Tonic ubiquitylation controls T‐cell receptor:CD3 complex expression during T‐cell development

Expression of the T‐cell receptor (TCR):CD3 complex is tightly regulated during T‐cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3ε proline‐rich sequence, Lck, c‐Cbl, and SLAP, which collectively trigger the dynamin‐dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3ζ‐monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T‐cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T‐cell development.     

Co‐expression of NF‐κB‐p65 and phosphorylated NF‐κB‐p105 is associated with poor prognosis in surgically resectable non‐small cell lung cancer

Nuclear factor‐kappa B (NF‐κB) as a prognostic marker remains unclear in non‐small cell lung cancer (NSCLC). Here, we studied NF‐κB‐p65 (p65) expression and phosphorylated NF‐κB‐p105 (p‐p105) expression in NSCLC and correlated the finding with overall survival (OS) and clinicopathological features. A total of 186 archival samples from patients with surgically resectable NSCLC were probed with p65 and p‐p105 (Ser 932). The p65‐positive expression and p‐p105‐positive expression were defined as distinct nuclear p65 and cytoplasmic p‐p105 labelling in at least 1% of tumour cells, respectively. The positive staining of p65 alone, p‐p105 alone and co‐expression of p65 and p‐p105 were observed in 61 (32.8%), 90 (48.4%) and 35 (18.8%) patients, respectively. Co‐expression of p65 and p‐p105 but not of either p65 or p‐p105 alone was associated with a poor prognosis. Patients with co‐expression of p65 and p‐p105 had a shorter OS than others, median OS 26.5 months versus 64.1 months, HR 1.85 (95% CI: 1.18–2.91), P = 0.007. There was no statistically significant association between clinicopathological characteristics and either p65 or p‐p105 alone or co‐expression of p65 and p‐p105. This indicates that co‐expression of p65 and p‐p105 was a poor prognostic factor, and pathologic studies of NF‐κB expression could include multiple pathway components in NSCLC.     

Thrombin stimulates RPE cell motility by PKC‐ζ‐ and NF‐κB‐dependent gene expression of MCP‐1 and CINC‐1/GRO chemokines

Retinal pigment epithelial cells (RPE) are the major cell type involved in the pathogenesis of proliferative vitreoretinopathy (PVR), which involves the epithelial‐mesenchymal transition, proliferation, and directional migration of transformed RPE cells to the vitreous upon RPE exposure to serum components, thrombin among them. Although the aqueous humor and vitreous of PVR patients contain high levels of chemokines, their possible involvement in PVR development has not been explored. We here analyzed the effect of thrombin on chemokine gene expression and its correlation with RPE cell migration using rat RPE cells in culture as a model system. We demonstrated that thrombin induces RPE cell migration through the dose‐dependent stimulation of MCP1 and GRO expression/release, and the autocrine activation of CXCR‐2 and CCR‐2 chemokine receptors. Whereas inhibition of CXCR2 by Sb‐225002 and of CCR2 by Rs‐504393 partially prevented hirudin‐sensitive cell migration, the joint inhibition of these receptors abolished thrombin effect, suggesting the contribution of distinct but coincident mechanisms. Thrombin effects were not modified by Ro‐32‐0432 inhibition of conventional/novel PKC isoenzymes or by the MAPkinase pathway inhibitor U0126. MCP1 and GRO expression/secretion, and cell migration were completely prevented by the inhibitory PKC‐ζ pseudosubstrate and by the nuclear factor‐kappa B (NF‐κB) inhibitor BAY11‐7082, but not by wortmannin inhibition of PI3K. Results show that signaling pathways leading to RPE cell migration differ from the MEK–ERK–PI3K‐mediated promotion RPE of cell proliferation, both of which concur at the activation of PKC‐ζ. J. Cell. Biochem. © 2010 Wiley‐Liss, Inc.     

