USP4 targets TAK1 to downregulate TNFα-induced NF-κB activation

Lys63-linked polyubiquitination of transforming growth factor-β-activated kinase 1 (TAK1) has an important role in tumor necrosis factor-α (TNFα)-induced NF-κB activation. Using a functional genomic approach, we have identified ubiquitin-specific peptidase 4 (USP4) as a deubiquitinase for TAK1. USP4 deubiquitinates TAK1 in vitro and in vivo. TNFα induces association of USP4 with TAK1 to deubiquitinate TAK1 and downregulate TAK1-mediated NF-κB activation. Overexpression of USP4 wild type, but not deuibiquitinase-deficient C311A mutant, inhibits both TNFα- and TAK1/TAB1 co-overexpression-induced TAK1 polyubiquitination and NF-κB activation. Notably, knockdown of USP4 in HeLa cells enhances TNFα-induced TAK1 polyubiquitination, IκB kinase phosphorylation, IκBα phosphorylation and ubiquitination, as well as NF-κB-dependent gene expression. Moreover, USP4 negatively regulates IL-1β-, LPS- and TGFβ-induced NF-κB activation. Together, our results demonstrate that USP4 serves as a critical control to downregulate TNFα-induced NF-κB activation through deubiquitinating TAK1.     

Involvement of interleukin-1β mediated nuclear factor κB signalling pathways to down-regulate prostate-specific antigen and cell proliferation in LNCaP prostate cancer cells

Involvement of NF-κB (nuclear factor κB) mediated by IL-1β (interleukin-1β) on cell proliferation and PSA (prostate-specific antigen) production of LNCaP prostate cell lines and the possible cross-talk with Akt (also known as protein kinase B) signalling pathway has been investigated. NF-κB and Akt were analysed by Western blotting from LNCaP cells treated by IL-1β before proliferation and PSA production were measured. IL-1β inhibited proliferation and decreased PSA production. The Akt pathway was not sensitive, whereas NF-κB phosphorylation occurred as a result of treatment. PSA production and proliferation of LNCaP cells were down-regulated by NF-κB mediated by IL-1β promoting anti-apoptotic signalling and co-suppressor factors of PSA expression. IL-1β through NF-κB activation provides a rationale for therapeutic approaches in the anticancer treatment of prostate.     

Glycyrrhizic acid (GA), a triterpenoid saponin glycoside alleviates ultraviolet-B irradiation-induced photoaging in human dermal fibroblasts

Glycyrrhizic acid (GA), a triterpenoid saponin glycoside from the roots and rhizomes of licorice is used in traditional and modern medicine for the treatment of numerous medical conditions including skin diseases and beauty care product. In the present study, we investigated the effect of GA against ultraviolet B (UVB) irradiation-induced photoaging in human dermal fibroblasts (HDFs) and its possible mechanism of action. HDFs were subjected to photoaging by sub-toxic dose of UVB (10 mj/cm(2)) irradiation. Cell viability, matrix metalloproteinase 1 (MMP1), pro-collagen 1, cellular and nuclear morphology, cell cycle, intracellular reactive oxygen species (ROS), caspase 3 and hyaluronidase inhibition assays were performed. Western blotting was used to evaluate the expression of NF-kappa B (NF-κB) and cytochrome-C proteins. GA treatment significantly inhibited photoaging. It achieved this by reducing ROS, NF-κB, cytochrome c, caspase 3 levels and inhibiting hyaluronidase enzyme. The main mechanism seems to be, most likely by blocking MMP1 activation by modulating NF-κB signaling. These findings may be useful for development of natural and safe photoprotective agents against UVB irradiation.     

Cyr61 increases matrix metalloproteinase-3 expression and cell motility in human oral squamous cell carcinoma cells

Oral squamous cell carcinoma (OSCC) has a striking tendency to migrate and metastasize. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. However, the effects of Cyr61 on human OSCC cells are largely unknown. In this study, we found that Cyr61 increased the migration and the expression of matrix metalloproteinases-3 (MMP)-3 in human OSCC cells. αvβ5 or α6β1 monoclonal antibody (mAb), focal adhesion kinase (FAK) inhibitor, and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) inhibited the Cyr61-induced increase of the migration and MMP-3 up-regulation of OSCC cells. Cyr61 stimulation increased the phosphorylation of FAK, MEK, and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors suppressed the cell migration and MMP-3 expression enhanced by Cyr61. Moreover, Cyr61 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-3 promoter. Taken together, our results indicate that Cyr61 enhances the migration of OSCC cells by increasing MMP-3 expression through the αvβ3 or α6β1 integrin receptor, FAK, MEK, ERK, and NF-κB signal transduction pathway.     

