The proinflammatory LTB4/BLT1 signal axis confers resistance to TGF-ß1-induced growth inhibition by targeting Smad3 linker region.

Leukotriene B4 (LTB4) is a potent pro-inflammatory eicosanoid that is derived from arachidonic acid, and its signaling is known to have a tumor-promoting role in several cancer types. In this study, we investigated whether enhanced LTB4 signaling confers resistance to the cytostatic transforming growth factor-ß1 (TGF-ß1) response. We found that LTB4 pretreatment or ectopic expression of BLT1, a high affinity LTB4 receptor, fully abrogated TGF-ß1-induced cell cycle arrest and expression of p15INK4B and p27KIP1. Mechanism study revealed that LTB4-mediated suppression of TGF-ß1-induced Smad3 activation and growth inhibition was due to enhanced phosphorylation of Smad3 linker region (pSmad3L) through activation of BLT1-NAD(P)H oxidase (NOX)-reactive oxygen species (ROS)-epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3-K)-extracellular signal-activated kinase1/2 (ERK1/2)-linked signaling cascade. Furthermore, the LTB4/BLT1 signaling pathway leading to pSmad3L was constitutively activated in breast cancer cells and was correlated with TGF-ß1-resistant growth of the cells in vitro and in vivo. In human breast cancer tissues, the expression level of pSmad3L (Thr179) had a positive correlation with BLT1 expression. Collectively, our data demonstrate for the first time that the induction of pSmad3L through BLT1-NOX-ROS-EGFR-PI3K-ERK1/2 signaling pathway is a key mechanism by which LTB4 blocks the anti-proliferative responses of TGF-ß1, providing a novel mechanistic insight into the connection between enhanced inflammatory signal and cancer cell growth.

Kaempferol Suppresses Transforming Growth Factor-ß1-Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-179.

Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-ß1 (TGF-ß1). In human A549 non-small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-ß1-induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-ß1-mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-ß1-mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-ß1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-ß1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-ß1-induced EMT and cell migration. Furthermore, Akt1 was required for TGF-ß1-mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-ß1-induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-ß1-induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol.

A nuclear factor kappa B-derived inhibitor tripeptide inhibits UVB-induced photoaging process.

BACKGROUND: Ultraviolet (UV) irradiation on the skin induces photoaging which is characterized by keratinocyte hyperproliferation, generation of coarse wrinkles, worse of laxity and roughness. Upon UV irradiation, nuclear factor kappa B (NF-κB) is activated which plays a key role in signaling pathway leading to inflammation cascade and this activation stimulates expression of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1alpha (IL-1α) and a stress response gene cyclooxygenase-2 (COX-2). In addition, activation of NF-κB up-regulates the expression of matrix metalloprotease-1 (MMP-1) and consequently collagen in dermis is degraded. OBJECTIVE: In this study, the effects of a NF-κB-derived inhibitor tripeptide on the UVB-induced photoaging and inflammation were investigated in vitro and in vivo. METHODS: A NF-κB-derived inhibitor tripeptide (NF-κB-DVH) was synthesized based on the sequence of dimerization region of the subunit p65 of NF-κB. Its inhibitory activity was confirmed using chromatin immunoprecipitation assay and in situ proximate ligation assay. The effects of anti-photoaging and anti-inflammation were analyzed by Enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunochemistry. RESULTS: NF-κB-DVH significantly decreased UV-induced expression of TNF-α, IL-1α, MMP-1 and COX-2 while increased production of type I procollagen. CONCLUSION: Results showed NF-κB-DVH had strong anti-inflammatory activity probably by inhibiting NF-κB activation pathway and suggested to be used as a novel agent for anti-photoaging.

14-3-3 σ is a new target up-regulated by transforming growth factor-ß1 through a Smad3-dependent mechanism.

The seven members of the human 14-3-3 family play crucial roles in a diverse range of cellular responses including cell cycle progression, DNA damage checkpoint, and apoptosis. One particular isoform, 14-3-3 σ, the p53 target gene, is a unique tumor suppressor. We here report 14-3-3 σ as a transforming growth factor-beta (TGF-ß) target gene. In mammary epithelial cells, TGF-ß selectively induced expression of 14-3-3 σ at both mRNA and protein levels, and this induction was dependent on Smad3 not on p53. In addition, blockade of non-canonical Smad-independent pathways, including MAP kinases and Rho GTPases, did not affect the TGF-ß1-induced 14-3-3 σ expression. Our data provides the first evidence that 14-3-3 σ is a Smad3-dependent target gene of TGF-ß1.

Smad7 sensitizes A549 lung cancer cells to cisplatin-induced apoptosis through heme oxygenase-1 inhibition.

