miR-221/222 induce instability of p53 By downregulating deubiquitinase YOD1 in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by the impaired differentiation and uncontrolled proliferation of myeloid blasts. Tumor suppressor p53 is often downregulated in AML cells via ubiquitination-mediated degradation. While the role of E3 ligase MDM2 in p53 ubiquitination is well-accepted, little is known about the involvement of deubiquitinases (DUBs). Herein, we found that the expression of YOD1, among several DUBs, is substantially reduced in blood cells from AML patients. We identified that YOD1 deubiqutinated and stabilized p53 through interaction via N-terminus of p53 and OTU domain of YOD1. In addition, expression levels of YOD1 were suppressed by elevated miR-221/222 in AML cells through binding to the 3' untranslated region of YOD1, as verified by reporter gene assays. Treatment of cells with miR-221/222 mimics and inhibitors yielded the expected effects on YOD1 expressions, in agreement with the negative correlation observed between the expression levels of miR-221/222 and YOD1 in AML cells. Finally, overexpression of YOD1 stabilized p53, upregulated pro-apoptotic p53 downstream genes, and increased the sensitivity of AML cells to FLT3 inhibitors remarkably. Collectively, our study identified a pathway connecting miR-221/222, YOD1, and p53 in AML. Targeting miR-221/222 and stimulating YOD1 activity may improve the therapeutic effects of FLT3 inhibitors in patients with AML.

Effects of Blood Transfusion on Hepatic Ischemia-Reperfusion Injury-Induced Renal Tubular Injury.

OBJECTIVES: Hepatic ischemia-reperfusion injury and transfusion of red blood cells in liver surgery are wellknown risk factors to induce acute tubular injury. Transfusion of stored red blood cells may affect hepatic ischemia-reperfusion injury-induced acute tubular injury. Here, we hypothesized whether preischemic (due to increased severity of hepatic injury) and postischemic (due to renal uptake of free heme and iron) transfusion of stored red blood cells may potentiate acute tubular injury in rats subjected to hepatic ischemia-reperfusion injury. MATERIALS AND METHODS: Sprague Dawley rats (n = 24) were divided into 4 groups: sham operation (sham group), hepatic ischemia-reperfusion injury only (injury-only group), red blood cell transfusion before hepatic ischemia-reperfusion injury (preinjury transfusion group), and red blood celltransfusion after hepatic ischemia-reperfusion injury (postinjury transfusion group). Partial hepatic ischemia was induced for 90 minutes, with reperfusion allowed for 12 hours. Hepatic and renal tubular injury markers, renal mRNA levels of oxidant stress markers, and inflammatory markers were assessed. Renal cortex samples were examined under hematoxylin and eosin staining for tubular histopathologic score and immunohistochemical staining forinflammatory cells. RESULTS: With regard to hepatic and renal tubular injury markers, serum alanine aminotransferase, serum urea nitrogen, and histopathologic scores were increased in the preinjury and postinjury transfusion groups versus injury-only group, with moderate to strong correlation between alanine aminotransferase and tubular injury markers. Renal oxidative stress markers (heme oxygenase-1 and neutrophil gelatinaseassociated lipocalin) were correlated with increased alanine aminotransferase, with upregulation of oxidant stress markers in the preinjury transfusion group versus sham group (all markers), as well as in the injury-only and postinjury transfusion groups (heme oxygenase-1 only). We observed no changes in renal inflammatory responses among the groups. CONCLUSIONS: Preischemic transfusion potentiated acute tubular injury without triggering renal inflammatory responses. Exacerbation of hepatic injury may induce acute tubular injury via renal oxidant stress.

Ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 inhibits inflammation by regulating Nur77 stability.

