Renliang Wu

Glycogen synthase kinase 3β induces apoptosis in cancer cells through increase of survivin nuclear localization

Glycogen synthase kinase 3beta (GSK3beta) regulates numerous signaling pathways that control a wide range of cellular processes, including cell proliferation, differentiation, apoptosis and metabolism. We report a novel function of GSK3beta: It interacts with the inhibitor-of-apoptosis protein (IAP) survivin to modulate its expression, thus regulating apoptosis in human lung cancer cells. A co-immunoprecipitation assay revealed that GSK3beta can bind survivin. Activation of GSK3beta induced translocation of survivin from the cytoplasm to the nucleus, resulting in G1 cell-cycle arrest and apoptosis, as well as sensitization to the chemotherapeutic drug doxorubicin. In contrast, inactivation of GSK3beta, either by transfection of a dominant-negative mutant inhibitor DN-GSK3beta or with selective inhibitor LiCl, increased cytoplasmic survivin expression, leading to cell-cycle progression and resistance to apoptosis. These results identify a pro-apoptotic role for GSK3beta in cancer cells, through its modulation of survivin in subcellular redistribution. This new role suggests that there is a potential for pharmacologic activation of GSK3beta to enhance treatment of cancer patients, including those with resistance.

Cigarette smoke extract induces activation of β-catenin/TCF signaling through inhibiting GSK3β in human alveolar epithelial cell line

Cigarette smoke is known to have various injurious and cytotoxic effects on alveolar epithelial cells. However, the mechanism about the effects caused by cigarette smoke on alveolar epithelial cells remains unclear. In the present study, we first validated that cigarette smoke extract (CSE) impaired the viability of alveolar epithelial cells (A549 cells) and resulted in some morphological changes. Next, we found that glycogen synthase kinase 3beta (GSK3beta) was highly expressed in A549 cells, and CSE significantly inhibited GSK3beta by reducing GSK3beta expression and increasing inactive phosphorylated GSK3beta. It was also observed that CSE promoted beta-catenin accumulation and nuclear translocation, and further activated beta-catenin/TCF signaling. Finally, we demonstrated that GSK3beta over-expression promoted the degradation of beta-catenin and abolished beta-catenin/TCF transcriptional activity that was induced by CSE in alveolar epithelial cells. These results suggest that CSE induces the activation of beta-catenin/TCF signaling through inhibiting GSK3beta, implying a possible mechanism responsible for the injurious and cytotoxic effects on alveolar epithelial cell caused by cigarette smoke.

IQGAP1 activates Tcf signal independent of Rac1 and Cdc42 in injury and repair of bronchial epithelial cells

The process of injury and repair involves spreading, migration and cell proliferation. The functions of Rho GTPases and their effector IQGAP1 are poor known in this process of airway epithelium. In the present study, we employed a widely used in vitro model by scratching a monolayer of BECs. We found that scratching induced decreasing of the GTP-bound Rac1 and Cdc42, but increasing the amounts of IQGAP1 at different time points. Next, we confirmed that IQGAP1 interacted with the constitutively active Rac1 (Rac1(V12)) and Cdc42 (Cdc42(V12)) rather than the dominant negative Rac1 (Rac1(N17)) and Cdc42 (Cdc42(N17)). Over-expressions of wild type (WT) IQGAP1 and its mutant (T1050AX2), which was defective to interact with Rho GTPases, induced translocation of beta-catenin from the cytoplasm into the nucleus. These results activated Tcf/Lef and increased the expression levels of its target genes of c-myc and cyclin D1. Likewise, the amounts of c-myc and cyclin D1 increased after scratching. Our results suggested that IQGAP1 mediated cell proliferation through activating Tcf in a manner independent of Rac1 and Cdc42 in wound repair of BECs.

Bleomycin-induced nuclear factor-κB activation in human bronchial epithelial cells involves the phosphorylation of glycogen synthase kinase 3β

Nuclear factor-kappaB (NF-kappaB) plays a central role in the development of bleomycin (BLM) lung toxicity, but the regulatory mechanisms are still unknown. In the present study, we investigated the cytotoxic effect of BLM on cultured human bronchial epithelial cells (BECs) and first confirmed that BLM induced the transcriptional activation of NF-kappaB signaling in BECs. We also found that BLM activated Akt (protein kinase B, PKB) and increased the phosphorylation level of glycogen synthase kinase 3beta (GSK3beta). GSK3beta is known to be a key downstream target of Akt, and LY294002, the PI3K (phosphatidylinositol 3-kinase)/Akt inhibitor, which promoted the dephosphorylation of GSK3beta, significantly attenuated BLM-induced NF-kappaB activation. Next, we further observed that constitutively active GSK3beta stabilized the inhibitor of NF-kappaB (IkappaBalpha), inhibited p65 nuclear translocation and partially blocked BLM-induced NF-kappaB activation. Importantly, a co-immunoprecipitation assay revealed that GSK3beta formed a complex with IkappaBalpha, while GSK3beta phosphorylation caused by BLM led to their dissociation. These results suggest that BLM can induce the activation of NF-kappaB signaling in BECs and this process is tightly associated with the phosphorylation status of GSK3beta, implying a possible regulatory mechanism of NF-kappaB signaling in BECs during the toxic lung injury induced by BLM.

