Jab1 Knockdown Research Articles

Reactive Oxygen Species-Mediated c-Jun Activation Domain-Binding Protein-1 (Jab1) Promotes Immune Escape in Hyperleukocytic Acute Myeloid Leukemia

Background: Hyperleukocytic acute myeloid leukemia (HL-AML) is a group of clonal hematopoietic stem cell diseases. Our previous studies have confirmed that Jab1 mediated by reactive oxygen species (ROS) plays a role in the pathogenesis of AML as a transcription coactivator of AP-1/JUN. Here, we reveal the specific regulatory mechanism of immune escape in HL-AML by Jab1, which serves as a new therapeutic target for HL-AML. Methods: The relationship between Jab1 expression and immune cell subtypes in clinical bone marrow samples were analyzed by RT-qPCR. Lentiviral knockout of Jab1 was combined with transcriptomics and chromatin co-precipitation to evaluate the transcriptional regulatory mechanisms involved, and bioinformatics to identify molecular interactions. The changes of each immunophenotype were analyzed by flow cytometry. Results: Compared with healthy controls and AML, Jab1 was highly expressed in HL-AML and was associated with a poor prognosis. However, in HL-AML patients, the proportion of CD8+ T cells was significantly decreased, while the proportion of regulatory T cells was slightly increased, and the secretion of cytokines IL-2, IL-4, IFN-γ, and IFN-α decreased. In H2O2-induced oxidative stress, ROS levels promote the entry of c-Jun into the nucleus, while Jab1 knockdown blocks c-Jun nuclear expression and transcriptional activity. Mechanically, Jab1 regulates leukemic immunosuppressive target LILRB4 rather than PDL1/PDL2. As a coactivator of transcription factor c-Jun, Jab1 cannot directly regulate the transcription of LILRB4 but enhances the stability of LILRB4 mRNA. By high-throughput screening, it was found that N6-methyladenosine modification directly recognized the LILRB4 mRNA site. Finally, it was concluded that Jab1/IGF2BPs/LILRB4 signal axis was involved in disease progression in HL-AML. Conclusions: We provided compelling evidence to demonstrate that Jab1 under oxidative stress is associated with tumorigenesis and poor prognosis of AML, which can serve as a target to develop therapeutics for cancer treatment.

Signaling of MK2 sustains robust AP1 activity for triple negative breast cancer tumorigenesis through direct phosphorylation of JAB1

Triple negative breast cancer (TNBC) cells are generally more invasive than estrogen receptor-positive (ER + ) breast cancer cells. Consistent with the importance of activator protein 1 (AP1) transcription factors in invasion, AP1 activity is much higher in TNBC lines than ER + lines. In TNBC cells, robust AP1 activity is facilitated by both ERK and p38MAPK signaling pathways. While ERK signaling pathway regulates AP1 activity by controlling the abundance of AP1 transcription factors, p38MAPK signaling pathway does it by enhancing AP1 binding to AP1 sites without altering their abundance. Here, we show that p38MAPK regulation of AP1 activity involves both MAPKAPK2 (MK2) and JAB1, a known JUN-binding protein. MK2 not only interacts with JAB1 but also directly phosphorylates JAB1 at Ser177 in TNBC cells. Interestingly, Ser177 phosphorylation does not affect JAB1 and JUN interaction. Instead, interfering with p38MAPK signaling pathway or introducing an S to A point mutation at Ser177 of JAB1 reduces JUN recruitment to the AP1 sites in cyclin D1, urokinase plasminogen activator (uPA) and uPA receptor promoters. Moreover, knockdown of JAB1 diminishes >60% of AP1 transcriptional activity in TNBC cells. Taken together, these results indicate that MK2-mediated phosphorylation of JAB1 facilitates JUN recruitment to AP1 sites, thus augmenting AP1 activity. In line with the role of JAB1 in AP1 activity, silencing JAB1 leads to dramatic reduction in TNBC cell growth, in vitro invasion and in vivo tumor outgrowth. This study suggests that the p38MAPK-MK2 signaling pathway promotes TNBC tumorigenesis by sustaining robust AP1 activity.