Nur77 (NR4A1) plays an important role in various inflammatory responses. Nur77 is rapidly degraded in cells and its protein level is critically controlled. Although few E3 ligases regulating the Nur77 protein have been defined, the deubiquitinase (DUB) responsible for Nur77 stability has not been reported to date. We identified ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 (OTUB1) as a DUB that stabilizes Nur77 by preventing its proteasomal degradation. We found that OTUB1 interacted with Nur77 to deubiquitinate it, thereby stabilizing Nur77 in an Asp88-dependent manner. This suggests that OTUB1 targets Nur77 for deubiquitination via a non-canonical mechanism. Functionally, OTUB1 inhibited TNFα-induced IL-6 production by promoting Nur77 protein stability. OTUB1 modulated the stability of Nur77 as a counterpart of tripartite motif 13 (Trim13). That is, OTUB1 reduced the ubiquitination and degradation of Nur77 potentiated by Trim13. In addition, this DUB also inhibited IL-6 production, which was further amplified by Trim13 in TNFα-induced responses. These findings suggest that OTUB1 is an important regulator of Nur77 stability and plays a role in controlling the inflammatory response.

The orphan nuclear receptor NR4A1 promotes FcεRI-stimulated mast cell activation and anaphylaxis by counteracting the inhibitory LKB1/AMPK axis.

BACKGROUND: Nuclear receptor subfamily 4 group A member 1 (NR4A1), an orphan nuclear receptor, has been implicated in several biological events such as metabolism, apoptosis, and inflammation. Recent studies indicate a potential role for NR4A1 in mast cells, yet its role in allergic responses remains largely unknown. OBJECTIVES: The aim of this study was to clarify the role of NR4A1 in mast cell activation and anaphylaxis. METHODS: To evaluate the function of NR4A1 in mast cells, the impacts of siRNA knockdown, gene knockout, adenoviral overexpression, and pharmacological inhibition of NR4A1 on FcεRI signaling and effector functions in mouse bone marrow-derived mast cells (BMMCs) in vitro and on anaphylactic responses in vivo were evaluated. RESULTS: Knockdown or knockout of NR4A1 markedly suppressed degranulation and lipid mediator production by FcεRI-crosslinked BMMCs, while its overexpression augmented these responses. Treatment with a NR4A1 antagonist also blocked mast cell activation to a similar extent as NR4A1 knockdown or knockout. Moreover, mast cell-specific NR4A1-deficient mice displayed dampened anaphylactic responses in vivo. Mechanistically, NR4A1 promoted FcεRI signaling by counteracting the liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK) axis. Following FcεRI crosslinking, NR4A1 bound to the LKB1/AMPK complex and sequestered it in the nucleus, thereby promoting FcεRI downstream signaling pathways. Silencing or knockout of LKB1/AMPK largely abrogated the effect of NR4A1 on mast cell activation. Additionally, NR4A1 facilitated spleen tyrosine kinase activation independently of LKB1/AMPK. CONCLUSIONS: Nuclear receptor subfamily 4 group A member 1 positively regulates mast cell activation by antagonizing the LKB1-AMPK-dependent negative regulatory axis. This finding may provide a novel therapeutic strategy for the development of anti-allergic compounds.

The E3 ubiquitin ligase Trim13 regulates Nur77 stability via casein kinase 2α.

Nur77 is a member of the NR4A subfamily of nuclear receptors and has been shown to regulate various biological processes such as apoptosis and inflammation. Here, we show that Nur77 ubiquitination is mediated by the tripartite motif 13 (Trim13), a RING-type E3 ubiquitin ligase. The interaction between Nur77 and Trim13 was confirmed by co-immunoprecipitation. Moreover, we found that Lys539 in Nur77 ubiquitination is targeted for Trim13, which leads to Nur77 degradation. The Trim13-mediated ubiquitination of Nur77 was optimal in the presence of the E2 enzyme UbcH5. Importantly, in addition to Trim13-mediated ubiquitination, the stability of Nur77 was also regulated by casein kinase 2α (CK2α). Pharmacological inhibition of CK2 markedly increased Nur77 levels, whereas overexpression of CK2α, but not its inactive mutant, dramatically decreased Nur77 levels by promoting Nur77 ubiquitination. CK2α phosphorylated Ser154 in Nur77 and thereby regulated Nur77 protein levels by promoting its ubiquitin-mediated degradation. Importantly, we also show that degradation of Nur77 is involved in TNFα-mediated IL-6 production via CK2α and Trim13. Taken together, these results suggest that the sequential phosphorylation and ubiquitination of Nur77 controls its degradation, and provide a therapeutic approach for regulating Nur77 activity through the CK2α-Trim13 axis as a mechanism to control the inflammatory response.

