Recombination Signal Binding Protein Jκ Research Articles

MiR-223-3p attenuates M1 macrophage polarization via suppressing the Notch signaling pathway and NLRP3-mediated pyroptosis in experimental autoimmune uveitis

Autoimmune uveitis is an intraocular inflammatory disease with a high blindness rate in developed countries such as the United States. It is pressing to comprehend the pathogenesis of autoimmune uveitis and develop novel schemes for its treatment. In the present research, we demonstrated that the Notch signaling pathway was activated, and the level of miR-223-3p was significantly reduced in rats with experimental autoimmune uveitis (EAU) compared with the level of normal rats. To investigate the relationship between miR-223-3p and Notch signaling, EAU rats received miR-223-3p-carrying lentivirus, miR-223-3p vector-carrying lentivirus (miR-223-3p-N), and γ-secretase inhibitor (DAPT), respectively. The results of Q-PCR, immunological experiments, and flow cytometry analysis all support the hypothesis that both miR-223-3p and DAPT, a Notch signaling pathway inhibitor, had similar inhibitory effects on the EAU pathological process. That is to say, they could both inhibit the activation of the Notch signaling pathway via modulating recombination signal binding protein-Jκ (RBPJ) to restore the polarization imbalance of M/M2 macrophages in EAU rats. In addition, miR-223-3p could also inhibit NLRP3 inflammasome activation and inflammasome-induced pyroptosis in ocular tissues. Taken together, our findings indicate that miR-223-3p serves as an important regulator in M1 macrophage polarization and pyroptosis, thereby alleviating the inflammatory response in uveitis.

Myeloid-specific blockade of notch signaling alleviates dopaminergic neurodegeneration in Parkinson's disease by dominantly regulating resident microglia activation through NF-κB signaling.

Yolk sac-derived microglia and peripheral monocyte-derived macrophages play a key role during Parkinson's disease (PD) progression. However, the regulatory mechanism of microglia/macrophage activation and function in PD pathogenesis remains unclear. Recombination signal-binding protein Jκ (RBP-J)-mediated Notch signaling regulates macrophage development and activation. In this study, with an 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride-induced acute murine PD model, we found that Notch signaling was activated in amoeboid microglia accompanied by a decrease in tyrosine hydroxylase (TH)-positive neurons. Furthermore, using myeloid-specific RBP-J knockout (RBP-JcKO) mice combined with a PD model, our results showed that myeloid-specific disruption of RBP-J alleviated dopaminergic neurodegeneration and improved locomotor activity. Fluorescence-activated cell sorting (FACS) analysis showed that the number of infiltrated inflammatory macrophages and activated major histocompatibility complex (MHC) II+ microglia decreased in RBP-JcKO mice compared with control mice. Moreover, to block monocyte recruitment by using chemokine (C-C motif) receptor 2 (CCR2) knockout mice, the effect of RBP-J deficiency on dopaminergic neurodegeneration was not affected, indicating that Notch signaling might regulate neuroinflammation independent of CCR2+ monocyte infiltration. Notably, when microglia were depleted with the PLX5622 formulated diet, we found that myeloid-specific RBP-J knockout resulted in more TH+ neurons and fewer activated microglia. Ex vitro experiments demonstrated that RBP-J deficiency in microglia might reduce inflammatory factor secretion, TH+ neuron apoptosis, and p65 nuclear translocation. Collectively, our study first revealed that RBP-J-mediated Notch signaling might participate in PD progression by mainly regulating microglia activation through nuclear factor kappa-B (NF-κB) signaling.

Cellular senescence primes liver fibrosis regression through Notch-EZH2.