JAB1 Promotes High Glucose-Induced Inflammation and Extracellular Matrix Deposition in Glomerular Mesangial Cells by Regulating Angiopoietin-Like Protein 2

Diabetic or hyperglycaemic conditions stimulate the inflammatory response, excessive accumulation of extracellular matrix, and result in glomerulosclerosis, a scarring process of diabetic nephropathy. c-Jun activation domain-binding protein 1 (JAB1) functions as a regulator of pathways involved in cellular apoptosis and proliferation. The role of JAB1 in diabetic nephropathy was investigated in this study. Firstly, glomerular mesangial cells (GMCs) were treated with high glucose, and high glucose conditions induced up-regulation of JAB1 in the GMCs. Moreover, IL-6, TNF-α, MCP-1, and IL-1β were also elevated in high glucose-induced GMCs. Secondly, silencing of JAB1 reduced the levels of IL-6, TNF-α, MCP-1, and IL-1β in high glucose-induced GMCs. In addition, silencing of JAB1 attenuated the high glucose-induced decrease of superoxide dismutase (SOD) and the increase of reactive oxygen species (ROS) and malondialdehyde (MDA). The increased TGF-β1, collagen I, collagen IV, and fibronectin levels in high glucose-induced GMCs were restored by knockdown of JAB1. Thirdly, angiopoietin-like protein 2 (ANGPTL2) expression was reduced by JAB1. Over-expression of ANGPTL2 weakened the JAB1 silence-induced decrease of IL-6, TNF-α, MCP-1, IL-1β, TGF-β1, collagen I, collagen IV, and fibronectin. In conclusion, silencing of JAB1 reduced extracellular matrix deposition and suppressed inflammation in high glucose-induced GMCs through down-regulation of ANGPTL2.

JAB1 promotes palmitate-induced insulin resistance via ERK pathway in hepatocytes.

Insulin resistance (IR) is the primary pathological mechanism underlying Type 2 diabetes mellitus (T2DM). Many researches have reported the relationship between chronic inflammation and IR, while the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway is rapidly activated in inflammatory conditions. However, the functional role of ERK1/2 in IR remains to be identified. We here reported that C-Jun activation domain-binding protein-1 (JAB1) was upregulated in IR. In addition, we showed that depletion of JAB1 led to recovery of insulin sensitivity. Given the fact that JAB1 played as an activator of ERK1/2, we assumed JAB1 was involved in IR through ERK pathway. So we assessed the effects of JAB1 knockdown in palmitate acid (PA) treated HepG2 cells. Importantly, JAB1 siRNA blocked the effect of PA-induced activation of ERK1/2. Furthermore, silencing of JAB1 could reduce the release of inflammatory factors, facilitate hepatic glucose uptake and improve lipid metabolism. All these data implicated that JAB1 knockdown might alleviate PA-induced IR through ERK pathway in hepatocytes.

MicroRNA-17 acts as a tumor chemosensitizer by targeting JAB1/CSN5 in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is the breast cancer subtype with the poorest prognosis. Evidence indicates that aberrant JAB1/CSN5 expression is associated with advanced tumor stage and poor prognosis in breast cancer. In this study, we evaluated expression of JAB1 in TNBC and potential mechanisms regulating this expression. We found that miR-17 expression was lower in TNBC than in normal breast tissue, and miR-17 expression in patients with TNBC was associated with a good prognosis. Furthermore, JAB1 expression was regulated by miR-17 in TNBC cells, and mice with miR-17-overexpressing tumors had less tumor growth and lower tumor JAB1 expression than control mice. We also demonstrated that miR-17 suppressed JAB1's oncogenic function, leading to tumor growth inhibition and sensitizing TNBC cells to chemotherapy treatment. JAB1 knockdown in TNBC cells mimicked the effect of miR-17 overexpression and led to significant decreases in cell proliferation, colony formation, and migration, increased p27 expression, and enhanced cisplatin sensitivity. Our findings suggest that miR-17 acts as a tumor suppressor by directly targeting JAB1 in TNBC; this may lead to novel therapeutic targets and strategies for treating TNBC patients.

Jab1 Silencing Inhibits Proliferation and Sensitizes to Cisplatin in Biliary Tract Cancer

