activity of gremlin, a novel vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) agonist, against UV-induced skin cell damages. We show that gremlin activated VEGFR2 and significantly inhibited UV-induced death and apoptosis of skin keratinocytes and fibroblasts. Pharmacological inhibition or shRNA-mediated knockdown of VEGFR2 almost abolished gremlin-mediated cytoprotection against UV in the skin cells. Further studies showed that gremlin activated VEGFR2 downstream NF-E2-related factor 2 (Nrf2) signaling, which appeared required for subsequent skin cell protection. Nrf2 shRNA knockdown or S40T dominant negative mutation largely inhibited gremlin-mediated skin cell protection against UV. At last, we show that gremlin dramatically inhibited UV-induced ROS production and DNA SSB formation in skin keratinocytes and fibroblasts. We conclude that gremlin protects skin cells from UV damages via activating VEGFR2-Nrf2 signaling. Gremlin could be further tested as a novel anti-UV skin protectant.