Smad7, an inhibitory Smad, acts as a key regulator forming autoinhibitory feedback loop in transforming growth factor-beta (TGF-ß) signaling. However, a growing body of evidences suggests that Smad7 is capable of apoptotic function. In the present study, we have demonstrated a proapoptotic function of Smad7 as a negative regulator of survival protein heme oxygenase-1 (HO-1). The HO-1 protein level was elevated in cisplatin-resistant A549 human lung cancer cells and blockade of HO-1 activation sensitized the cells to apoptosis. Interestingly, overexpression of Smad7 decreased HO-1 gene expression and its enzymatic activity. Notably, Smad7 reduced Akt activity and infection with adenovirus expressing a constitutively active form of the Akt reversed the inhibitory effects of Smad7 to HO-1, indicating a negative action mechanism of Smad7 to Akt-HO-1-linked survival pathway. Consistently, Smad7 sensitized A549 cells to cisplatin-induced apoptosis and these effects were dependent on HO-1 and Akt inhibition. Based on these findings, we suggest that targeting Smad7 may be an efficient strategy for overcoming drug-resistance in cancer therapy.

Genipin up-regulates heme oxygenase-1 via PI3-kinase-JNK1/2-Nrf2 signaling pathway to enhance the anti-inflammatory capacity in RAW264.7 macrophages.

Genipin, an aglycon of geniposide, has been reported to exhibit diverse pharmacological functions such as antitumor and anti-inflammatory effects. This study aimed to elucidate the anti-inflammatory mechanism of genipin, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. In RAW264.7 cells, genipin increased HO-1 expression and its enzyme activity via a NF-E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway. These effects were significantly inhibited by exposure to the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, or by expression of a dominant negative mutant of PI 3-kinase. Additional experiments showed that the activation of c-Jun NH(2)-terminal kinase 1/2 (JNK1/2) is required for genipin-induced phosphorylation and nuclear translocation of Nrf2 and antioxidant response element (ARE)-driven induction of HO-1, and acts as a downstream effector of PI 3-kinase. Furthermore, functional significance of HO-1 induction was revealed by genipin-mediated inhibition of lipopolysaccharide-stimulated inducible nitric oxide synthase expression or cyclooxygenase-2 promoter activity, the response was reversed by the blocking of HO-1 protein synthesis or HO-1 enzyme activity. Therefore, identification of PI 3-kinase-JNK1/2-Nrf2-linked signaling cascade in genipin-mediated HO-1 expression defines the signaling event that could participate in genipin-mediated anti-inflammatory response.

Fyn mediates transforming growth factor-beta1-induced down-regulation of E-cadherin in human A549 lung cancer cells.

Transforming growth factor-beta (TGF-ß) signaling positively contributes to the regulation of tumor metastasis. However, the underlying molecular mechanisms are less well defined. We here show that Fyn, a member of Src family tyrosine kinases, plays a critical role in mediating TGF-ß1-induced down-regulation of E-cadherin in human A549 lung cancer cells. Blockade of Fyn with siRNA knockdown or ligand-binding defective mutant significantly lowered the ability of TGF-ß1 to repress E-cadherin expression. Furthermore, our results demonstrated that Fyn facilitates TGF-ß1-mediated suppression of E-cadherin through p38 kinase-dependent induction of Snail. Collectively, our findings identify a Fyn-p38-Snail cascade as a new signaling pathway mediating oncogenic TGF-ß function.

The inhibitory effect of raloxifene on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells is mediated through a ROS/p38 MAPK/CREB pathway to the up-regulation of heme oxygenase-1 independent of estrogen receptor.

In this study, we demonstrate that raloxifene, a selective estrogen receptor modulator, is a potent inducer of the anti-inflammatory enzyme heme oxygenase-1 (HO-1). In RAW264.7 macrophages, raloxifene induced HO-1 mRNA and protein expression. Pretreatment of ICI182780, an estrogen receptor (ER) antagonist or knock-down of endogenous ERα or ERß gene by RNA interference failed to reverse raloxifene-mediated HO-1 induction, indicating an estrogen receptor-independent mechanism. Interestingly, the raloxifene-induced HO-1 expression was suppressed by reactive oxygen species (ROS) scavengers, including glutathione, TEMPO, Me(2)SO, 1,10-phenanthroline, or allopurinol. In addition, buthionine sulfoximine, an inhibitor of reduced glutathione synthesis, or Fe(2+)/Cu(2+) ions enhanced the positive effect of raloxifene on HO-1 expression. Consistent with these findings, raloxifene induced production of intracellular ROS and increased xanthine oxidase activity in vitro. Additional experiments revealed the involvement of mitogen-activated protein kinase (MAPK) kinase6 and p38 MAPK in the up-regulation of HO-1 by raloxifene and identified p38 MAPK as a downstream effector of ROS. Furthermore, the ROS-p38 MAPK cascade targeted the transcription factor cAMP-responsive element-binding protein (CREB). Finally, the functional significance of HO-1 induction was revealed by raloxifene-mediated inhibition of inducible nitric oxide synthase expression and nitric oxide production, a response reversed by the inhibition of HO-1 protein synthesis or blockade of p38 MAPK or xanthine oxidase activity. Therefore, identification of ROS-p38 MAPK-CREB-linked cascade as cellular relays in raloxifene-mediated HO-1 expression defines the signaling events that could participate in raloxifene-mediated anti-inflammatory response.