PurposeJab1 is a coactivator of c-Jun that enhances the transcriptional function of c-Jun. Jab1 is frequently overexpressed in various cancers and is associatedwith poor prognosis of cancer patients. Thus, Jab1 could be a potential therapeutic target in cancer. However, the role of Jab1 in biliary tract cancer (BTC) has not been studied.Materials and MethodsWe performed in vitro and in vivo experiments to evaluate the therapeutic potential ofJab1 inhibition in BTC.ResultsAmong 8 BTC cell lines, many showed higher Jab1 expression levels. In addition, Jab1 silencing by siRNA increased p27 expression levels. SNU478 and HuCCT-1 cells exhibited profound Jab1 knockdown and increased p27 expression by Jab1-specific siRNA transfection. Jab1 silencing induced anti-proliferative and anti-migratory effects and resulted in G1 cell cycle arrest in SNU478 and HuCCT-1 cells. In addition, Jab1 silencing potentiated the anti-proliferative and anti-migratory effects of cisplatin by increasing DNA damage. Interestingly,Jab1 knockdown increased PTEN protein half-life, resulting in increased PTEN expression. In the HuCCT-1 mouse xenograft model, stable knockdown of Jab1 by shRNA also showed anti-proliferative effects in vivo, with decreased Ki-67 expression and AKT phosphorylation and increased Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling and p27 expression.ConclusionJab1 knockdown demonstrated anti-proliferative and anti-migratory effects in BTC cells by increasing DNA damage and stabilizing PTEN, resulting in G1 cell cycle arrest. In addition, Jab1 silencing potentiated the anti-proliferative effects of cisplatin. Our data suggest that Jab1 may be a potential therapeutic target in BTC that is worthy of further investigations.

Interferon-τ regulates prostaglandin release in goat endometrial stromal cells via JAB1 - unfolded protein response pathway

Prostaglandins (PGs) are major products of the uterine endometrium, and they are critical for recognition of pregnancy in ruminants. During the peri-implantation period of pregnancy, interferon tau (IFN-τ) plays an important role in the regulation of endometrial PGs synthesis, but the underlying mechanisms remain poorly understood. In this work, the results demonstrated that IFN-τ increased the PGE2/PGF2α ratio, up-regulated the expression of JAB1 and activated the unfolded protein response (UPR). Knockdown of JAB1 reduced the PGE2/PGF2α ratio and inhibited the expression of UPR markers in endometrial stromal cells (ESCs) under IFN-τ treatment. Pre-treatment with endoplasmic reticulum (ER) stress activator thapsigargin (Tg) activated UPR and restored the PGE2/PGF2α ratio in shJAB1 groups under IFN-τ treatment. In conclusion, our results indicated that IFN-τ regulated the PGE2/PGF2α ratio via cooperation between JAB1 and UPR, and the reduction of JAB1 led to the down-regulation of the PGE2/PGF2α ratio, which inhibits UPR, and thus is harmful to early pregnancy. Activation of UPR could restore JAB1 reduction, resulting in a reduced PGE2/PGF2α ratio. These findings extend our understanding and may provide new insights into the mechanism of IFN-τ regulation of PG secretion in ESCs and the biological functions of JAB1 and UPR.

Abstract A24: The transcriptional regulator JAB1 is a novel regulator of osteosarcoma development

Abstract Osteosarcoma is the most common malignant primary bone tumor. Osteosarcoma patients with metastases have a survival rate of only 25%. Therefore, a greater understanding of the pathogenesis of osteosarcoma is needed for better diagnosis and treatment. An emerging player in tumorigenesis is the transcriptional cofactor JAB (also named CSN5, COPS5), through its effects on cell proliferation, differentiation, survival, and cell cycle progression. Our laboratory has recently shown that Jab1 plays critical roles in the successive steps of mouse skeletal development by affecting master developmental regulators Runx2 and Sox9 in a context-dependent manner. As an essential component of the highly conserved COP9 signalosome complex, JAB1 is over-expressed in many different types of cancers, including breast and lung, but has not been studied in bone cancer. Thus, the goal of this study is to determine the potential role of JAB1 in the pathogenesis of osteosarcoma. We first determined, by immunohistochemistry, that JAB1 was highly expressed in over 75% of fifty-one osteosarcoma patients’ biopsy samples. Next, to determine whether down-regulating JAB1 expression in osteosarcoma reduces tumorigenesis, we knocked down JAB1 expression in a highly metastatic osteosarcoma cell line 143B, using shRNA lentiviral vectors. The JAB1 knockdown cells exhibited significantly reduced cell proliferation, colony formation, and motility compared with the control cells. Therefore, these results suggest that down-regulating JAB1 expression in osteosarcoma might reduce tumorigenesis. We will determine whether the down-regulation of JAB1 in 143B cells reduces tumorigenesis in vivo in xenograft experiments. On the other hand, to study the consequence of JAB1 over-expression in vivo, we have generated Col1a1-JAB1 transgenic mouse lines using a well-characterized 2.3kb Col1a1 promoter to over-express JAB1 in osteoblasts specifically. Previous mouse genetic studies have shown that p53 is a critical factor in the development of osteosarcoma, and that JAB1 is a negative regulator of p53 activity in other cancers in cell lines. To test whether increased JAB1 over-expression promotes bone tumorigenesis in vivo in a p53-dependent manner, we crossed Cola1-JAB1 transgenic mice, which had no gross abnormality and no osteosarcoma occurrence, with p53+/- heterozygous mice. Impressively, our preliminary study so far indicates that significantly more Cola1-JAB1; p53+/- double transgenic mice developed solid tumors on limbs than p53+/- control mice at the 11-month time point. Thus, our study provided a novel mouse model to study the JAB1-p53 interaction in osteosarcoma development. Ultimately, new therapies based on controlling the JAB1-mediated tumorigenesis will open new avenues for treating osteosarcoma and other cancers. Citation Format: Lindsay Bashur, Robin Elliott, Guang Zhou. The transcriptional regulator JAB1 is a novel regulator of osteosarcoma development. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A24.