Proteomic characterization of endogenous substrates of mammalian ubiquitin ligase Hrd1.

BACKGROUND: Endoplasmic reticulum (ER)-associated degradation (ERAD) regulates protein homeostasis in the secretory pathway by targeting misfolded or unassembled proteins for degradation by the proteasome. Hrd1 is a conserved multi-spanning membrane bound ubiquitin ligase required for ubiquitination of many aberrant ER proteins, but few endogenous substrates of Hrd1 have been identified to date. METHODS: Using a SILAC-based quantitative proteomic approach combined with CRISPR-mediated gene silencing, we searched for endogenous physiological substrates of Hrd1. We used RNA microarray, immunoblotting, cycloheximide chase combined with chemical genetics to define the role of Hrd1 in regulating the stability of endogenous ERAD substrates. RESULTS: We identified 58 proteins whose levels are consistently upregulated in Hrd1 null HEK293 cells. Many of these proteins function in pathways involved in stress adaptation or immune surveillance. We validated OS9, a lectin required for ERAD of glycoproteins as a highly upregulated protein in Hrd1 deficient cells. Moreover, the abundance of OS9 is inversely correlated with Hrd1 level in clinical synovium samples isolated from osteoarthritis and rheumatoid arthritis patients. Intriguingly, immunoblotting detects two OS9 variants, both of which are upregulated when Hrd1 is inactivated. However, only one of these variants is subject to proteasome dependent degradation that requires Hrd1 and the AAA (ATPase associated with diverse cellular activities) ATPase p97. The stability of the other variant on the other hand is influenced by a lysosomal inhibitor. CONCLUSION: Hrd1 regulates the stability of proteins involved in ER stress response and immune activation by both proteasome dependent and independent mechanisms.

Effects of Intraneural Injection of Dexmedetomidine in Combination With Ropivacaine in Rat Sciatic Nerve Block.

BACKGROUND AND OBJECTIVES: Dexmedetomidine is known to have neural protection effect via attenuation of inflammatory responses induced by local anesthetics. We investigated whether intraneural dexmedetomidine is effective for attenuating or preventing neural injury resulting from inadvertent intraneural injection of local anesthetic. METHODS: Rats were randomly divided, and left sciatic nerve was surgically exposed. The rats received no injection (control group) or intraneural injections of 0.2 mL of normal saline (saline group), 0.2 mL of 0.5% ropivacaine (ropivacaine group), or 0.2 mL of 0.5% ropivacaine and 0.5 µg/kg of dexmedetomidine (ropivacaine plus dexmedetomidine group). Interleukin (IL)-6 and IL-1ß messenger RNA (mRNA) levels were detected at 60 minutes after intraneural injection in experiment 1 (5 per group). Sensory and motor functions were assessed until the return of normal sensory and motor functions, and histopathological and ultrastructure analysis were performed at 4 weeks after intraneural injection in experiment 2 (8 per group). RESULTS: Dexmedetomidine with ropivacaine better enhanced sensory and motor blockade than ropivacaine alone. IL-6 (3.2 ± 1.0 vs 5.9 ± 2.1), IL-1ß (1.1 ± 0.1 vs 2.2 ± 0.7) levels, scores of axon and myelinated fiber degeneration (1 [0-2] vs 2 [1-3]), and demyelinated fiber percentages (20.1 ± 10.4 vs 48.3 ± 12.7) were lower in the ropivacaine plus dexmedetomidine group than in the ropivacaine group. No animals showed any signs of permanent neurological deficit. CONCLUSIONS: Intraneural dexmedetomidine has sensory and motor blockade-enhancing effects, anti-inflammatory properties, and protective effects against neural injury. These findings suggest that dexmedetomidine as an adjuvant has beneficial effects in rat when intraneural injection of local anesthetic occurs.