Cellular senescence plays a pivotal role in wound healing. At the initiation of liver fibrosis regression, accumulated senescent cells were detected and genes of senescence were upregulated. Flow cytometry combined with single-cell RNA sequencing analyses revealed that most of senescent cells were liver nonparenchymal cells. Removing senescent cells by dasatinib and quercetin (DQ), alleviated hepatic cellular senescence, impeded fibrosis regression, and disrupted liver sinusoids. Clearance of senescent cells not only decreased senescent macrophages but also shrank the proportion of anti-inflammatory M2 macrophages through apoptotic pathway. Subsequently, macrophages were depleted by clodronate, which diminished hepatic senescent cells and impaired fibrosis regression. Mechanistically, the change of the epigenetic regulator enhancer of zeste homolog2 (EZH2) accompanied with the emergence of hepatic senescent cells while liver fibrosis regressed. Blocking EZH2 signaling by EPZ6438 reduced hepatic senescent cells and macrophages, decelerating liver fibrosis regression. Moreover, the promoter region of EZH2 was transcriptionally suppressed by Notch-Hes1 (hairy and enhancer of split 1) signaling. Disruption of Notch in macrophages using Lyz2 (lysozyme 2) Cre-RBP-J (recombination signal binding protein Jκ) f/f transgenic mice, enhanced hepatic cellular senescence, and facilitated fibrosis regression by upregulating EZH2 and blocking EZH2 abrogated the above effects caused by Notch deficiency. Ultimately, adopting Notch inhibitor Ly3039478 or exosome-mediated RBP-J decoy oligodeoxynucleotides accelerated liver fibrosis regression by augmenting hepatic cellular senescence.

POFUT1 promotes gastric cancer progression through Notch/Wnt dual signaling pathways dependent on the parafibromin-NICD1-β-catenin complex.

Aberrant glycosylation performed by glycosyltransferases is a leading cause of gastric cancer (GC). Protein O-fucosyltransferase 1 (POFUT1) expression is increased in GC specimens and cells. In this study, the biological effects and mechanisms of POFUT1 underlying the development of GC were investigated. POFUT1 downregulated and upregulated GC cells were established. The effects of POFUT1 on cell proliferation, metastasis and apoptosis were examined using cell counting kit-8 (CCK8) assay, transwell assay, and flow cytometry. Subcutaneous xenograft tumor models were established followed by immunohistochemistry staining of resected tumors. Facilitating modulators and transcription factors were detected by western blot, immunofluorescence, luciferase reporter assay, and co-immunoprecipitation. POFUT1 played a pro-oncogenic role both in vivo and in vitro, which promoted proliferation and metastasis, as well as inhibited apoptosis in GC cells. POFUT1 promoted Cyclin D3 expression and inhibited the expression of apoptotic proteins, such as Bcl-2-associated X protein (Bax) and cleaved caspase 3, facilitating tumor growth. Moreover, POFUT1 accelerated matrix metalloproteases expression and attenuated E-cadherin expression, contributing to GC metastasis. In addition, POFUT1 expression promoted the expression and nuclear translocation of Notch1 intracellular domain (NICD1) and β-catenin and inhibited β-catenin phosphorylation degradation, accompanied by the activation of recombination signal binding protein-Jκ (RBP-J) and T-cell factor (TCF) transcription factors, respectively. It is notable that parafibromin integrated NICD1 and β-catenin, enabling the concerted activation of Wnt and Notch signaling targeted proteins. These observations indicated that POFUT1 promoted GC development through activation of Notch and Wnt signaling pathways, which depended on the parafibromin-NICD1-β-catenin complex. This work provides new evidence for the further diagnosis and treatment of GC.

Hsa_circ_0001535 Regulates Colorectal Cancer Progression via the miR-433-3p/RBPJ Axis.