Long non‐coding RNA DBH‐AS1 promotes cancer progression in diffuse large B‐cell lymphoma by targeting FN1 via RNA‐binding protein BUD13

Diffuse large B‐cell lymphoma (DLBC) is a subtype of lymphoma with the worst prognosis. Existing treatment methods are not effective enough due to its high occurrence of metastasis. Therefore, identification of effective therapeutic targets is becoming increasingly important. In this research, long non‐coding RNA dopamine β hydroxylase antisense RNA 1 (DBH‐AS1) was found to be upregulated in DLBC tissues and cells. Knockdown of DBH‐AS1 suppressed the proliferation, migration, and invasion of cancer cells. Afterwards, RNA‐binding protein BUD13 homolog (BUD13) was found to be upregulated in cancer tissues and cells while binding to DBH‐AS1. Fibronectin 1 (FN1) was the downstream messenger RNA (mRNA) of BUD13. FN1 was upregulated in DLBC and was positively correlated with DBH‐AS1. Further rescue assays proved that DBH‐AS1 mediated FN1 expression by recruiting BUD13. In the meantime, BUD13 stabilized FN1 mRNA to promote FN1 expression. In this way, DBH‐AS1/BUD13/FN1 axis was confirmed. A set of rescue assays proved that DBH‐AS1 regulated DLBC progression via BUD13 and FN1. The function and mechanism of DBH‐AS1 were investigated for the first time in DLBC. DBH‐AS1 might become a therapeutic target in lymphoma treatment in the future.     

Down‐regulation of Notch‐1 and Jagged‐1 inhibits prostate cancer cell growth,migration and invasion,and induces apoptosis via inactivation of Akt,mTOR, and NF‐κB signaling pathways

Notch signaling is involved in a variety of cellular processes, such as cell fate specification, differentiation, proliferation, and survival. Notch‐1 over‐expression has been reported in prostate cancer metastases. Likewise, Notch ligand Jagged‐1 was found to be over‐expressed in metastatic prostate cancer compared to localized prostate cancer or benign prostatic tissues, suggesting the biological significance of Notch signaling in prostate cancer progression. However, the mechanistic role of Notch signaling and the consequence of its down‐regulation in prostate cancer have not been fully elucidated. Using multiple cellular and molecular approaches such as MTT assay, apoptosis assay, gene transfection, real‐time RT‐PCR, Western blotting, migration, invasion assay and ELISA, we found that down‐regulation of Notch‐1 or Jagged‐1 was mechanistically associated with inhibition of cell growth, migration, invasion and induction of apoptosis in prostate cancer cells, which was mediated via inactivation of Akt, mTOR, and NF‐κB signaling. Consistent with these results, we found that the down‐regulation of Notch‐1 or Jagged‐1 led to decreased expression and the activity of NF‐κB downstream genes such as MMP‐9, VEGF, and uPA, contributing to the inhibition of cell migration and invasion. Taken together, we conclude that the down‐regulation of Notch‐1 or Jagged‐1 mediated inhibition of cell growth, migration and invasion, and the induction of apoptosis was in part due to inactivation of Akt, mTOR, and NF‐κB signaling pathways. Our results further suggest that inactivation of Notch signaling pathways by innovative strategies could be a potential targeted approach for the treatment of metastatic prostate cancer. J. Cell. Biochem. 109: 726–736, 2010. © 2010 Wiley‐Liss, Inc.     

p97/VCP promotes Cullin‐RING‐ubiquitin‐ligase/proteasome‐dependent degradation of IκBα and the preceding liberation of RelA from ubiquitinated IκBα

Cullin‐RING‐ubiquitin‐ligase (CRL)‐dependent ubiquitination of the nuclear factor kappa B (NF‐κB) inhibitor IκBα and its subsequent degradation by the proteasome usually precede NF‐κB/RelA nuclear activity. Through removal of the CRL‐activating modification of their cullin subunit with the ubiquitin (Ub)‐like modifier NEDD8, the COP9 signalosome (CSN) opposes CRL Ub‐ligase activity. While RelA phosphorylation was observed to mediate NF‐κB activation independent of Ub‐proteasome‐pathway (UPP)‐dependent turnover of IκBα in some studies, a strict requirement of the p97/VCP ATPase for both, IκBα degradation and NF‐κB activation, was reported in others. In this study, we thus aimed to reconcile the mechanism for tumour necrosis factor (TNF)‐induced NF‐κB activation. We found that inducible phosphorylation of RelA is accomplished in an IKK‐complex‐dependent manner within the NF‐κB/RelA‐IκBα‐complex contemporaneous with the phosphorylation of IκBα, and that RelA phosphorylation is not sufficient to dissociate NF‐κB/RelA from IκBα. Subsequent to CRL‐dependent IκBα ubiquitination functional p97/VCP is essentially required for efficient liberation of (phosphorylated) RelA from IκBα, preceding p97/VCP‐promoted timely and efficient degradation of IκBα as well as simultaneous NF‐κB/RelA nuclear translocation. Collectively, our data add new facets to the knowledge about maintenance of IκBα and RelA expression, likely depending on p97/VCP‐supported scheduled basal NF‐κB activity, and the mechanism of TNF‐induced NF‐κB activation.     

