Identification of Clusterin Domain Involved in NF-��B Pathway Regulation

Clusterin (CLU) is a ubiquitous protein that has been implicated in tumorigenesis, apoptosis, inflammation, and cell proliferation. We and others have previously shown that CLU is an inhibitor of the NF-κB pathway. However, the exact form of CLU and the region(s) of CLU involved in this effect were unknown. Using newly generated molecular constructs encoding for CLU and various regions of the molecule, we demonstrated that the presecretory form of CLU (psCLU) form bears the NF-κB regulatory activity. Sequence comparison analysis showed sequence motif identity between CLU and β-transducin repeat-containing protein (β-TrCP), a main E3 ubiquitin ligase involved in IκB-α degradation. These homologies were localized in the disulfide constraint region of CLU. We generated a specific molecular construct of this region, named ΔCLU, and showed that it has the same NF-κB regulatory activity as CLU. Neither the α-chain nor the β-chain of CLU had any NF-κB regulatory activity. Furthermore, we showed that following tumor necrosis factor-α stimulation of transfected cells, we could co-immunoprecipitate phospho-IκB-α with ΔCLU. Moreover, we showed that ΔCLU could localize both in the cytoplasm and in the nucleus. These results demonstrate the identification of a new CLU activity site involved in NF-κB pathway regulation.     

Chafuroside B,an Oolong Tea Polyphenol,Ameliorates UVB-Induced DNA Damage and Generation of Photo-immunosuppression Related Mediators in Human Keratinocytes

Chafuroside B was recently isolated as a new polyphenolic constituent of oolong tea leaves. However, the effects of chafuroside B on skin function have not been examined. In this study, we investigated the protective effects of chafuroside B against UVB-induced DNA damage, apoptosis and generation of photo-immunosuppression related mediators in cultured normal human epidermal keratinocytes (NHEK). Chafuroside B at 1 µM attenuated both UVB-induced apoptosis, evaluated in terms of caspase-3/7 activity, and UVB-induced DNA damage, evaluated in terms of formation of cyclobutane pyrimidine dimers (CPD), in NHEK exposed to UVB (20 mJ/cm²). In addition, chafuroside B at 0.3 or 1 µM suppressed the UVB-induced production of interleukin (IL)-10, tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE₂), as determined by ELISA, and conversely enhanced IL-12 mRNA expression and production, as measured by RT-PCR and ELISA. Further, chafuroside B at 1 µM also suppressed UVB-induced expression of receptor activator of nuclear factor κB ligand (RANKL) mRNA. These results indicate that chafuroside B promotes repair of UVB-induced DNA damage and ameliorates the generation of IL-10, TNF-α, PGE₂, and RANKL, all of which are UVB-induced immunosuppression related mediators. These effects of chafuroside B may be mediated at least in part through induction of IL-12 synthesis in human keratinocytes. Because chafuroside B might have practical value as a photoprotective agent, a further study of the in vivo effects of chafuroside B seems warranted.     

Ubiquitin-specific Peptidase 21 Inhibits Tumor Necrosis Factor ��-induced Nuclear Factor ��B Activation via Binding to and Deubiquitinating Receptor-interacting Protein 1

Ubiquitination and deubiquitination of receptor-interacting protein 1 (RIP1) play an important role in the positive and negative regulation of the tumor necrosis factor α (TNFα)-induced nuclear factor κB (NF-κB) activation. Using a combination of functional genomic and proteomic approaches, we have identified ubiquitin-specific peptidase 21 (USP21) as a deubiquitinase for RIP1. USP21 is constitutively associated with RIP1 and deubiquitinates RIP1 in vitro and in vivo. Notably, knockdown of USP21 in HeLa cells enhances TNFα-induced RIP1 ubiquitination, IκB kinase β (IKKβ), and NF-κB phosphorylation, inhibitor of NF-κB α (IκBα) phosphorylation and ubiquitination, as well as NF-κB-dependent gene expression. Therefore, our results demonstrate that USP21 plays an important role in the down-regulation of TNFα-induced NF-κB activation through deubiquitinating RIP1.     