Preischemic transfusion of old packed RBCs exacerbates early-phase warm hepatic ischemia reperfusion injury in rats.

BACKGROUND: Hepatic innate immune cells are considered to play a central role in the early phase of hepatic ischemia reperfusion (IR) injury. Transfusion of old red blood cells (RBCs) is known to prime immune cells, and transfusion before IR may exacerbate liver injury because of the expected hyperresponsiveness of immune cells. MATERIALS AND METHODS: Twenty-four Sprague-Dawley rats were divided into four groups: sham operation (Sham); hepatic IR only (IR Control); and two transfusion groups, preischemic (Pre-T) and postischemic (Post-T), in which allogeneic RBCs stored for 2 weeks were transfused before hepatic IR or after reperfusion, respectively. Partial hepatic ischemia was induced for 90 min, and reperfusion was allowed for 120 min. Serum alanine transaminase levels, area of necrosis, and apoptotic cells were then assessed. Inflammatory (tumor necrosis factor alpha, interleukin 1 beta [IL-1ß], IL-6, IL-10, and cyclooxygenase 2) and oxidative mediators (heme oxygenase 1, superoxide dismutase, and glutathione peroxidase 1) were assessed for elucidating the relevant mechanisms underlying the hepatic injury. RESULTS: Pre-T, but not Post-T, showed increased serum alanine transaminase levels than IR Control (P < 0.05). Area of necrosis was more severe in Pre-T than in IR Control or Post-T (P < 0.01), and apoptotic cells were also more abundant in Pre-T than in IR Control (P < 0.01). tumor necrosis factor alpha and IL-6 levels were higher in Pre-T than in IR Control or Post-T (P < 0.05), with no significant difference in cytoprotective protein levels. CONCLUSIONS: Preischemic transfusion of old RBCs aggravated hepatic injury. Inflammatory cytokines seemed to play a crucial role in liver injury exacerbation. Our results indicate that transfusion before hepatic ischemia may be detrimental.

Methionine sulfoxide reductase A protects against lipopolysaccharide-induced septic shock via negative regulation of the proinflammatory responses.

Methionine sulfoxide reductase A (MsrA) is a major antioxidant enzyme that specifically catalyzes the reduction of methionine S-sulfoxide. In this study, we used MsrA gene-knockout (MsrA-/-) mice and bone marrow-derived macrophages (BMDMs) to investigate the role of MsrA in the regulation of inflammatory responses induced by lipopolysaccharide (LPS). MsrA-/- mice were more susceptible to LPS-induced lethal shock than wild-type (MsrA+/+) mice. Serum levels of the proinflammatory cytokines IL-6 and TNF-α induced by LPS were higher in MsrA-/- than in MsrA+/+ mice. MsrA deficiency in the BMDMs also increased the LPS-induced cytotoxicity as well as TNF-α level. Basal and LPS-induced reactive oxygen species (ROS) levels were higher in MsrA-/- than in MsrA+/+ BMDMs. Phosphorylation levels of p38, JNK, and ERK were higher in MsrA-/- than in MsrA+/+ BMDMs in response to LPS, suggesting that MsrA deficiency increases MAPK activation. Furthermore, MsrA deficiency increased the expression and nuclear translocation of NF-κB and the expression of inducible nitric oxide synthase, a target gene of NF-κB, in response to LPS. Taken together, our results suggest that MsrA protects against LPS-induced septic shock, and negatively regulates proinflammatory responses via inhibition of the ROS-MAPK-NF-κB signaling pathways.

Ovarian tumor domain-containing protein 1 deubiquitinates and stabilizes p53.