A large number of studies have shown that circular RNAs (circRNAs) are of great significance in the occurrence and development of colorectal cancer (CRC). The purpose of this study was to explore the mechanism of circ_0001535 in CRC. The expressions of circ_0001535, miR-433-3p and recombination signal-binding protein Jκ (RBPJ) mRNA and protein in CRC tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. The effect of circ_0001535 on cell proliferation was detected using the Cell Counting Kit-8 (CCK-8) assay, colony formation assay and 5-ethynyl-2'-deoxyuridine (EdU) assay. The effects of circ_0001535 on migration, invasion, angiogenesis and apoptosis were investigated by wound healing assay, transwell assay, tube formation assay and flow cytometry, respectively. The interactions between miR-433-3p and circ_0001535 or RBPJ were studied using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Xenograft tumor assay was performed to verify the role of circ_0001535 in tumor growth in vivo. The results showed that circ_0001535 and RBPJ mRNA expression levels were up-regulated and miR-433-3p was down-regulated in CRC tissues and cells. Circ_0001535 knockdown inhibited cell proliferation, migration, invasion, angiogenesis as well as promoted apoptosis in CRC cells. After analysis, it was found that circ_0001535 acted as a competing endogenous RNA (ceRNA) to inhibit miR-433-3p and then up-regulate RBPJ in CRC cells. In addition, in vivo experiment had shown that circ_0001535 knockdown inhibited tumor growth by up-regulating miR-433-3p and inhibiting RBPJ expression. The circ_0001535/miR-433-3p/ RBPJ axis plays a catalytic role in the progression of CRC, which may provide new insights into the molecular mechanism of CRC.

Myeloid-specific blockade of Notch signaling alleviates murine pulmonary fibrosis through regulating monocyte-derived Ly6clo MHCIIhi alveolar macrophages recruitment and TGF-β secretion.

Macrophages in lung, including resident alveolar macrophages (AMs) and interstitial macrophages (IMs), and monocyte-derived macrophages, play important roles in pulmonary fibrosis (PF), but mechanisms underlying their differential regulation remain unclear. Recombination signal-binding protein Jκ (RBP-J)-mediated Notch signaling regulates macrophage development and phenotype. Here, using bleomycin-induced fibrosis model combined with myeloid-specific RBP-J disruption (RBP-JcKO ) mouse, we investigated the role of Notch signaling in macrophages during PF. Compared with the control, RBP-JcKO mice exhibited alleviated lung fibrosis as manifested by reduced collagen deposition and inflammation, and decreased TGF-β production. FACS analysis suggested that decreased Ly6clo MHCIIhi AMs might make the major contribution to attenuated fibrogenesis in RBP-JcKO mice, probably by reduced inflammatory factor release and enhanced matrix metalloproteinases expression. Using clodronate-mediated macrophage depletion in RBP-JckO mice, we demonstrated that embryonic-derived AMs play negligible role in lung fibrosis, which was further supported by adoptive transfer experiments. Moreover, on CCR2 knockout background, the effect of RBP-J deficiency on fibrogenesis was not elicited, suggesting that Notch regulated monocyte-derived AMs. Co-culture experiment showed that monocyte-derived AMs from RBP-JcKO mice exhibit reduced myofibroblast activation due to decreased TGF-β secretion. In conclusion, monocyte-derived Ly6clo MHCIIhi AMs, which are regulated by RBP-J-mediated Notch signaling, play an essential role in lung fibrosis.

P0014MYELOID-SPECIFIC TARGETING OF NOTCH AMELIORATES RENAL FIBOSIS VIA REDUCED INFILTRATION AND ACTIVATION OF BONE MARROW-DERIVED MACROPHAGE

Abstract Background and Aims Macrophages play critical roles in renal fibrosis. However, macrophages exhibit ontogenic and functional heterogeneities, and which population of macrophages contributes to renal fibrosis and the underlying mechanisms remain unclear. Method In this study, we genetically targeted Notch signaling by disrupting the transcription factor recombination signal binding protein-Jκ (RBP-J), to reveal its role in regulation of macrophages during the unilateral ureteral obstruction (UUO)-induced murine renal fibrosis. Results Myeloid-specific disruption of RBP-J attenuated renal fibrosis with reduced extracellular matrix deposition and myofibroblast activation, as well as attenuated epithelial-mesenchymal transition, likely owing to the reduced expression of TGF-β. Meanwhile, RBP-J deletion significantly hampered macrophage infiltration and activation in fibrotic kidney, although their proliferation appeared unaltered. By using macrophage clearance experiment, we found that kidney resident macrophages made negligible contribution, but bone marrow (BM)-derived macrophages played a major role in renal fibrogenesis. Further mechanistic analyses showed that Notch blockade reduced monocyte emigration from BM by down-regulating CCR2 expression. Finally, we found that myeloid-specific Notch activation aggravated renal fibrosis, which was mediated by CCR2+ macrophages infiltration. Conclusion In summary, our data have unveiled that myeloid-specific targeting of Notch could ameliorate renal fibrosis by regulating BM-derived macrophages recruitment and activation, providing a novel strategy for intervention of this disease.