NF-��B Functions in Stromal Fibroblasts to Regulate Early Postnatal Muscle Development

Classical NF-κB activity functions as an inhibitor of the skeletal muscle myogenic program. Recent findings reveal that even in newborn RelA/p65^−/− mice, myofiber numbers are increased over that of wild type mice, suggesting that NF-κB may be a contributing factor in early postnatal skeletal muscle development. Here we show that in addition to p65 deficiency, repression of NF-κB with the IκBα-SR transdominant inhibitor or with muscle-specific deletion of IKKβ resulted in similar increases in total fiber numbers as well as an up-regulation of myogenic gene products. Upon further characterization of early postnatal muscle, we observed that NF-κB activity progressively declines within the first few weeks of development. At birth, the majority of this activity is compartmentalized to muscle fibers, but by neonatal day 8 NF-κB activity from the myofibers diminishes, and instead, stromal fibroblasts become the main cellular compartment within the muscle that contains active NF-κB. We find that NF-κB functions in these fibroblasts to regulate inducible nitric-oxide synthase expression, which we show is important for myoblast fusion during the growth and maturation process of skeletal muscle. Together, these data broaden our understanding of NF-κB during development by showing that in addition to its role as a negative regulator of myogenesis, NF-κB also regulates nitric-oxide synthase expression within stromal fibroblasts to stimulate myoblast fusion and muscle hypertrophy.     

Histone Deacetylase Inhibitors Activate NF-��B in Human Leukemia Cells through an ATM/NEMO-related Pathway

Mechanisms underlying histone deacetylase inhibitor (HDACI)-mediated NF-κB activation were investigated in human leukemia cells. Exposure of U937 and other leukemia cells to LBH-589 induced reactive oxygen species (ROS) followed by single strand (XRCC1) and double strand (γ-H2AX) DNA breaks. Notably, LBH-589 lethality was markedly attenuated by small interfering RNA (siRNA) knockdown of the DNA damage-linked histone, H1.2. LBH-589 triggered p65/RelA activation, NF-κB-dependent induction of Mn-SOD2, and ROS elimination. Interference with LBH-589-mediated NF-κB activation (e.g. in IκBα super-repressor transfected cells) diminished HDACI-mediated Mn-SOD2 induction and increased ROS accumulation, DNA damage, and apoptosis. The Mn-SOD2 mimetic TBAP (manganese(III)-tetrakis 4-benzoic acid porphyrin) prevented HDACI-induced ROS and NF-κB activation while dramatically attenuating DNA damage and cell death. In contrast, TRAF2 siRNA knockdown, targeting receptor-mediated NF-κB activation, blocked TNFα- but not HDACI-mediated NF-κB activation and lethality. Consistent with ROS-mediated DNA damage, LBH-589 exposure activated ATM (on serine 1981) and increased its association with NEMO. Significantly, siRNA NEMO or ATM knockdown blocked HDACI-mediated NF-κB activation, resulting in diminished MnSOD2 induction and enhanced oxidative DNA damage and cell death. In accord with the recently described DNA damage/ATM/NEMO pathway, SUMOylation site mutant NEMO (K277A or K309A) cells exposed to LBH-589 displayed diminished ATM/NEMO association, NEMO and p65/RelA nuclear localization/activation, and MnSOD2 up-regulation. These events were accompanied by increased ROS production, γ-H2AX formation, and cell death. Together, these findings indicate that in human leukemia cells, HDACIs activate the cytoprotective NF-κB pathway through an ATM/NEMO/SUMOylation-dependent process involving the induction of ROS and DNA damage and suggest that blocking NF-κB activation via the atypical ATM/NEMO nuclear pathway can enhance HDACI antileukemic activity.     