Ubiquitination and deubiquitination pathways play important roles in the regulation of p53 stability and activity. p53 is ubiquitinated and destabilized by E3 ubiquitin ligases and is deubiquitinated and stabilized by deubiquitinases (DUBs). We screened ovarian tumor (OTU) subfamily proteins to identify novel DUBs that stabilized p53. OTU domain-containing protein 1 (OTUD1) is a DUB belonging to the OTU family; however, its substrates and its role in cells are unknown. Here, we used an overexpression and knockdown system to show that OTUD1 is a novel regulator of p53 stability. OTUD1 overexpression increased p53 stability, whereas OTUD1 knockdown decreased p53 stability. Moreover, we observed that OTUD1 directly interacted with p53. Our results showed that OTUD1 deubiquitinated p53 and that functional OTUD1 was required for p53 stabilization. The deubiquitination activity of OTUD1 was necessary for p53 stabilization, as confirmed using an inactive OTUD1 mutant (C320S OTUD1 mutant). We also found that wild-type OTUD1 upregulated p21 and Mdm2 expression but inactive OTUD1 mutant did not. Furthermore, OTUD1 significantly suppressed colony formation. Next, we confirmed that OTUD1 overexpression increased the cleavage of caspase-3 and PARP and subsequently increased apoptosis. Together, these results suggest that OTUD1 is a novel regulator of p53 stability and activity.

Trim13 Potentiates Toll-Like Receptor 2-Mediated Nuclear Factor κB Activation via K29-Linked Polyubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6.

Ubiquitination is a versatile post-translational modification involved in nuclear factor-κB (NF-κB) activation of Toll-like receptor (TLR) signaling. Here, we demonstrated that Trim13, an E3 ubiquitin ligase, is up-regulated in macrophages upon stimulation with TLR2 ligand. Knockdown of Trim13 attenuated TLR2-mediated production of cytokines/chemokines and formation of foam cells as well as activation of NF-κB. Trim13 interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6) and potentiates NF-κB activity via ubiquitination of TRAF6. Overexpression of inactive mutant (C10/13A) or really interesting new gene (RING) deletion mutant of Trim13 did not potentiate ubiquitination of TRAF6 or activation of NF-κB. These results suggest that the effects of Trim13 are dependent on its E3 ligase activity. Trim13 used K29-linked polyubiquitin chains for TRAF6 ubiquitination to promote NF-κB activity and thus potentiated activation of TLR2-mediated immune responses. Our data identify Trim13 as a positive regulator of NF-κB activation and suggest that K29-linked polyubiquitination is a specific ubiquitin-linked pattern involved in the control of TLR2 signaling.

Anticancer effect of luteolin is mediated by downregulation of TAM receptor tyrosine kinases, but not interleukin-8, in non-small cell lung cancer cells.

TAM receptor tyrosine kinases (RTKs), Tyro3, Axl and MerTK, transduce diverse signals responsible for cell survival, growth, proliferation and anti-apoptosis. In the present study, we demonstrated the effect of luteolin, a flavonoid with antioxidant, anti-inflammatory and anticancer activities, on the expression and activation of TAM RTKs and the association with its cytotoxicity in non-small cell lung cancer (NSCLC) cells. We observed the cytotoxic effect of luteolin in parental A549 and H460 cells as well as in cisplatin-resistant A549/CisR and H460/CisR cells. Exposure of these cells to luteolin also resulted in a dose­dependent decrease in clonogenic ability. Next, luteolin was found to decrease the protein levels of all three TAM RTKs in the A549 and A549/CisR cells in a dose­dependent manner. In a similar manner, in H460 and H460/CisR cells, the protein levels of Axl and Tyro3 were decreased following luteolin treatment. In addition, Axl promoter activity was decreased by luteolin, indicating that luteolin suppresses Axl expression at the transcriptional level. We next found that luteolin abrogated Axl phosphorylation in response to growth arrest-specific 6 (Gas6), its ligand, implying the inhibitory effect of luteolin on Gas6-induced Axl activation. Ectopic expression of Axl was observed to attenuate the antiproliferative effect of luteolin, while knockdown of the Axl protein level using a gold nanoparticle-assisted gene delivery system increased its cytotoxicity. In contrast to the inhibitory effect of luteolin on the expression of TAM RTKs, interleukin-8 (IL-8) production was not decreased by luteolin in H460 and H460/CisR cells, while IL-8 production/cell was increased. Collectively, our data suggest that TAM RTKs, but not IL-8, are promising therapeutic targets of luteolin to abrogate cell proliferation and to overcome chemoresistance in NSCLC cells.