Protection against UVB-induced damages in human dermal fibroblasts: efficacy of tricin isolated from enzyme-treated Zizania latifolia extract

This study was undertaken to determine the effects of enzyme-treated Zizania latifolia (ETZL) and of its major compound tricin on skin photo-aging and to investigate the mechanisms involved. It was found ETZL and tricin suppressed matrix metalloproteinase (MMP) production and increased type I-procollagen production in UVB-irradiated human dermal fibroblasts (HDFs). Furthermore, ETZL and tricin significantly up-regulated the expressions of the antioxidant enzymes HO-1 and SOD1, reduced UVB-induced reactive oxygen species (ROS) generation and mitogen-activated protein kinase (MAPK) induction by ROS and thereby attenuated activator protein-1 (AP-1) expression. In addition, ETZL and tricin both reduced the phosphorylations of IκBα and IKKα/ß and κB blocked the nuclear translocation of nuclear factor-κB (NF-κB) p65. These results show that ETZL have skin protective effects against UVB and suggest tricin as major efficacious material in ETZL protecting skin photoaging.     

Combined effects of interleukin-1α and transforming growth factor-β1 on modulation of human cardiac fibroblast function

During cardiac remodeling, cardiac fibroblasts (CF) are influenced by increased levels of interleukin-1α (IL-1α) and transforming growth factor-β1 (TGFβ1). The present study investigated the interaction between these two important cytokines on function of human CF and their differentiation to myofibroblasts (CMF). CF were isolated from human atrial appendage and exposed to IL-1α and/or TGFβ1 (both 0.1 ng/ml). mRNA expression levels of selected genes were determined after 6–24 h by real-time RT-PCR, while protein levels were analyzed at 24–48 h by ELISA or western blot. Activation of canonical signaling pathways (NFκB, Smad3, p38 MAPK) was determined by western blotting. Differentiation to CMF was examined by collagen gel contraction assays. Exposure of CF to IL-1α alone enhanced levels of IL-6, IL-8, matrix metalloproteinase-3 (MMP3) and collagen III (COL3A1), but reduced the CMF markers α-smooth muscle actin (αSMA) and connective tissue growth factor (CTGF/CCN2). By contrast, TGFβ1 alone had minor effects on IL-6, IL-8 and MMP3 levels, but significantly increased levels of the CMF markers αSMA, CTGF, COL1A1 and COL3A1. Co-stimulation with both IL-1α and TGFβ1 increased MMP3 expression synergistically. Furthermore, while TGFβ1 had no effect on IL-1α-induced IL-6 or IL-8 levels, co-stimulation inhibited the TGFβ1-induced increase in αSMA and blocked the gel contraction caused by TGFβ1. Combining IL-1α and TGFβ1 had no apparent effect on their canonical signaling pathways. In conclusion, IL-1α and TGFβ1 act synergistically to stimulate MMP3 expression in CF. Moreover, IL-1α has a dominant inhibitory effect on the phenotypic switch of CF to CMF induced by TGFβ1.     

Role of vitronectin and fibronectin receptors in oral mucosal and dermal myofibroblast differentiation

Background information. The activation of fibroblasts into myofibroblasts is a crucial event in healing that is linked to remodelling and scar formation, therefore we determined whether regulation of myofibroblast differentiation via integrins might affect wound healing responses in populations of patient‐matched HOFs (human oral fibroblasts) compared with HDFs (human dermal fibroblasts). Results. Both the HOF and HDF cell types underwent TGF‐β1 (transforming growth factor‐β1)‐induced myofibroblastic differentiation [upregulation of the expression of α‐sma (α‐smooth muscle actin)], although analysis of unstimulated cells indicated that HOFs contained higher basal levels of α‐sma than HDFs (P<0.05). Functional blocking antibodies against the integrin subunits α5 (fibronectin) or αv (vitronectin) were used to determine whether the effects of TGF‐β1 were regulated via integrin signalling pathways. α‐sma expression in both HOFs and HDFs was down‐regulated by antibodies against both α5 and αv. Functionally, TGF‐β1 inhibited cell migration in an in vitro wound model and increased the contraction of collagen gels. Greater contraction was evident for HOFs compared with HDFs, both with and without stimulation by TGF‐β1 (P<0.05). When TGF‐β1‐stimulated cells were incubated with blocking antibodies against α5 and αv, gel contraction was decreased to that of non‐stimulated cells; however, blocking αv or α5 could not restore cellular migration in both HOFs and HDFs. Conclusions. Despite intrinsic differences in their basal state, the cellular events associated with TGF‐β1‐induced myofibroblastic differentiation are common to both HOFs and HDFs, and appear to require differential integrin usage; up‐regulation of α‐sma expression and increases in collagen gel contraction are vitronectin‐ and fibronectin‐receptor‐dependent processes, whereas wound re‐population is not.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