ROS-Mediated Jab1-Thioredoxin Interaction in Acute Monocytic Leukemia

SUMMARYIncreased production of reactive oxygen species (ROS) or an inefficient antioxidant system leads to oxidative stress, which influences hematopoietic cell function in acute myeloid leukemia (AML); however, the mechanisms of this stress remain poorly understood. Investigations were carried out on matched samples from the same 58 patients with AML-M5 (33 males and 25 females) who experienced a relapse after treatment. They had a median age of 43.90 ± 17.39 years (range, 14-77 years) at diagnosis and 44.86 ± 16.28 years at relapse. The mean white blood cell (WBC) counts were 16.58 x 109/L at diagnosis and 24.41 x 109/L at relapse (P < 0.05). Here, our studies showed that abnormally high ROS levels and low antioxidant enzyme capacity were characteristic of AML-M5 at both diagnosis and relapse. It is worth mentioning Jab1 and Trx protein levels were higher in all leukemia cell lines tested (THP-1, U937, and MOLM-13 cells), as well as in leukemic cells derived from AML-M5 patients, than in CD34+ cells from healthy donors (Figure 1A-D). A chi-square test revealed that patients with high Jab1 levels were less sensitive to Ara-c-based chemotherapy (Figure 1E). Consistently, high gene expression levels of thioredoxin (Trx) and Jab1 were associated with poor survival rates in AML-M5 patients(Figure 1F-H). Moreover, stimulating AML-M5 cells with a low concentration of hydrogen peroxide increased Jab1 and Trx expression. Additionally, depletion of Jab1 inhibited cellular proliferation and invasion, which was accompanied by a decrease in Trx expression.Mechanistically, we demonstrate that Jab1 controls a major antioxidative cellular molecule Trx by interacting with and enhancing its transcriptional levels through AP-1 binding sites. To assess whether Jab1 and Trx interact we performed co-immunoprecipitation assays and demonstrated that Jab1 and Trx proteins interacted specifically with each other using endogenous proteins or ectopically expressed tagged-proteins, in U937 and THP-1 cells (Figure 2A and B). Furthermore, our results demonstrate that Jab1 knockdown remarkably reduced Trx levels (Figure 2C). Inversely, Jab1 overexpression induced higher endogenous Trx levels (Figure 2D). However, neither Trx silencing nor Trx overexpression affected Jab1 levels. Thus, our findings indicate that Jab1 is an upstream regulator and positively controls Trx expression in AML-M5 cells. To further delineate whether Jab1 regulates transcriptionally Trx, we performed a luciferase reporter assays with the human Trx promoter. Our data demonstrated that transactivation of Trx expression is driven by Jab1 through the AP-1 binding site (Figure 2E-H). In conclusion, these results elucidate a novel Jab1 and Trx axis, which is a critical cellular process in the pathobiology of AML-M5 and could promotes AML-M5 relapse. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 1714: The role and mechanism of JAB1 as a therapeutic target in biliary tract cancer