Alpha B‐crystallin promotes the invasion and metastasis of gastric cancer via NF‐κB‐induced epithelial‐mesenchymal transition

Alpha B‐crystallin (CRYAB) is overexpressed in a variety of cancers. However, little is known about its specific function and regulatory mechanism in gastric cancer. Here, we first explore the role of CRYAB in gastric cancer progression and metastasis. The expression of CRYAB was determined by western blot and immunohistochemistry in gastric cancer tissues. Besides, methods including stably transfected against CRYAB into gastric cancer cells, western blot, migration and invasion assays in vitro and metastasis assay in vivo were also conducted. The expression of CRYAB is up‐regulated in gastric cancer tissues compared with matched normal tissues. High expression level of CRYAB is closely correlated with cancer metastasis and shorter survival time in patients with gastric cancer. Additionally, CRYAB silencing significantly suppresses epithelial‐mesenchymal transition (EMT), migration and invasion of gastric cancer cells in vitro and in vivo, whereas CRYAB overexpression dramatically reverses these events. Mechanically, CRYAB facilitates gastric cancer cells invasion and metastasis via nuclear factor‐κ‐gene binding (NF‐κB)‐regulated EMT. These findings suggest that CRYAB expression predicts a poor prognosis in patients with gastric cancer. Besides, CRYAB contributes to gastric cancer cells migration and invasion via EMT, mediated by the NF‐κB signalling pathway, thus possibly providing a novel therapeutic target for gastric cancer.     

TNIP1‐mediated TNF‐α/NF‐κB signalling cascade sustains glioma cell proliferation

As a malignant tumour of the central nervous system, glioma exhibits high incidence and poor prognosis. Although TNIP1 and the TNF‐α/NF‐κB axis play key roles in immune diseases and inflammatory responses, their relationship and role in glioma remain unknown. Here, we revealed high levels of TNIP1 and TNF‐α/NF‐κB in glioma tissue. Glioma cell proliferation was activated with TNF‐α treatment and showed extreme sensitivity to the TNF receptor antagonist. Furthermore, loss of TNIP1 disbanded the A20 complex responsible for IκB degradation and NF‐κB nucleus translocation, and consequently erased TNFα‐induced glioma cell proliferation. Thus, our investigation uncovered a vital function of the TNIP1‐mediated TNF‐α/NF‐κB axis in glioma cell proliferation and provides novel insight into glioma pathology and diagnosis.     

The possible roles of B‐cell novel protein‐1 (BCNP1) in cellular signalling pathways and in cancer

B‐cell novel protein‐1 (BCNP1) or Family member of 129C (FAM129C) was identified as a B‐cell‐specific plasma‐membrane protein. Bioinformatics analysis predicted that BCNP1 might be heavily phosphorylated. The BCNP1 protein contains a pleckstrin homology (PH) domain, two proline‐rich (PR) regions and a Leucine Zipper (LZ) domain suggesting that it may be involved in protein‐protein interactions. Using The Cancer Genome Atlas (TCGA) data sets, we investigated the correlation of alteration of the BCNP1 copy‐number changes and mutations in several cancer types. We also investigated the function of BCNP1 in cellular signalling pathways. We found that BCNP1 is highly altered in some types of cancers and that BCNP1 copy‐number changes and mutations co‐occur with other molecular alteration events for TP53 (tumour protein P53), PIK3CA (Phosphatidylinositol‐4,5‐Bisphosphate 3‐Kinase, Catalytic Subunit Alpha), MAPK1 (mitogen‐activated protein kinase‐1; ERK: extracellular signal regulated kinase), KRAS (Kirsten rat sarcoma viral oncogene homolog) and AKT2 (V‐Akt Murine Thymoma Viral Oncogene Homolog 2). We also found that PI3K (Phoshoinositide 3‐kinase) inhibition and p38 MAPK (p38 mitogen‐activated protein kinase) activation leads to reduction in phosphorylation of BCNP1 at serine residues, suggesting that BCNP1 phosphorylation is PI3K and p38MAPK dependent and that it might be involved in cancer. Its degradation depends on a proteasome‐mediated pathway.     