TRIF is a regulator of TLR2-induced foam cell formation.

The activation of toll-like receptor 2 (TLR2) stimulates foam cell formation, which is a key early event in the process of atherosclerosis. In the present study, the role of toll/interleukin-1 receptor-domain-containing adaptor-inducing interferon-ß (TRIF) in TLR2-mediated foam cell formation was investigated, and the importance of monocyte chemoattractant protein­1 (MCP­1), tissue factor (TF) and lectin­like oxidized low­density lipoprotein receptor­1 (Lox­1) were examined. Treatment of Raw 264.7 cells with the TLR2 agonist. Pam3CSK4, increased the gene expression of TRIF in a time­dependent manner (RT­PCR). The induced gene expression of TRIF stimulated by TLR2 was not observed in TLR2­knockout mice­derived bone marrow­derived macrophages (BMDMs). Pam3CSK4 increased the mRNA expression of TRIF in the wild­type BMDMs, but not in the TLR2­knockout BMDMs. Knockdown of the expression of TRIF using small interfering RNA decreased Pam3CSK4­induced foam cell formation (combination of oil­red O and hematoxylin staining), suggesting a role of TRIF. Stimulation of TLR2 increased the expression levels of various genes, which are known to control atherosclerosis, including MCP­1, TF and Lox­1. The knockdown of TRIF also attenuated the Pam3CSK4­induced expression of these genes. In addition, a reduction in TRIF affected the Pam3CSK4­induced protein expression of MCP­1 (EIA). Taken together, the results of the present study suggested that TRIF regulated foam cell formation via regulation of the expression levels of MCP­1, TF and Lox­1.

Regulation of PHLDA1 Expression by JAK2-ERK1/2-STAT3 Signaling Pathway.

Toll-like receptor 2 (TLR2)-mediated signaling cascades and gene regulation are mainly involved in diseases, such as immunity and inflammation. In this study, microarray analysis was performed using bone marrow-derived macrophages (BMDM) and Raw 264.7 cells to identify novel proteins involved in the TLR2-mediated cellular response. We found that pleckstrin homology-like domain family, member 1 (PHLDA1) is a novel gene up-regulated by TLR2 stimulation and determined the unique signaling pathway for its expression. Treatment with TLR2 agonist Pam3 CSK4 increased mRNA, protein, and fluorescence staining of PHLDA1. Induction of PHLDA1 by TLR2 stimulation disappeared from TLR2 KO mice-derived BMDM. Among janus kinase (JAK) family members, JAK2 was involved in TLR2-stimulated PHLDA1 expression. Signal transducer and activator of transcription 3 (STAT3) also participated in PHLDA1 expression downstream of the JAK2. Interestingly, ERK1/2 was an intermediate between JAK2 and STAT3. In silico analysis revealed the presence of highly conserved γ-activated sites within mouse PHLDA1 promoter and confirmed the JAK2-STAT3 pathway is important to Pam3 CSK4 -induced PHLDA1 transcription. These findings suggest that the JAK2-ERK1/2-STAT3 pathway is an important signaling pathway for PHLDA1 expression and that these proteins may play a critical role in eliciting TLR2-mediated immune and inflammatory response.

Curcumin-induced downregulation of Axl receptor tyrosine kinase inhibits cell proliferation and circumvents chemoresistance in non-small lung cancer cells.