Anti-inflammatory activity of n-propyl gallate through down-regulation of NF-κB and JNK pathways.

The present study aimed to assess anti-inflammatory activity and underlying mechanism of n-propyl gallate, the n-propyl ester of gallic acid. n-Propyl gallate was shown to contain anti-inflammatory activity using two experimental animal models, acetic acid-induced permeability model in mice, and air pouch model in rats. It suppressed production of nitric oxide and induction of inducible nitric oxide synthase and cyclooxygenase-2 in the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. It was able to diminish reactive oxygen species level elevated in the LPS-stimulated RAW264.7 macrophage cells. It also suppressed gelatinolytic activity of matrix metalloproteinase-9 enhanced in the LPS-stimulated RAW264.7 macrophage cells. It inhibited inhibitory κB-α degradation and enhanced NF-κB promoter activity in the stimulated macrophage cells. It was able to suppress phosphorylation of c-Jun NH(2)-terminal kinase 1/2 (JNK1/2) and activation of c-Jun promoter activity in the stimulated macrophage cells. In brief, n-propyl gallate possesses anti-inflammatory activity via down-regulation of NF-κB and JNK pathways.

Up-regulation of heme oxygenase-1 expression through CaMKII-ERK1/2-Nrf2 signaling mediates the anti-inflammatory effect of bisdemethoxycurcumin in LPS-stimulated macrophages.

We have identified a novel anti-inflammatory signaling pathway that leads to the expression of heme oxygenase-1 (HO-1) in response to bisdemethoxycurcumin (BDMC), an analog of curcumin. Treatment with BDMC suppressed inducible nitric oxide synthase expression and nitric oxide (NO) production by down-regulating NF-kappaB activity in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. These effects were reversed by blocking HO-1 activity or expression. The signaling pathway involved in BDMC-mediated HO-1 induction included Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase 1/2 (ERK1/2). BDMC induced phosphorylation of ERK1/2 in a CaMKII-dependent manner. Pretreatment with the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor, U0126, inhibited CaMKII-induced stimulation of HO-1 promoter activity, suggesting that ERK1/2 is a downstream mediator of CaMKII in BDMC signaling to HO-1 expression. Furthermore, the CaMKII-ERK1/2 cascade targets the transcription factor, NF-E2-related factor-2 (Nrf2). Finally, inhibition of the Ca(2+)-CaMKII-ERK1/2-linked cascade attenuated significantly suppression by BDMC of LPS-induced iNOS expression and subsequent NO production. Collectively, our findings identify a Ca(2+)/calmodulin-CaMKII-ERK1/2-Nrf2 cascade as a novel anti-inflammatory pathway mediating BDMC signaling to HO-1 expression in macrophages.

14-3-3 sigma and 14-3-3 zeta plays an opposite role in cell growth inhibition mediated by transforming growth factor-beta 1.

The expression of 14-3-3 proteins is dysregulated in various types of cancer. This study was undertaken to investigate the effects of 14-3-3 zeta and 14-3-3 sigma on cell growth inhibition mediated by transforming growth factor-beta 1 (TGF-beta1). Mouse mammary epithelial cells (Eph4) that are transformed with oncogenic c-H-Ras (EpRas) and no longer sensitive to TGF-beta1-mediated growth inhibition displayed increased expression of 14-3-3 zeta and decreased expression of 14-3-3 sigma compared with parental Eph4 cells. Using small interfering RNA-mediated knockdown and overexpression of 14-3-3 sigma or 14-3-3 zeta, we showed that 14-3-3 sigma is required for TGF-beta1-mediated growth inhibition whereas 14-3-3 zeta negatively modulates this growth inhibitory response. Notably, overexpression of 14-3-3 zeta increased the level of Smad3 protein that is phosphorylated at linker regions and cannot mediate the TGF-beta1 growth inhibitory response. Consistent with this finding, mutation of the 14-3-3 zeta phosphorylation sites in Smad3 markedly reduced the 14-3-3 zeta-mediated inhibition of TGF-beta1-induced p15 promoter-reporter activity and cell cycle arrest, suggesting that these residues are critical targets of 14-3-3 zeta in the suppression of TGF-beta1-mediated growth. Taken together, our findings indicate that dysregulation of 14-3-3 sigma or 14-3-3 zeta contributes to TGF-beta1 resistance in cancer cells.