Nitric oxide inhibits ultraviolet B-induced murine keratinocyte apoptosis by regulating apoptotic signaling cascades

Cytotoxic effects of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) are considered to be one of the major causes of inflammatory diseases. On the other hand, protective effects of NO on toxic insults-induced cellular damage/apoptosis have been demonstrated recently. Ultraviolet B (UVB)-induced apoptosis of epidermal keratinocytes leads to skin inflammation and photoageing. However, it has not been elucidated what kind of effects NO has on UVB-induced keratinocyte apoptosis. Thus, in the present study, we investigated the problem and demonstrated that NO from NO donor suppressed UVB-induced apoptosis of murine keratinocytes. In addition, NO significantly suppressed activities of caspase 3, caspase 8 and caspase 9 that had been upregulated by UVB radiation. NO also suppressed p53 expression that had been upregulated by UVB radiation and upregulated Bcl-2 expression that had been downregulated by UVB radiation. These findings suggested that NO might suppress UVB-induced keratinocyte apoptosis by regulating apoptotic signaling cascades in p53, Bcl-2, caspase3, caspase 8 and caspase 9.     

GSK-3 Represses Growth Factor-inducible Genes by Inhibiting NF-��B in Quiescent Cells

GSK-3 is active in the absence of growth factor stimulation and generally acts to induce apoptosis or inhibit cell proliferation. We previously identified a subset of growth factor-inducible genes that can also be induced in quiescent T98G cells solely by inhibition of GSK-3 in the absence of growth factor stimulation. Computational predictions verified by chromatin immunoprecipitation assays identified NF-κB binding sites in the upstream regions of 75% of the genes regulated by GSK-3. p50 bound to most of these sites in quiescent cells, and for one-third of the genes, binding of p65 to the predicted sites increased upon inhibition of GSK-3. The functional role of p65 in gene induction following inhibition of GSK-3 was demonstrated by RNA interference experiments. Furthermore, inhibition of GSK-3 in quiescent cells resulted in activation of IκB kinase, leading to phosphorylation and degradation of IκBα and nuclear translocation of p65 and p50. Taken together, these results indicate that the high levels of GSK-3 activity in quiescent cells repress gene expression by negatively regulating NF-κB through inhibition of IκB kinase. This inhibition of NF-κB is consistent with the role of GSK-3 in the induction of apoptosis or cell cycle arrest in cells deprived of growth factors.     

Natural proteasome inhibitor celastrol suppresses androgen-independent prostate cancer progression by modulating apoptotic proteins and NF-kappaB

Background

Celastrol is a natural proteasome inhibitor that exhibits promising anti-tumor effects in human malignancies, especially the androgen-independent prostate cancer (AIPC) with constitutive NF-κB activation. Celastrol induces apoptosis by means of proteasome inhibition and suppresses prostate tumor growth. However, the detailed mechanism of action remains elusive. In the current study, we aim to test the hypothesis that celastrol suppresses AIPC progression via inhibiting the constitutive NF-κB activity as well as modulating the Bcl-2 family proteins.

Methodology/Principal Findings

We examined the efficacy of celastrol both in vitro and in vivo, and evaluated the role of NF-κB in celastrol-mediated AIPC regression. We found that celastrol inhibited cell proliferation in all three AIPC cell lines (PC-3, DU145 and CL1), with IC₅₀ in the range of 1–2 µM. Celastrol also suppressed cell migration and invasion. Celastrol significantly induced apoptosis as evidenced by increased sub-G1 population, caspase activation and PARP cleavage. Moreover, celastrol promoted cleavage of the anti-apoptotic protein Mcl-1 and activated the pro-apoptotic protein Noxa. In addition, celastrol rapidly blocked cytosolic IκBα degradation and nuclear translocation of RelA. Likewise, celastrol inhibited the expression of multiple NF-κB target genes that are involved in proliferation, invasion and anti-apoptosis. Celastrol suppressed AIPC tumor progression by inhibiting proliferation, increasing apoptosis and decreasing angiogenesis, in PC-3 xenograft model in nude mouse. Furthermore, increased cellular IκBα and inhibited expression of various NF-κB target genes were observed in tumor tissues.

Conclusions/Significance

Our data suggest that, via targeting the proteasome, celastrol suppresses proliferation, invasion and angiogenesis by inducing the apoptotic machinery and attenuating constitutive NF-κB activity in AIPC both in vitro and in vivo. Celastrol as an active ingredient of traditional herbal medicine could thus be developed as a new therapeutic agent for hormone-refractory prostate cancer.     