Abstract Background: JAB1 (c-Jun activation domain binding protein-1) is a c-Jun coactivator, also known as COP9 signalosome subunit 5 (CSN5). Jab1 has an important role in cell proliferation and apoptosis. Overexpression of Jab1 is correlated with poor prognosis in various cancers. Biliary tract cancer (BTC) has a poor prognosis with a huge unmet medical need. The role of Jab1 has not been studied in BTC. We investigated the role and mechanism of Jab1 as a potential therapeutic target in BTC. Methods: We used 8 kinds of BTC cells and designed Jab1 siRNA. MTT assay and colony formation assay were done to determine growth inhibitory effect of Jab1 knockdown. Cell cycle analysis was done by FACS Calibur flow cytometer and cell migration was evaluated by wound healing assay. We used cycloheximide chase assay for measuring of protein half-life. Results: BTC cell lines showed high level of Jab1 expression. Among them, SNU478, SNU869 and SNU 1196 were indicated with especially higher level of Jab1 expression. Cell growth and proliferation of BTC cells were decreased by Jab1 knockdown. Depletion of Jab1 induced G1 arrest, as well as decreased CyclinD/CyclinA and increased p27. Cell migration was also inhibited by Jab1 knockdown. Inhibition of Jab1 showed the decrease of pSrc, pAkt. Interestingly, depletion of Jab1 led to the elevation of PTEN protein levels without change of PTEN mRNA levels. PTEN half-life was longer in Jab1 siRNA-transfected cells. Suppression of Jab1 increased the sensitivity of BTC cells to the cytotoxic chemotherapeutic agent. Conclusion: Suppression of Jab1 shows antitumor effects in BTC cells by inhibiting cell proliferation, migration, cell cycle and increase of chemosensitivity. Taken together, Jab1 might be a potential therapeutic target in BTC. Citation Format: Ah-Rong Nam, Kyo Hwa Kang, Ji Eun Park, Ju-Hee Bang, Ling Jin, Mei Hua Jin, Tae Yong Kim, Sae-Won Han, Sang-Hyun Song, Seock-Ah Im, Tae-You Kim, Do-Youn Oh, Yung-Jue Bang. The role and mechanism of JAB1 as a therapeutic target in biliary tract cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1714. doi:10.1158/1538-7445.AM2015-1714

Abstract 1825: Jab1/Csn5 a new target in the resistant mechanism to HER2-targeted therapies for breast cancer

Abstract Her2 positive (Her2+) breast cancer (BC) accounts for 20% of all breast cancer subtypes and is associated with high risk of death. Trastuzumab (Ttzm), first FDA-approved targeted therapy for BC, represents a key milestone in the personalized treatment of Her2+ metastatic disease (MBC). However, 75% of Her2+ MBC are or become resistant to Ttzm and experience relapse or disease progression. This suggests that tumors acquire or possess an intrinsic mechanism of resistance that prevents from Her2 inhibition. Therefore, it is important to identify novel pathway to overcome the resistance. Recent studies have proposed various potential mechanisms leading to the resistance, including p27 rapid degradation. Jab1/Csn5 a novel candidate oncogene that we identified contributes to the progression of BC and is correlated with poor prognosis. Our study demonstrated that Jab1/Csn5 degrades p27 in MBC. These findings suggest that Jab1 overexpression contributes to Ttzm-resistance by facilitating p27-degradation. Jab1/Csn5 is overexpressed in 50% of primary and 90% of MBC while its expression is low or absent in normal adult breast tissues. We previously identified that high expression of Jab1 is associated with shorter progression-free survival in MBC patients. In this study, we showed that the knockdown of Jab1 sensitizes to Ttzm-treatment. Mechanistically, we found that Jab1/Csn5 overexpression is significantly correlated with the activation of Akt pathway in Her2+ MBC and xenograft models. Interestingly, activated Akt, due to PTEN-loss or PI3K activating mutation has been widely implicated in the potential mechanism to Ttzm-resistance. Therefore, our results suggest that targeting Jab1/Csn5 overcomes the resistance to Ttzm via interfering with PI3K/Akt pathway. Our study identifies Jab1 as a novel contributor to Ttzm-resistance and elucidates its potential mechanisms of actions. Jab1 could be used as a predictive marker of tumor response to Ttzm, which could assist clinicians in selecting the best treatment modalities for patients with MBC. Note: This abstract was not presented at the meeting. Citation Format: Francois X. Claret, Thuy Vu, Terry J. Shackleford, Jennifer Allensworth, Qingxiu Zhang, Ling Tian, Ronghua Zhang. Jab1/Csn5 a new target in the resistant mechanism to HER2-targeted therapies for breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1825. doi:10.1158/1538-7445.AM2014-1825