NOTCH Signaling via WNT Regulates the Proliferation of Alternative, CCR2-Independent Tumor-Associated Macrophages in Hepatocellular Carcinoma

Tumor-associated macrophages (TAM) play pivotal roles in tumor progression and metastasis, but the contribution and regulation of different macrophage populations remain unclear. Here we show that Notch signaling plays distinct roles in regulating different TAM subsets in hepatocellular carcinoma (HCC). Myeloid-specific NOTCH blockade by conditional disruption of recombination signal binding protein Jκ (RBPj cKO) significantly delayed the growth of subcutaneously inoculated Lewis lung carcinoma (LLC), but accelerated orthotopically inoculated hepatic Hepa1-6 tumors in mice. In contrast to subcutaneous LLC, RBPj cKO significantly increased the number of TAMs in hepatic Hepa1-6 tumors despite impeded differentiation of monocyte-derived TAMs (moTAM). The dominating TAMs in orthotopic HCC manifested properties of Kupffer cells (KC) and hence are tentatively named KC-like TAMs (kclTAM). The increased proliferation of RBPj cKO kclTAMs was maintained even in Ccr2 -/- mice, in which moTAMs were genetically blocked. NOTCH signaling blockade accelerated proliferation of kclTAMs via enhanced β-catenin-dependent WNT signaling, which also downregulated IL12 and upregulated IL10 expression by kclTAMs likely through c-MYC. In addition, myeloid-specific RBPj cKO facilitated hepatic metastasis of colorectal cancer but suppressed lung metastasis in mice, suggesting that the phenotype of RBPj cKO in promoting tumor growth was liver-specific. In patient-derived HCC biopsies, NOTCH signaling negatively correlated with WNT activation in CD68+ macrophages, which positively correlated with advanced HCC stages. Therefore, NOTCH blockade impedes the differentiation of moTAMs, but upregulates Wnt/β-catenin signaling to promote the proliferation and protumor cytokine production of kclTAMs, facilitating HCC progression and hepatic metastasis of colorectal cancer. SIGNIFICANCE: These findings highlight the role of NOTCH and WNT signaling in regulating TAMs in hepatocellular carcinoma.

RBPJ polymorphisms associated with cerebral infarction diseases in Chinese Han population: A Clinical Trial/Experimental Study (CONSORT Compliant).

Trial design:Cerebral small vessel diseases (CSVDs) are a group of brain pathological processes involving cerebral small arteries, brain venules, and capillaries. The recombination signal-binding protein Jκ (RBPJ) is implicated in the pathogenesis of these diseases but its actual roles need confirmation. The aim of this work was to evaluate variations in RBPJ gene for their possible associations with the disease.Methods:The RBPJ gene was sequenced for 400 patients with cerebral infarction disease and 600 normal controls. The statistical analyses and Hardy–Weinberg equilibrium tests of the patients and control populations were conducted using the SPSS software (version 19.0) and Plink (version 1.9), Haploview software, and online software SNPSpD.Results:We characterized variants rs2871198, rs1397731, rs3822223, rs2077777, rs2270226, and rs2788861 within or near the RBPJ gene. The genetic heterozygosity of rs2871198, rs1397731, rs3822223, rs2077777, and rs2270226 was very high. Statistical analysis showed that the variants rs2270226 and rs2077777 in the gene were associated with the risk of cerebral infarction diseases in the Chinese Han population.Conclusions:rs2270226 and rs2077777 in the RBPJ gene were associated with the risk of cerebral infarction diseases in the Chinese Han population.

RBPJ mediates uterine repair in the mouse and is reduced in women with recurrent pregnancy loss.