p120 Modulates LPS-Induced NF-κB Activation Partially through RhoA in Bronchial Epithelial Cells

p120-Catenin (p120) is an adherens junction protein recognized to regulate cell-cell adhesion. Emerging evidence indicates that p120 may also play an important role in inflammatory responses, and the regulatory mechanisms are still unknown. In the present study, we showed that p120 was associated with airway inflammation. p120 downregulation induced nuclear factor-κB (NF-κB) activation, accompanied with IκBα degradation, p65 nuclear translocation, and increased expression of interleukin-8 (IL-8) in lipopolysaccharide (LPS)- treated C57BL mice and human bronchial epithelial cells (BECs). Moreover, we first found that p120 directly coprecipitated with RhoA in BECs. After LPS stimulation, although total RhoA and p120-bound RhoA were unchanged, RhoA activity was increased. Y27632, a ROCK inhibitor, could partially inhibit nuclear translocation of p65. Overexpression of p120 inactivated RhoA and NF-κB in BECs, whereas p120 loss significantly increased RhoA activity, p65 nuclear translocation, and IL-8 expression. Taken together, our study supports the regulatory role of p120 in airway inflammation and reveals that p120 may modulate NF-κB signaling partially through RhoA.

Involvement of p120 in LPS-induced NF-κB activation and IL-8 production in human bronchial epithelial cells

P120-catenin (p120), a prototypic member of a subfamily of Armadillo repeat domain (Arm domain) proteins, not only participates in cell-cell adhesion, but also mediates inflammatory responses in the skin. In the present study, we demonstrated the effect of p120 on lipopolysaccharide (LPS)-induced inflammatory responses in human bronchial epithelial cells (BECs). We first confirmed that p120 expression was significantly reduced after LPS stimulation in BECs, the p65 subunit of nuclear factor-kappaB (NF-kappaB) nuclear translocation was promoted and NF-kappaB activity was rapidly induced. Moreover, the expression level of interleukin-8 (IL-8) increased after LPS treatment. Over-expression of p120 attenuated LPS-stimulated NF-kappaB reporter gene expression and IL-8 mRNA expression and protein synthesis. On the contrary, transfection with p120 small interfering RNA (siRNA) significantly elevated LPS-stimulated NF-kappaB transcriptional activity, p65 nuclear translocation and IL-8 expression. Collectively, these results indicate an anti-inflammatory effect of p120 in BECs, through its modulation of NF-kappaB signaling.

Cigarette smoke extract-induced p120-mediated NF-κB activation in human epithelial cells is dependent on the RhoA/ROCK pathway

Cigarette smoke exposure is a major cause of chronic obstructive pulmonary disease (COPD), but the underlying molecular inflammatory mechanisms remain poorly understood. Previous studies have found that smoke disrupts cell-cell adhesion by inducing epithelial barrier damage to the adherens junction proteins, primarily E-cadherin (E-cad) and p120-catenin (p120). Recently, the anti-inflammatory role of p120 has drawn increasing attention. In this study, we demonstrate that p120 has a role in the cigarette smoke extract-induced inflammatory response, presumably by regulating NF-κB signaling activation. Mechanistically, we show that p120-mediated NF-κB signaling activation in airway epithelial inflammation is partially RhoA dependent and is independent of E-cad. These results provide novel evidence for the role of p120 in the anti-inflammatory response.

p120-Catenin modulating nuclear factor-κB activation is partially RhoA/ROCKdependent in scratch injury

p120‐catenin (p120) is known as a cadherin‐associated protein that participates in tumor metastasis and invasion, as well as an anti‐inflammatory mediator. Recently, its anti‐inflammatory role is drawing increasing attention, but the regulatory mechanisms are still unknown. Here, we report that p120 modulated inflammatory responses partially depends on RhoA/ROCK pathway in scratch‐induced injury in human bronchial epithelial cells (BECs). For the first time, we found that p120 was significantly reduced in BECs after scratching, which could induce interleukin‐8 (IL‐8) production through nuclear factor‐κB (NF‐κB) activation accompanied with IκBα phosphorylation. Over‐expression of p120 3A could inhibit NF‐κB activation and IL‐8 mRNA expression and protein synthesis after scratching, while p120 knockdown by small interfering RNA could promote NF‐κB activation and IL‐8 mRNA expression and protein synthesis after scratching. Furthermore, we found that RhoA was the binding partner of p120 in BECs. Although total RhoA and p120‐binded RhoA remained unchanged, the RhoA activity was increased after scratching. Chemical blockade of RhoA/ROCK signaling (Y27632) inhibited scratch‐induced nuclear translocation of NF‐κB p65. Over‐expression of p120 3A attenuated scratch‐induced RhoA activation, whereas silence of p120 significantly elevated scratch‐induced RhoA activation in BCEs. Conclusively, these results indicate an anti‐inflammatory effect of p120 in bronchial epithelial cells through its modulation of NF‐κB signaling depending on RhoA/ROCK pathway.