Wenming Chu

High basal STAT4 balanced by STAT1 induction to control type 1 interferon effects in natural killer cells

The best-characterized type 1 interferon (IFN) signaling pathway depends on signal transducer and activator of transcription 1 (STAT1) and STAT2. The cytokines can, however, conditionally activate all STATs. Regulation of their access to particular signaling pathways is poorly understood. STAT4 is important for IFN-γ induction, and NK cells are major producers of this cytokine. We report that NK cells have high basal STAT4 levels and sensitivity to type 1 IFN–mediated STAT4 activation for IFN-γ production. Increases in STAT1, driven during viral infection by either type 1 IFN or IFN-γ, are associated with decreased STAT4 access. Both STAT1 and STAT2 are important for antiviral defense, but STAT1 has a unique role in protecting against sustained NK cell IFN-γ production and resulting disease. The regulation occurs with an NK cell type 1 IFN receptor switch from a STAT4 to a STAT1 association. Thus, a fundamental characteristic of NK cells is high STAT4 bound to the type 1 IFN receptor. The conditions of infection result in STAT1 induction with displacement of STAT4. These studies elucidate the critical role of STAT4 levels in predisposing selection of specific signaling pathways, define the biological importance of regulation within particular cell lineages, and provide mechanistic insights for how this is accomplished in vivo.

SIRT1 confers protection against UVB- and H2O2-induced cell death via modulation of p53 and JNK in cultured skin keratinocytes

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.

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.

AMP-activated Protein Kinase Contributes to UV- and H2O2-induced Apoptosis in Human Skin 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.

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.

Galpha(i1) and Galpha(i3) are required for epidermal growth factor-mediated activation of the Akt-mTORC1 pathway.

Two members of the Gαi family of G proteins form complexes with EGFR and the adaptor protein Gab1 to mediate activation of Akt. Double Duty The activation of the serine-threonine kinase Akt and of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) are important for cellular responses to growth factors, including epidermal growth factor (EGF); however, exactly how the EGF receptor (EGFR) stimulates activation of Akt is unclear. Cao et al. provide evidence of a role for the heterotrimeric guanine nucleotide–binding proteins (G proteins) Gαi1 and Gαi3, better known as transducers of G protein–coupled receptor signaling, in this process. Both G proteins formed complexes with EGFR and the adaptor protein Gab1 and were required for the phosphorylation of Gab1, its interaction with the p85 subunit of phosphatidylinositol 3-kinase (PI3K), and for the phosphorylation of downstream targets of Akt and mTORC1. Further, loss of both Gαi1 and Gαi3 impaired the migration, survival, and growth of fibroblasts in response to EGF. Together, these data suggest that these members of the Gαi family of G proteins are required for EGF-mediated activation of Akt and mTORC1, providing further evidence of a role for G proteins in mediating signaling from receptor tyrosine kinases. The precise mechanism whereby epidermal growth factor (EGF) activates the serine-threonine kinase Akt and the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) remains elusive. Here, we report that the α subunits of the heterotrimeric guanine nucleotide–binding proteins (G proteins) Gαi1 and Gαi3 are critical for this activation process. Both Gαi1 and Gαi3 formed complexes with growth factor receptor binding 2 (Grb2)–associated binding protein 1 (Gab1) and the EGF receptor (EGFR) and were required for the phosphorylation of Gab1 and its subsequent interaction with the p85 subunit of phosphatidylinositol 3-kinase in response to EGF. Loss of Gαi1 and Gαi3 severely impaired the activation of Akt and of p70 S6 kinase and 4E-BP1, downstream targets of mTORC1, in response to EGF, heparin-binding EGF-like growth factor, and transforming growth factor α, but not insulin, insulin-like growth factor, or platelet-derived growth factor. In addition, ablation of Gαi1 and Gαi3 largely inhibited EGF-induced cell growth, migration, and survival, and the accumulation of cyclin D1. Overall, this study suggests that Gαi1 and Gαi3 lie downstream of EGFR, but upstream of Gab1-mediated activation of Akt and mTORC1, thus revealing a role for Gαi proteins in mediating EGFR signaling.

Priming with EGFR tyrosine kinase inhibitor and EGF sensitizes ovarian cancer cells to respond to chemotherapeutical drugs.

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 EGFRs 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.

ATP-sensitive potassium channel: A novel target for protection against UV-induced human skin cell damage

Ultraviolet radiation (UV) induces cell damages leading to skin photoaging and skin cancer. ATP‐sensitive potassium (KATP) channel openers (KCOs) have been shown to exert significant myocardial preservation and neuroprotection in vitro and in vivo, and yet the potential role of those KCOs in protection against UV‐induced skin cell damage is unknown. We investigated the effects of pinacidil and diazoxide, two classical KCOs, on UV‐induced cell death using cultured human keratinocytes (HaCat cells). Here, we demonstrated for the first time that Kir 6.1, Kir 6.2 and SUR2 subunits of KATP channels are functionally expressed in HaCaT cells and both non‐selective KATP channel opener pinacidil and mitoKATP (mitochondrial KATP) channel opener diazoxide attenuated UV‐induced keratinocytes cell death. The protective effects were abolished by both non‐selective KATP channel blocker glibenclamide and selective mitoKATP channel blocker 5‐hydroxydecanoate (5‐HD). Also, activation of KATP channel with pinacidil or diazoxide resulted in suppressive effects on UV‐induced MAPK activation and reactive oxygen species (ROS) production. Unexpectedly, we found that the level of intracellular ROS was slightly elevated in HaCaT cells when treated with pinacidil or diazoxide alone. Furthermore, UV‐induced mitochondrial membrane potential loss, cytochrome c release and ultimately apoptotic cell death were also inhibited by preconditioning with pinacidil and diazoxide, and their effects were reversed by glibenclamide and 5‐HD. Taken together, we contend that mitoKATP is likely to contribute the protection against UV‐induced keratinocytes cell damage. Our findings suggest that KATP openers such as pinacidil and diazoxide may be utilized to prevent from UV‐induced skin aging. J. Cell. Physiol. 212: 252–263, 2007.

Signaling Pathways Leading to the Activation of IKK and MAPK by Thymosin α1

Abstract:  Thymosin alpha 1 (Tα1) has therapeutic potential in the treatment of infectious diseases and cancer. However, the exact molecular pathways for Tα1 action are not fully understood. We found that Tα1 induces the production of interleukin‐6 (IL‐6), IL‐10, and IL‐12 in murine bone marrow‐derived macrophages (BMDMs) through IKK and MAPK pathways. Tα1 triggers the activation of AP‐1 and the phosphorylation of JNK and p38. Inhibition of p38 impairs IL‐6 production in response to Tα1. Further, TRAF6 is involved in the activation of JNK and IRAK4 is involved for the activation of IKK and PKCζ in a Tα1‐induced system. Loss of IRAK4 largely blocked induction of IL‐6. Thus, our studies define early signal events that are critical for the Tα1‐induced immune responses.