Unexplained recurrent pregnancy loss (uRPL) is associated with repeated embryo loss and endometrial repair with elevated endometrial expression of inflammatory cytokines, including IFN-γ. Notch signaling through its transcription factor recombination signal binding protein Jκ (RBPJ) regulates mechanisms including the immune response and repair after tissue injury. Initially, null mutation of RBPJ in the mouse uterus ( Pgrcre/+Rbpjf/f; Rbpj c-KO) results in subfertility, but we have found that these mice become infertile after pregnancy as a result of dysfunctional postpartum uterine repair, including delayed endometrial epithelial and myometrial regeneration. RNA sequencing of postpartum uterine repair sites revealed global up-regulation of inflammatory pathways, including IFN signaling. Consistent with elevated IFN-γ, macrophages were recruited and polarized toward an M1-cytotoxic phenotype, which is associated with preventing repair and promoting further tissue injury. Through embryo transfer experiments, we show that dysfunctional postpartum repair directly impairs future embryo implantation in Rbpj c-KO mice. Last, we clinically correlated our findings from the Rbpj c-KO mouse in women diagnosed with uRPL. Reduced RBPJ in women with uRPL was associated with increased levels of IFN-γ. The data, taken together, indicate that RBPJ regulates inflammation during endometrial repair, which is essential for future pregnancy potential, and its dysregulation may serve as an unidentified contributor to uRPL in women.-Strug, M. R., Su, R.-W., Kim, T. H., Mauriello, A., Ticconi, C., Lessey, B. A., Young, S. L., Lim, J. M., Jeong, J.-W., Fazleabas, A. T. RBPJ mediates uterine repair in the mouse and is reduced in women with recurrent pregnancy loss.

RBPJ inhibition impairs the growth of lung cancer.

The exact effects of the modulation of Notch signaling pathway on cell growth have been shown to depend on tumor cell type. Recombination signal-binding protein Jκ (RBPJ) is a key transcription factor downstream of receptor activation in Notch signaling pathway. Here, we evaluated the effects of RBPJ inhibition on the growth of lung cancer cells. We found that a short hairpin interfering RNA (shRNA) for RBPJ efficiently inhibited RBPJ expression in lung cancer cells, resulting in a significant decrease in the cell growth. Further analyses showed that RBPJ inhibition altered the levels of its downstream targets, including p21, p27, CDK2, Hes1, Bcl-2, and SKP2, to prevent the cells from growing. Our data thus suggest that shRNA intervention of RBPJ expression could be a promising therapeutic approach for treating human lung cancer.

Myeloid-specific disruption of recombination signal binding protein Jκ ameliorates hepatic fibrosis by attenuating inflammation through cylindromatosis in mice.

Macrophages play multidimensional roles in hepatic fibrosis, but their control has not been fully understood. The Notch pathway mediated by recombination signal binding protein Jκ (RBP-J), the transcription factor transactivated by signals from four mammalian Notch receptors, is implicated in macrophage activation and plasticity. In this study, by using mouse hepatic fibrosis models, we show that myeloid-specific disruption of RBP-J resulted in attenuated fibrosis. The activation of hepatic stellate cells and production of profibrotic factors including platelet-derived growth factor (PDGF)-B and transforming growth factor beta1 (TGF-β1) reduced significantly in myeloid-specific RBP-J deficient mice. The infiltration of inflammatory cells and production of proinflammatory factors were reduced in liver of myeloid-specific RBP-J-deficient mice during fibrosis. In RBP-J-deficient macrophages, the nuclear factor kappa B (NF-κB) activation was remarkably attenuated as compared with the control. This could be attributed to the up-regulation of cylindromatosis (CYLD), a negative regulator of NF-κB, in Notch signal-compromised macrophages, because the knockdown of CYLD in RBP-J-deficient macrophages or overexpression of p65 in RBP-J knockdown cells both restored NF-κB activation and the production of proinflammatory and/or profibrotic factors by macrophages. In human hepatic fibrosis biopsies, stronger Notch activation is correlated with more severe fibrosis, which is accompanied by a lower level of CYLD but irrespective of etiological reasons. RBP-J-mediated Notch signaling is required for macrophages to promote hepatic fibrosis by up-regulation of NF-κB activation through CYLD.