Abstract P5-08-02: New target in the resistant mechanism to trastuzumab

Abstract Her2 positive (Her2+) breast cancer (BC) accounts for 18-20% of all breast cancer subtypes and is associated with high risk of death. Trastuzumab, the first Food and Drug Administration-approved targeted therapy for BC, represents a key milestone in the personalized treatment of Her2+ metastatic disease (Her2+ MBC). However, the median duration of response is less than a year, due to either primary or acquired resistance to the therapy. In addition, there is currently no conclusive biomarker for the response of patients to trastuzumab. Therefore, understanding the development of resistance to trastuzumab is of our interest. Recent studies have proposed various potential mechanisms leading to the resistance, including p27 rapid degradation. Previously, our study and other research demonstrated that Jab1 degrades p27 in breast cancer. These findings suggest that Jab1 over-expression contributes to trastuzumab resistance by facilitating p27 degradation. Jab1/CSN5 (c-Jun activation domain-binding protein 1) is over-expressed in 50% of primary and 90% of metastatic breast cancers, while its expression is low or absent in normal adult breast tissues. We previously identified that high expression of Jab1 is associated with shorter progression-free survival in breast cancer patients. In this study, our preliminary data showed that the knockdown of Jab1 sensitizes the breast cancer cells to trastuzumab treatment in a dose-dependent manner. Mechanistically, we found that Jab1 over-expression is significantly correlated with the activation of Akt pathway in Her2+ breast cancer cells and xenograft model. Interestingly, activated Akt, due to PTEN loss or PI3K activating mutation has been widely implicated in the potential mechanism to trastuzumab resistance. Therefore, our results suggest that targeting Jab1 overcomes the resistance to trastuzumab via interfering with PI3K/Akt pathway. In general, our study identifies Jab1 as a novel contributor to trastuzumab resistance and elucidates its potential mechanisms of actions. The successful completion of the study can potentially be translated to the clinic for the benefit of patients refractory to the therapy. Also our study suggests Jab1 expression level can be used as a predictive marker for trastuzumab treatment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-02.

Jab1/CSN5 Negatively Regulates p27 and Plays a Role in the Pathogenesis of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignancy most common in East Asia and Africa. Aberrant expression of Jab1/CSN5, a negative regulator of the cell-cycle inhibitor p27, is correlated with reduced p27 expression and associated with advanced tumor stage and poor prognosis in several human cancers. In this study, we examined the functional relationship between Jab1 and p27 protein expression in NPC. Immunohistochemical analysis showed an inverse association between Jab1 and p27 in NPC tissue samples, and overexpression of Jab1 correlated with poor survival in patients with NPC. Mechanistically, Jab1 and p27 were found to interact directly in NPC cells, with Jab1 mediating p27 degradation in a proteasome-dependent manner. Knockdown of Jab1 resulted in a remarkable increase in p27 levels and inhibition of cell proliferation, indicating that Jab1 targets p27 for degradation, thereby controlling its stability. Jab1 depletion also enhanced the antitumor effects of cisplatin in NPC cells. Together, our findings suggest that Jab1 overexpression plays an important role in the pathogenesis of NPC through Jab1-mediated p27 degradation. Jab1 therefore represents a novel diagnostic marker and therapeutic target in patients with NPC.

C‐Jun activating binding protein 1 binds to the IgA receptor and modulates protein levels of FcαRI and FcRγ‐chain

The receptor for IgA, FcalphaRI or CD89, is expressed on myeloid cells and can trigger phagocytosis, tumor cell lysis, and release of inflammatory mediators. These functions critically depend on the associated FcR gamma-chain; however, some biological functions, like receptor internalization, are solely mediated by FcalphaRI alpha-chain. Little is known as to how FcalphaRI regulates these processes and the FcalphaRI intracellular domain does not contain recognized signalling motifs. We searched for associating proteins and identified c-Jun activating binding protein 1 (JAB1) as a binding partner specifically for FcalphaRI. We found increased FcalphaRI surface expression after ectopic expression of JAB1 as well as diminished protein levels of total FcR gamma-chain levels after JAB1 knock-down. These data functionally link JAB1 with controlling protein expression levels of FcalphaRI-FcR gamma-chain protein complex.

Jab1 regulates levels of endothelin type A and B receptors by promoting ubiquitination and degradation