Disabled‐1 is down‐regulated in clinical breast cancer and regulates cell apoptosis through NF‐κB/Bcl‐2/caspase‐9

Disabled‐1 (Dab1) is best known as an adaptor protein regulating neuron migration and lamination during development. However, the exact function of Dab1 in breast cancer is unknown. In this study, we examined the expression of Dab1 in 38 breast cancer paraffin sections, as well as 60 paired frozen breast cancer and their adjacent tissues. Our results showed that Dab1 was reduced in breast cancer, and its compromised expression correlated with triple negative breast cancer phenotype, poor differentiation, as well as lymph node metastasis. Functional analysis in breast cancer cell lines demonstrated that Dab1 promoted cell apoptosis, which, at least partially, depended on its regulation of NF‐κB/Bcl‐2/caspase‐9 pathway. Our study strongly suggests that Dab1 may be a potential tumour suppressor gene in breast cancer.     

Pinitol attenuates LPS‐induced pneumonia in experimental animals: Possible role via inhibition of the TLR‐4 and NF‐κB/IκBα signaling cascade pathway

Pneumonia is a chronic disorder of the respiratory system associated with worsening quality of life and a significant economic burden. Pinitol, a plant cyclic polyol, has been documented for immune‐inflammatory potential. The aim of present investigation was to evaluate the potential and possible mechanism of action of pinitol against lipopolysaccharide (LPS)‐induced pneumonia in the experimental animal model. Pneumonia was induced in Sprague‐Dawley rats by intratracheal administration of LPS (2 mg/kg). Animals were treated with either vehicle or dexamethasone or pinitol (5 or 10 or 20 mg/kg). Potential of pinitol against LPS‐induced pulmonary insult was assessed based on behavioral, biochemical, molecular, and ultrastructural studies. Intratracheal instillation of LPS induced significant (P < .05) inflammatory infiltration in bronchoalveolar lavage fluid (BALF) and lung tissue reflected by elevated pleural effusion volume, lung edema, BALF polymorphonuclear leukocytes count and lung myeloperoxidase levels, which was attenuated by pinitol (10 and 20 mg/kg) administration. Pinitol also markedly (P < .05) inhibited LPS‐induced alterations in electrocardiographic, hemodynamic changes, right ventricular, and lung function tests. The LPS‐induced downregulated nuclear factor erythroid 2–related factor 2 (Nrf‐2) and heme oxygenase‐1 (HO‐1), whereas upregulated transforming growth factor‐β (TGF‐β), tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3), and inducible nitric oxide synthase (iNOs) lung messenger RNA expressions were significantly (P < .05) inhibited by pinitol. Western blot analysis suggested pinitol markedly (P < .05) decreased nuclear factor‐κB (NF‐κB), inhibitor of nuclear factor κB (IkBα), toll‐like receptor 4 (TLR‐4), and cyclooxygenase‐II (COX‐II) protein expressions in the lung. These findings were further supported by histological and ultrastructural analyses of lung tissue that show pinitol significantly (P < .05) ameliorates LPS‐induced aberrations in lung tissue. In conclusion, pinitol attenuated LPS‐induced pneumonia via inhibition of TLR‐4 to downregulate the NF‐κB/IκBα signaling cascade and thus ameliorated the production of proinflammatory cytokines (TNF‐α, ILs, NLRP3, and TGF‐β), inflammatory mediators (COX‐II and iNOs) and elevated oxidative stress (Nrf‐2 and HO‐1).