Lung cancer is still in the first place in terms of both incidence and mortality. In the present study, we demonstrated the effect of curcumin, a phytochemical of the plant Curcuma longa, on expression and activation of Axl receptor tyrosine kinase (RTK) which plays an important role in cell survival, proliferation and anti-apoptosis. Curcumin treatment of non-small cell lung cancer (NSCLC) A549 and H460 cells, was found to decrease Axl protein as well as mRNA levels in a dose- and time-dependent manner. Axl promoter activity was also reduced by curcumin, indicating that curcumin downregulates Axl expression at the transcriptional level. Moreover, Axl phosphorylation in response to binding of its ligand, Gas6, was abrogated by curcumin, suggesting the inhibitory effect of curcumin on Gas6-induced Axl activation. We next found cytotoxic effect of cucumin on both the parental A549 and H460 cells, and their variants which are resistant to cisplatin (A549/CisR and H460/CisR) and paclitaxel (A549/TR and H460/TR). Exposure of these cells to curcumin resulted in dose-dependent decline of cell viability and clonogenic ability. It is further observed that the anti-proliferative effect of curcumin on A549 cells overexpressing Axl protein was reduced, while that on H460 cells transfected Axl specific siRNA was augmented, confirming that curcumin inhibits cell proliferation via downregulation of Axl expression. In addition, curcumin was found to cause the induction of p21, a cyclin-dependent kinase inhibitor, and reduction of X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptotic molecule, in parental H460 cells as well as chemoresistant cells, H460/CisR and H460/TR. Taken together, our data imply that Axl RTK is a novel target of curcumin through which it exerts anti-proliferative effect in both parental and chemoresistant NSCLC cells.

TLR2 deficiency attenuates skeletal muscle atrophy in mice.

Oxidative stress and inflammation are associated with skeletal muscle atrophy. Because the activation of toll-like receptor (TLR) 2 induces oxidative stress and inflammation, TLR2 may be directly linked to skeletal muscle atrophy. This study examined the role of TLR2 in skeletal muscle atrophy in wild-type (WT) and TLR2 knockout (KO) mice. Immobilization for 2 weeks increased the expression of cytokine genes and the levels of carbonylated proteins and nitrotyrosine in the skeletal muscle, but these increases were lower in the TLR2 KO mice. Muscle weight loss and a reduction in treadmill running times induced by immobilization were also attenuated in TLR2 KO mice. Furthermore, immobilization increased the protein levels of forkhead box O 1/3, atrogin-1 and muscle ring finger 1 in the WT mice, which was attenuated in TLR2 KO mice. In addition, immobilization-associated increases in ubiquitinated protein levels were lower in the TLR2 KO mice. Immobilization increased the phosphorylation of Akt and p70S6K similarly in WT and KO mice. Furthermore, cardiotoxin injection into the skeletal muscle increased the protein levels of atrogin-1, interleukin-6, and nitrotyrosine and increased the levels of ubiquitinated proteins, although these levels were increased to a lesser extent in TLR2 KO mice. These results suggest that TLR2 is involved in skeletal muscle atrophy, and the inhibition of TLR2 offers a potential target for preventing skeletal muscle atrophy.

RGS2 suppresses breast cancer cell growth via a MCPIP1-dependent pathway.