Endothelin type A receptor (ET AR) plays an important role in some cardiovascular disorders where ET AR levels are increased. However, regulatory mechanisms for ET AR levels are unknown. Here, we identified Jun activation domain-binding protein 1 (Jab1) as an ET AR-interacting protein by yeast two-hybrid screening of human heart cDNA library using carboxyl terminal tail (C-tail) of ET AR as a bait. The interaction was confirmed by glutathione S-transferase pull-down assay, co-immunoprecipitation in HEK293T cells expressing ET AR-myc and FLAG-Jab1, and confocal microscopy. Jab1 knockdown increased whole cell and cell surface levels of ET AR and ET-1-induced ERK1/2 phosphorylation in HEK293T cells expressing ET AR, whereas Jab1 overexpression decreased them. Jab1 overexpression accelerated disappearance rate of ET AR after protein synthesis inhibition as an index of a degradation rate. ET AR was constitutively ubiquitinated, and the level of ubiquitination was enhanced by Jab1 overexpression. Long-term ET-1 stimulation markedly accelerated the rate of ET AR degradation and increased the amount of Jab1 bound to ET AR with a maximal level of 500% at 3 h. In the absence of ET-1 stimulation, the level of ET BR was lower than that of ET AR and the degradation rate of ET BR was markedly faster than that of ET AR. Notably, the amount of Jab1 bound to ET BR and ubiquitination level of ET BR were markedly higher than those for ET AR. Taken together, these results suggest that the amount of Jab1 bound to ETR regulates the degradation rate of ET AR and ET BR by modulating ubiquitination of these receptors, leading to changes in ET AR and ET BR levels.

Polyethylene Glycosylated Curcumin Conjugate Inhibits Pancreatic Cancer Cell Growth through Inactivation of Jab1

Jab1 (Jun activation domain binding protein 1), integrated into COP9 signalosome complex (CSN), induces protein instability of many tumor suppressors and cell cycle regulators and is therefore a novel target in cancer therapy. Curcumin, an inhibitor of Jab1/CSN-associated kinase(s), has been reported to suppress tumor growth; however, curcumin is highly hydrophobic, and this feature prevents its usage as an antitumor drug. To increase the solubility and targeted delivery, we generated a water-soluble polyethylene glycol (PEG)-conjugated curcumin system, in which curcumin is covalently linked to PEG(35kD). PEGylated curcumin showed much greater reduction of cell growth than free curcumin in pancreatic cancer cells. Cells treated with PEGylated curcumin had increased arrest at the mitotic phase with the formation of abnormal multinucleated cells, indicating that this compound affects cell cycle progression, which may contribute to cell growth inhibition. The stabilities of Jab1 target proteins were also examined. PEGylated curcumin increased protein stability of these proteins in pancreatic cancer cells and directly inhibited the activity of Jab1/CSN-associated kinases. Moreover, the inhibitory effect of PEGylated curcumin on cell proliferation was blunted in pancreatic cancer cells with Jab1 knockdown. The results suggest that PEGylated curcumin inhibits cell proliferation through suppression of Jab1/CSN activity. More importantly, the new compound sensitized pancreatic cancer cells to gemcitabine-induced apoptosis and cell proliferation inhibitory effects. Collectively, the PEGylated curcumin conjugate has much more potent effects on pancreatic cancer cell growth inhibition than free curcumin. The current study provides a biologic rationale to treat patients with pancreatic adenocarcinoma with the nontoxic phytochemical conjugated to PEG for systemic delivery.

JAB1 accelerates mitochondrial apoptosis by interaction with proapoptotic BclGs

The Bcl-2 family of proteins is the key regulators of cell apoptosis at the mitochondria level. The BH3-only pro-apoptotic member BclGs was unique among the family due to its highly specific expression in human testis and has been demonstrated to induce apoptosis dependent on the BH3 domain. However, the molecular mechanism of BclGs-induced apoptosis remains unclear. Here we show that overexpression of BclGs could induce Bax expression upregulation and translocation to mitochondria, cytochrome c release and activation of caspase-3. Moreover, we identified JAB1 as a novel BclGs-specific binding protein through a yeast two-hybrid screening in a human testis cDNA library. BclGs interacts with JAB1 both in vitro and in vivo. N-terminal region of BclGs (aa 1–67) was required for the interaction. Importantly, JAB1 and BclGs co-expression synergistically induces apoptosis. JAB1 could compete with Bcl-XL/Bcl-2 to bind to BclGs; thus, promote the apoptosis. RNAi-mediated knock-down of JAB1 results in the reduced proapoptotic activity of BclGs. Taken together, our results provided the first evidence that JAB1 is involved in the regulation of mitochondrial apoptotic pathway through specific interaction with BclGs.