IL-4 suppresses UVB-induced apoptosis in skin

In this study, cutaneous role of IL-4 in UVB-induced apoptosis was investigated using transgenic mice with skin-specific expression of IL-4 (IL-4 Tg mice). The transgenic mice did not show any gross clinical abnormalities. However, epidermis was thickened and increased MHC class II positive cells were detected as well as enhanced expression of inflammatory cytokines such as IL-1 and TNF-alpha in skin. In addition, histological analysis revealed increased infiltration of lymphocytes, acanthosis, hyperkeratosis, and parakeratosis in skin of IL-4 Tg mice. The physiological effect of IL-4 overexpression in skin against environmental stimulus such as UVB was investigated by irradiating wild-type and IL-4 Tg mice with UVB followed by evaluation of apoptosis. The result demonstrated suppressed apoptosis in epidermis of IL-4 Tg mice compared with wild-type mice. To further assess anti-apoptotic function of IL-4 in keratinocytes, stable cell clones were made where IL-4 was constitutively overexpressed and examined for UVB-induced apoptosis. The results showed that apoptosis was remarkably decreased in IL-4 over-expressing cell clones compared with that in mock transfected cells. Collectively, data presented here shows that IL-4 has an inhibitory effect against UVB-induced apoptosis in keratinocytes, suggesting that IL-4 may be an important regulator in cutaneous immunity against UVB.     

Protective Effect of Indole-3-Pyruvate against Ultraviolet B-Induced Damage to Cultured HaCaT Keratinocytes and the Skin of Hairless Mice

Previous investigations demonstrated that pyruvate protects human keratinocytes against cell damage stemming from exposure to ultraviolet B (UVB) radiation. This study endeavoured to elucidate the protective capacity of aromatic pyruvates (e.g., phenylpyruvate (PPyr), 4-hydroxyphenylpyruvate (HPPyr), and indole-3-pyruvate (IPyr)) against UVB-induced injury to skin cells, both in vitro and in vivo. Cultured human HaCaT keratinocytes were irradiated with UVB light (60 mJ/cm²) and maintained with or without test compounds (1–25 mM). In addition, the dorsal skin of hairless mice (HR-1) was treated with test compounds (100 µmol) and exposed to UVB light (1 J/cm²) for two times. The ability of the test compounds to ameliorate UVB-induced cytotoxicity and inflammation was then assessed. Aromatic pyruvates reduced cytotoxicity in UVB-irradiated HaCaT keratinocytes, and also diminished the expression of interleukin 1β (IL-1β) and interleukin 6 (IL-6). IPyr was more efficacious than either PPyr or HPPyr. Furthermore, only IPyr inhibited cyclooxygenase-2 (Cox-2) expression at both the mRNA and the protein level in UVB-treated keratinocytes. Topical application of IPyr to the dorsal skin of hairless mice reduced the severity of UVB-induced skin lesions, the augmentation of dermal thickness, and transepithelial water loss. Overproduction of IL-1β and IL-6 in response to UVB radiation was also suppressed in vivo by the topical administration of IPyr. These data strongly suggest that IPyr might find utility as a UVB-blocking reagent in therapeutic strategies to lessen UVB-induced inflammatory skin damage.     

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.     

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined.Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity.In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin.Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.     