Regulator of G protein signaling 2 (RGS2) is a member of a family of proteins that functions as a GTPase-activating protein (GAP) for Gα subunits. RGS2 mRNA expression is lower in breast cancerous tissues than in normal tissues. In addition, expression of RGS2 is also lower in MCF7 (cancerous breast cells) than in MCF10A (normal breast cells). Here we investigated whether RGS2 inhibits growth of breast cancer cells. RGS2 overexpression in MCF7 cells inhibited epidermal growth factor- or serum-induced proliferation. In HEK293T cells expressing RGS2, cell growth was also significantly suppressed (In addition, exogenous expression of RGS2 in HEK293T cells resulted in the significant suppression of cell growth). These results suggest that RGS2 may have a tumor suppressor function. MG-132 treatment of MCF7 cells increased endogenous or exogenous RGS2 levels, suggesting a post-transcriptional regulatory mechanism that controls RGS2 protein levels. RGS2 protein was degraded polyubiquitinated the K71 residue, but stabilized by deubiquitinase monocyte chemotactic protein-induced protein 1 (MCPIP1), and not affected by dominant negative mutant (C157A) of MCPIP1. Gene expression profiling study showed that overexpression of RGS2 decreased levels of testis specific Y encoded like protein 5 (TSPYL5), which plays a causal role in breast oncogenesis. TSPYL5 protein expression was low in MCF10A and high in MCF7 cells, showing the opposite aspect to RGS2 expression. Additionally, RGS2 or MCPIP1 overexpression in MCF7 cells decreased TSPYL5 protein level, indicating that RGS2 stabilized by MCPIP1 have diminished TSPYL5 protein levels, thereby exerting an inhibitory effect of breast cancer cell growth.

Inhibitory effects of (-)-loliolide on cellular senescence in human dermal fibroblasts.

Cellular senescence influences tumor suppression and progress, tissue repair and regeneration, tissue and organismal aging, and age-related diseases. Aging intervention might be an advantageous target for prevention and treatment of diverse age-related diseases. In this study, we investigated whether (-)-loliolide purified from the crude extract of Polygonum aviculare exerted inhibitory activity against cellular senescence in human dermal fibroblasts (HDFs). (-)-Loliolide diminished senescence-associated ß-galactosidase activity (SA-ß-gal), the level of p21 protein, and the level of reactive oxygen species in senescent cells induced by adriamycin treatment. (-)-Loliolide also attenuated SA-ß-gal activity in HDFs under replicative senescence. These findings imply that (-)-loliolide rescues cellular senescence in HDFs and might be useful for the development of dietary supplements or cosmetics that ameliorate tissue aging or age-associated diseases.

Quercetin-3-O-ß-D-glucuronide isolated from Polygonum aviculare inhibits cellular senescence in human primary cells.

Cellular senescence is known to contribute to tissue aging, a variety of age-related diseases, tissue regeneration, and cancer. Therefore, aging intervention might be useful for prevention of aging as well as age-related disease. In this study, we investigated compounds from Polygonum aviculare to determine if they inhibited cellular senescence in human primary cells, human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs). Ten compounds from P. aviculare were purified and their inhibitory effects on adriamycin-induced cellular senescence were measured by observing senescence-associated ß-galactosidase (SA-ß-gal) activity and reactive oxygen species. Among them, compound 9 (quercetin-3-O-ß-D-glucuronide) showed inhibitory effects against cellular senescence in HDFs and HUVECs treated with adriamycin. Additionally, compound 9 rescued replicative senescence in HDFs and HUVECs. These data imply that compound 9 represses cellular senescence in human primary cells and might be useful for the development of dietary supplements or cosmetics that ameliorate tissue aging or aging-associated diseases.

Inhibitory effects of juglanin on cellular senescence in human dermal fibroblasts.

Cellular senescence contributes to tissue and organismal aging, tumor suppression and progress, tissue repair and regeneration, and age-related diseases. Thus, aging intervention might be a promising target for treatment and prevention of diverse age-related diseases. In the present study, we investigated whether juglanin purified from the crude extract of Polygonum aviculare exerted inhibitory activity against cellular senescence in human dermal fibroblasts (HDFs). Juglanin decreased senescence-associated ß-galactosidase activity (SA-ß-gal) and the level of reactive oxygen species in senescent cells induced by adriamycin treatment. Juglanin also repressed SA-ß-gal activity in HDFs under replicative senescence. These results suggest that juglanin represses cellular senescence in HDFs and might be useful for the development of dietary supplements or cosmetics that alleviate tissue aging or age-related diseases.