Myeloid-Specific Blockade of Notch Signaling by RBP-J Knockout Attenuates Spinal Cord Injury Accompanied by Compromised Inflammation Response in Mice.

The outcome of spinal cord injury (SCI) is determined by both neural cell-intrinsic survival pathways and tissue microenvironment-derived signals. Macrophages dominating the inflammatory responses in SCI possess both destructive and reparative potentials, according to their activation status. Notch signaling is involved in both cell survival and macrophage-mediated inflammation, but a comprehensive role of Notch signaling in SCI has been elusive. In this study, we compared the effects of general Notch blockade by a pharmaceutical γ-secretase inhibitor (GSI) and myeloid-specific Notch signal disruption by recombination signal binding protein Jκ (RBP-J) knockout on SCI. The administration of Notch signal inhibitor GSI resulted in worsened hind limb locomotion and exacerbated inflammation. However, mice lacking RBP-J, the critical transcription factor mediating signals from all four mammalian Notch receptors, in myeloid lineage displayed promoted functional recovery, attenuated glial scar formation, improved neuronal survival and axon regrowth, and mitigated inflammatory response after SCI. These benefits were accompanied by enhanced AKT activation in the lesion area after SCI. These findings demonstrate that abrogating Notch signal in myeloid cells ameliorates inflammation response post-SCI and promotes functional recovery, but general pharmaceutical Notch interception has opposite effects. Therefore, clinical intervention of Notch signaling in SCI needs to pinpoint myeloid lineage to avoid the counteractive effects of global inhibition.

Disruption of the transcription factor RBP-J results in osteopenia attributable to attenuated osteoclast differentiation

The transcription factor recombination signal binding protein-Jκ (RBP-J) is the critical transcription factor downstream to all four mammalian Notch receptors. Although it has been reported that Notch signaling pathway is involved in bone remodeling, the importance of RBP-J in osteoclastogenesis has not been fully explored. To investigate the role of RBP-J in osteoclastogenesis, we conditionally deleted RBP-J systemically in bone marrow (BM) or specifically in macrophages. We found that disruption of RBP-J in BM resulted in an obvious decrease in trabecular bone mass associated with an increase in osteoclasts, leading to osteopenia. Disruption of RBP-J in macrophages phenocopied the phenotypes of RBP-J deletion in BM with respect to osteoclastogenesis, suggesting that the osteopenia in RBP-J deficient mice is essentially resulted from increased osteoclastogenesis. Furthermore, we found that RBP-J deletion in osteoclasts resulted in a dramatic increase in tartrate-resistant acid phosphatase expression. These findings demonstrate a negatively role of RBP-J in the differentiation of osteoclasts and suggest that Notch pathway may be a new therapeutic target for bone diseases related to increased osteoclastogenesis.

Number of and distance between response elements in Kaposi’s sarcoma-associated herpesvirus ORF57 promoter influence its activation by replication and transcription activator and its repression by interferon regulatory factor 7

Kaposi's sarcoma-associated herpesvirus ORF57 expression is highly responsive to replication and transcription activator (RTA) and interferon regulatory factor 7 (IRF-7). Three RTA response elements (RREs) have been identified in the ORF57 promoter. Here, we show evidence of another functional RRE located between nt 82003 and 82081, which can complement the loss of RTA activation resulting from RRE1 deletion. Repeats of a recombination signal-binding protein Jkappa (RBP-Jkappa) site enhanced RTA activation, which could not be suppressed by IRF-7. Alteration of the distance between the RBP-Jkappa site and RRE2 modulated responsiveness to RTA and IRF-7. These results will help to elucidate the precise regulation of viral gene expression.