UVB-induced apoptosis in normal human keratinocytes: role of the erbB receptor family

Exposure of human keratinocytes to ultraviolet B (UVB) light leads to the activation of a variety of cell-surface receptors; however, the biologic consequences of these activated receptors are still unclear. It was previously reported that inhibition of cellular tyrosine kinase activity suppressed UVB-dependent effects in human skin. We confirmed that the same suppression of UVB-induced apoptosis occurs in normal human keratinocytes grown in culture. Furthermore, we sought to determine the role of erbB receptor tyrosine kinases in human keratinocytes following UVB irradiation. Using a specific inhibitor of the erbB family of tyrosine kinase receptors, DAPH, we investigated the effects of UVB-dependent activation of these receptors on keratinocyte biology. The addition of DAPH to keratinocytes resulted in the concentration-dependent protection of UVB-induced apoptosis. The protection from apoptosis was not due to the induction of keratinocyte differentiation, the loss of keratinocyte viability, or inhibition of the proliferative potential of keratinocytes by DAPH. The effect of DAPH on apoptosis was specific for UVB as it had no effect on bleomycin-induced apoptosis. Furthermore, the inhibition of UVB-induced apoptosis could also be observed using neutralizing antibodies to either erbB1 or erbB2. Finally, we demonstrated that DAPH could also inhibit UVB-induced apoptosis in an epidermal organotypic model system. These studies suggest an important role for the erbB receptors in UVB-induced apoptosis of human keratinocytes.     

Prolyl Hydroxylase EGLN3 Regulates Skeletal Myoblast Differentiation through an NF-��B-dependent Pathway

The egg-laying abnormal-9 (EGLN) prolyl hydroxylases have been shown to regulate the stability and thereby the activity of the α subunits of hypoxia-inducible factor (HIF) through its ability to catalyze their hydroxylation. We have previously shown that EGLN3 promotes differentiation of C2C12 skeletal myoblasts. However, the mechanism underlying this effect remains to be fully elucidated. Here, we report that exposure of C2C12 cells to dimethyl oxalylglycine (DMOG), desferrioxamine, and hypoxia, all inhibitors of prolyl hydroxylase activity, led to repression of C2C12 myogenic differentiation. Inactivation of HIF by expression of a HIF dominant-negative mutant or deletion of HIF-1α by RNA interference did not affect the inhibitory effect of DMOG, suggesting that the effect of DMOG is HIF-independent. Pharmacologic inactivation of EGLN3 hydroxylase resulted in activation of the canonical NF-κB pathway. The inhibitory effect of DMOG on myogenic differentiation was markedly impaired in C2C12 cells expressing a dominant-negative mutant of IκBα. Exogenous expression of wild-type EGLN3, but not its catalytically inactive mutant, significantly inhibited NF-κB activation induced by overexpressed TRAF2 or IκB kinase 2. In contrast, deletion of EGLN3 by small interfering RNAs led to activation of NF-κB. These data suggest that EGLN3 is a negative regulator of NF-κB, and its prolyl hydroxylase activity is required for this effect. Furthermore, wild-type EGLN3, but not its catalytically inactive mutant, potentiated myogenic differentiation. This study demonstrates a novel role for EGLN3 in the regulation of NF-κB and suggests that it is involved in mediating myogenic differentiation, which is HIF-independent.     

Aldose reductase in keratinocytes attenuates cellular apoptosis and senescence induced by UV radiation

Although aldose reductase (AR) has been implicated in the cellular response to oxidative stress, the role of AR in ultraviolet-B (UVB)-induced cellular injury has not been investigated. Here, we show that an increased expression of AR in human keratinocytes modulates UVB-induced apoptotic cell death and senescence. Overexpression of AR in HaCaT cells significantly attenuated UVB-induced cellular damage and apoptosis, with a decreased generation of reactive oxygen species (ROS) and aldehydes. Ablation of AR with small interfering RNA or inhibition of AR activity abolished these effects. We also show that increased AR activity suppressed UVB-induced activation of the p38 and c-Jun N-terminal kinases, but did not affect the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. Similarly, UVB-induced translocation of Bax and Bcl-2 to mitochondria and cytosol, respectively, was markedly attenuated in cells overexpressing AR. Knockdown or inhibition of AR activity in primary cultured keratinocytes enhanced UVB-induced cellular senescence and increased the level of a cell-cycle regulatory protein, p53. Finally, cellular apoptosis induced by UVB radiation was significantly reduced in the epidermis of transgenic mice overexpressing human AR. These findings suggest that AR plays an important role in the cellular response to oxidative stress by sequestering ROS and reactive aldehydes generated in keratinocytes.