miR-627/HMGB1/NF-κB regulatory loop modulates TGF-β1-induced pulmonary fibrosis

Pulmonary fibrosis (PF) is a fibroproliferative disease that can eventually lead to fatal lung failure. It is characterized by abnormal proliferation of fibroblasts, dysregulated fibroblast differentiation to myofibroblast, and disorganized collagen and extracellular matrix production, deposition and degradation. There is still a lack of effective treatment strategies for PF. Extracellular high-mobility group box protein 1 (HMGB1) induces PF through NF-κB-mediated TGF-β1 release. Herein, we first validate the suppressive effect of HMGB1 knockdown on TGF-β1-induced α-smooth muscle actin (α-SMA) and collagen I protein expression. In PF, miRNAs exert different effects through targeting various downstream target messenger RNAs. We searched an online database for dysregulated miRNAs in PF tissues; among them, miR-627 was predicted by online tools to target HMGB1 to inhibit its expression. miR-627 overexpression could partially reverse TGF-β1-induced normal human lung fibroblast proliferation, as well as α-SMA and collagen I protein expression. miR-627 inhibition could partially reverse the suppressive effect of HMGB1 knockdown on TGF-β1-induced α-SMA and collagen I protein expression through direct binding to the 3′-untranslated region of HMGB1. Moreover, miR-627/HMGB1 affected TGF-β1 release through RAGE/NF-κB signaling; miR-627/HMGB1 and RAGE/NF-κB signaling formed a regulatory loop to modulate TGF-β1-induced PF in vitro. In conclusion, miR-627 may be a potential agent that targets HMGB1 to inhibit its expression, thereby improving TGF-β1-induced PF in vitro.     

Stercurensin inhibits nuclear factor-κB-dependent inflammatory signals through attenuation of TAK1-TAB1 complex formation

We identified a chalcone, 2',4'-dihydroxy-6'-methoxy-3'-methylchalcone (stercurensin), as an active compound isolated from the leaves of Syzygium samarangense. In the present study, the anti-inflammatory effects and underlying mechanisms of stercurensin were examined using lipopolysaccharide (LPS)-stimulated RAW264.7 cells and mice. To determine the effects of stercurensin in vitro, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were analyzed by RT-PCR and immunoblotting. Nuclear factor-κB (NF-κB) activation and its upstream signaling cascades were also investigated using a dual-luciferase reporter assay, electrophoretic mobility shift assay, immunoblotting, immunofluorescence, and immunoprecipitation. To verify the effects of stercurensin in vivo, the mRNA expression levels of iNOS and COX-2 were evaluated in isolated mouse peritoneal macrophages by quantitative real-time PCR, and the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β were assessed in serum samples from mice using a Luminex system. Pretreatment with stercurensin reduced LPS-induced iNOS and COX-2 expression, thereby inhibiting nitric oxide (NO) and prostaglandin E(2) production, respectively. In addition, an inhibitory effect of stercurensin on NF-κB activation was shown by the recovery of LPS-induced inhibitor of κB (I-κB) degradation after blocking the transforming growth factor-β-activated kinase 1 (TAK1)/I-κB kinase signaling pathway. In mouse models, stercurensin negatively regulated NF-κB-dependent pro-inflammatory mediators and cytokines. These results demonstrate that stercurensin modulates NF-κB-dependent inflammatory pathways through the attenuation of TAK1-TAB1 complex formation. Our findings demonstrating the anti-inflammatory effects of stercurensin in vitro and in vivo will aid in understanding the pharmacology and mode of action of stercurensin.     

Dephosphorylation of Carma1 by PP2A negatively regulates T-cell activation

The Carma1-Bcl10-Malt1 (CBM) complex bridges T-cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF-κB pathway. NF-κB activation is triggered by PKCθ-dependent phosphorylation of Carma1 after TCR/CD28 co-stimulation. PKCθ-phosphorylated Carma1 was suggested to function as a molecular scaffold that recruits preassembled Bcl10-Malt1 complexes to the membrane. We have identified the serine-threonine protein phosphatase PP2A regulatory subunit Aα (PPP2R1A) as a novel interaction partner of Carma1. PPP2R1A is associated with Carma1 in resting as well as activated T cells in the context of the active CBM complex. By siRNA-mediated knockdown and in vitro dephosphorylation, we demonstrate that PP2A removes PKCθ-dependent phosphorylation of Ser645 in Carma1, and show that maintenance of this phosphorylation is correlated with increased T-cell activation. As a result of PP2A inactivation, we find that enhanced Carma1 S645 phosphorylation augments CBM complex formation, NF-κB activation and IL-2 or IFN-γ production after stimulation of Jurkat T cells or murine Th1 cells. Thus, our data define PP2A-mediated dephosphorylation of Carma1 as a critical step to limit T-cell activation and effector cytokine production.     

Overexpression of the human ZNF300 gene enhances growth and metastasis of cancer cells through activating NF-kB pathway

Zinc finger proteins (ZNF) play important roles in various physiological processes. Here we report that ZNF300, a novel zinc finger protein, identified specifically in humans, promotes tumour development by modulating the NF-κB pathway. Inflammatory factors were found to induce ZNF300 expression in HeLa cell line, and ZNF300 expression further enhanced NF-κB signalling by activating TRAF2 and physically interacting with IKKβ. Furthermore, ZNF300 overexpression increased ERK1/2 phosphorylation and the expression of c-myc, IL-6, and IL-8 but decreased the expression of p21(waf-1) and p27(Kip1) ; whose down-regulation led to the opposite effect. Most importantly, ZNF300 overexpression stimulated cancer cell proliferation in vitro and significantly enhanced tumour development and metastasis in mouse xenograft model, while knocking down ZNF300 led to the opposite effects. We have identified a novel function for ZNF300 in tumour development that may uniquely link inflammation and NF-κB to tumourigenesis in humans but not in mice.