HER-2/neu transcriptionally activates Jab1 expression via the AKT/β-catenin pathway in breast cancer cells

Jab1 is a co-activator of activating protein-1 (AP-1) transcription factor and the fifth subunit of the constitutive photomorphogenesis 9 (COP9) signalosome, which has been shown to mediate nuclear exportation and ubiquitin-dependent degradation of the tumor suppressor p27(Kip1). Jab1 is overexpressed in several types of human cancer. However, de-regulation of Jab1 gene expression in cancer cells is largely unclear. In this study, we reported that expression of Jab1 was stimulated by HER-2/neu oncogene via transcriptional activation. Promoter deletion and mutation analysis indicated that HER-2/neu stimulated Jab1 via the T cell factor (TCF) binding site located at the -380/-368 region of the human Jab1 promoter. DNA affinity precipitation assay and chromatin immunoprecipitation assay verified that binding of beta-catenin and TCF-4 to this consensus site was increased by HER-2/neu. In addition, dominant-negative mutant of TCF significantly attenuated the stimulatory effect of HER-2/neu. We also demonstrated that HER-2/neu increased beta-catenin/TCF-mediated Jab1 expression via the AKT signaling pathway because chemical inhibitor or dominant-negative mutant of AKT effectively attenuated the stimulatory action of HER-2/neu. IGF-I, which is a well-known AKT activator, also up-regulated the expression of Jab1 in NIH/3T3 and MCF-7 cells. Knockdown of Jab1 by small interfering RNA (siRNA) preferentially inhibited proliferation of HER-2/neu-overexpressing breast cancer cells. Taken together, our results suggest that HER-2/neu transcriptionally activates Jab1 expression to promote proliferation of breast cancer cells.

JAB1 Determines the Response of Rheumatoid Arthritis Synovial Fibroblasts to Tumor Necrosis Factor-α

Fibroblast-like synoviocytes (FLSs) of patients with rheumatoid arthritis (RA FLSs) exhibit prosurvival, rather than apoptotic, response to tumor necrosis factor (TNF)-alpha stimulation. Here, we show that JAB1 is a critical regulator of the TNF-alpha-mediated anti-apo-ptosis pathways in RA FLSs. We found that knockdown of JAB1 using small interfering (si)RNA led to restoration of the TNF-alpha-induced apoptosis response, reduction of nuclear factor-kappaB activity, delayed degradation of IkappaB-alpha, and inhibited phosphorylation of JNK. Analysis of the interactions of JAB1 by reciprocal co-immunoprecipitations and confocal microscopy revealed that JAB1 interacts with TNF receptor-associated-factor 2 (TRAF2). The generation of the anti-apoptotic signal on binding of TNF-alpha to the TNF receptor (TNFR)1 has been shown to be associated with the recruitment of TRAF2 to the TNFR1 in a process that requires ubiquitination of TRAF2 with lysine-63-linked polyubiquitin chains. We found that TNF-alpha stimulation of JAB1 siRNA-transfected RA FLSs failed to stimulate ubiquitination of TRAF2. Thus, we conclude that JAB1-regulated ubiquitination of TRAF2 is a novel mechanism whereby TNF-alpha can induce anti-apoptosis signaling and production of matrix metalloproteinases through activation of nuclear factor-kappaB and JNK in RA FLSs.

Jab1, a Novel Protease-activated Receptor-2 (PAR-2)-interacting Protein, Is Involved in PAR-2-induced Activation of Activator Protein-1

Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor for trypsin and tryptase, exerts important physiological and pathological functions in multiple systems. However, unlike PAR-1, the PAR-2-mediated intracellular signal transductions are hardly known. Here, using yeast two-hybrid screening with a human brain cDNA library, we identified an interacting partner of human PAR-2, the Jun activation domain-binding protein 1 (Jab1). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro, and by co-immunoprecipitation assays in vivo. Jab1 was also shown to be colocalized with PAR-2 in both transfected HEK293 cells and in normal primary human astrocytes by double immunofluorescence staining. Further experiments demonstrated that multiple intracellular domains of PAR-2 are required for the interaction with Jab1. We then showed that agonist stimulation of PAR-2 disrupted the interaction, which could be prevented by the inhibitor of receptor endocytosis phenylarsine oxide, but not by the lysosomal protease inhibitor ZPAD. Importantly, we found that activation of PAR-2 induced the redistribution of Jab1 from the plasma membrane to the cytosol, but did not influence expression of Jab1. Furthermore, Jab1 mediated PAR-2-induced c-Jun activation, which was followed by increased activation of activator protein-1. Loss-of-function studies, using Jab1 small interfering RNA, demonstrated that Jab1 knockdown blocked PAR-2-induced activator protein-1 activation. Taken together, our data demonstrate that Jab1 is an important effector that mediates a novel signal transduction pathway for PAR-2-dependent gene expression.