Decreased expression of glutaredoxin 1 is required for transforming growth factor-beta1-mediated epithelial-mesenchymal transition of EpRas mammary epithelial cells.

Transforming growth factor-beta (TGF-beta) is a cytokine important in inducing epithelial-mesenchymal transition (EMT), a crucial morphological event in a wide range of physiological and pathological cellular processes. In this study, we demonstrate that TGF-beta1 induces the EMT phenotype through decreasing the expression of the glutaredoxin 1 (Grx1) gene, an anti-oxidant enzyme, in H-Ras transformed EpH4 mammary epithelial cells (EpRas), but not in the parental EpH4 cells. TGF-beta1-induced reduction of Grx1 expression caused an increase of intracellular reactive oxygen species (ROS) in EpRas cells, and pre-treatment of the ROS scavenger N-acetylcysteine (NAC) inhibited TGF-beta1-induced EMT. Grx1-overexpressing EpRas cells showed a reduction in intracellular ROS generation and suppressed the expression of mesenchymal markers upon treatment of TGF-beta1. In addition, MEK/MAP kinase and phosphatidylinositol-3 kinase (PI3K) signaling were found to mediate the decrease in Grx1 expression upon TGF-beta1 treatment, depending on the presence of Ras protein. Thus our findings strongly suggest that TGF-beta1 promotes EMT by increasing intracellular ROS levels via down-regulation of the Grx1 gene in EpRas cells.

Up-regulation of heme oxygenase-1 expression through the Rac1/NADPH oxidase/ROS/p38 signaling cascade mediates the anti-inflammatory effect of 15-deoxy-delta 12,14-prostaglandin J2 in murine macrophages.

We investigated the signaling pathway that leads to the expression of heme oxygenase-1 (HO-1) in murine macrophages in response to 15-deoxy-delta 12,14-prostaglandin J2 (15dPGJ2). 15dPGJ2 caused dose- and time-dependent activation of Rac1, followed by a transient increase in reactive oxygen species (ROS) via NADPH oxidase, which leads to downstream activation of p38 kinase. Inhibition of 15dPGJ2-dependent HO-1 expression significantly attenuated suppression by 15dPGJ2 of LPS-induced iNOS expression and subsequent production of nitric oxide (NO). Our findings strongly suggest that 15dPGJ2 exerts its anti-inflammatory activity through the Rac1-NADPH oxidase-ROS-p38 signaling to the up-regulation of HO-1 in an in vitro inflammation model.

WITHDRAWN: Heme oxygenase-1 mediates the anti-inflammatory effect of propyl gallate in LPS-stimulated macrophages.

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The anti-inflammatory activity of Phellinus linteus (Berk. & M.A. Curt.) is mediated through the PKCdelta/Nrf2/ARE signaling to up-regulation of heme oxygenase-1.

It has been reported that heme oxygenase-1 (HO-1) mediates the anti-inflammatory activity of the n-BuOH subfraction (PL) prepared from fruiting bodies of Phellinus linteus. This continuing work aimed to elucidate the signaling pathway to the up-regulation of HO-1 by PL. In RAW264.7 macrophage cells, PL was able to enhance phosphorylation of protein kinase Cdelta (PKCdelta), but not PKCalpha/betaII, in a time-dependent manner. PL-induced HO-1 expression was dramatically released by GF109203X, a general inhibitor of PKC, and rottlerin, a specific PKCdelta inhibitor but not by Gö6976, a selective inhibitor for PKCalpha/beta. Additionally, PL treatment resulted in a marked increase in antioxidant response element (ARE)-driven transcriptional activity, which was dependent on PKCdelta but not PKCalpha. An increase by PL treatment in the ARE-driven transcriptional activity was further enhanced by Nrf2, whereas it was diminished by Keap1. Furthermore, pretreatment of rottlerin and overexpression of PKCdelta (K376R), a kinase-inactive form of PKCdelta, partly blocked the suppression by PL of nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and iNOS promoter activity, which were elevated in the lypopolysaccharide (LPS)-activated macrophages. Similarly, expression of matrix metalloproteinase-9 (MMP-9) and its promoter activity were suppressed by PL, which were dependent upon PKCdelta. The present findings indicate that Phellinus linteus gives rise to an anti-inflammatory activity though the PKCdelta/Nrf2/ARE signaling to the up-regulation of HO-1 in an in vitro inflammation model.