RUNX3 directly interacts with intracellular domain of Notch1 and suppresses Notch signaling in hepatocellular carcinoma cells

RUNX3 takes a strong suppressive effect in many tumors including hepatocellular carcinoma (HCC). HES-1, a downstream target of Notch signaling, is shown to be decreased in human HCC cell line SMMC7721 with RUNX3 gene transfection. Since Notch signaling is oncogenic in HCC, RUNX3 might exert its inhibitory effect in HCC partly through the suppression on Notch signaling. To investigate the possible mechanism of the down-regulation of HES-1 by RUNX3, we performed Western blot and reporter assay and found that RUNX3 suppressed intracellular domain of Notch1 (ICN1)-mediated transactivation of Notch signaling while it did not alter the expression of ICN1 and recombination signal binding protein-Jκ (RBP-J) in SMMC7721 cells. Besides, confocal microscopy, co-immunoprecipitation and GST pull-down assays showed that RUNX3 could co-localize with ICN1 and RBP-J, forming a complex with these two molecules in nucleus of SMMC7721 cells by its direct interaction with ICN1. Furthermore, RUNX3 was recruited to RBP-J recognition motif of HES-1 promoter, which was identified by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Taken together, these findings indicate that RUNX3 suppresses Notch signaling in HCC SMMC7721 cells by its interaction with ICN1 and thus recruitment to the RBP-J recognition motif of downstream genes of Notch signaling.

Disruption of the transcription factor recombination signal-binding protein-Jκ (RBP-J) leads to veno-occlusive disease and interfered liver regeneration in mice

Liver sinusoid (LS) endothelial cells (LSECs) support hepatocytes in resting livers and proliferate during liver regeneration to revascularize regenerated liver parenchyma. We report that recombination signal-binding protein-Jkappa (RBP-J), the critical transcription factor mediating Notch signaling, regulates both resting and regenerating LSECs. Conditional deletion of RBP-J resulted in LSEC proliferation and a veno-occlusive disease-like phenotype in the liver, as manifested by liver congestion, deposition of fibrin-like materials in LSs, edema in the space of Disse, and increased apoptosis of hepatocytes. Regeneration of liver was remarkably impaired, with reduced LSEC proliferation and destroyed sinusoidal structure. LSEC degeneration was obvious in the regenerating liver of RBP-J-deficient mice, with some LSECs losing cytoplasm, and organelles protruding into the remnant plasma-membrane of LSs to hamper the microcirculation and intensify veno-occlusive disease during liver regeneration. Hepatocytes were also degenerative, as shown by dilated endoplasmic reticulum, decreased proliferation, and increased apoptosis during liver regeneration. Molecular analyses revealed that the dynamic expression of several related molecules-such as vascular endothelial growth factor, vascular endothelial growth factor receptors 1 and 2, interleukin-6, and hepatocyte growth factor-was disturbed. Notch/RBP-J signaling may play dual roles in LSECs: in resting liver it represses proliferation, and in regenerating liver it supports proliferation and functional differentiation.

The latency-associated nuclear antigen encoded by Kaposi's sarcoma-associated herpesvirus activates two major essential Epstein-Barr virus latent promoters.

The latency-associated nuclear antigen (LANA) encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) is expressed in the majority of KSHV-infected cells and in cells coinfected with Epstein-Barr virus (EBV). In coinfected body cavity-based lymphomas (BCBLs), EBV latent membrane protein 1 (LMP1), which is essential for B-lymphocyte transformation, is expressed. EBNA2 upregulates the expression of LMP1 and other cellular genes through specific interactions with cellular transcription factors tethering EBNA2 to its responsive promoters. In coinfected BCBL cells, EBNA2 is not detected but LANA, which is constitutively expressed, contains motifs suggestive of potential transcriptional activity. Additionally, recent studies have shown that LANA is capable of activating cellular promoters. Therefore, we investigated whether LANA can affect transcription from two major EBV latent promoters. In this study, we demonstrated that LANA can efficiently transactivate both the LMP1 and C promoters in the human B-cell line BJAB as well as in the human embryonic kidney 293 cell line. Moreover, we demonstrated that specific domains of LANA containing the putative leucine zipper and the glutamic acid-rich region are highly effective in upregulating these viral promoters, while the amino-terminal region (435 amino acids) exhibited little or no transactivation activity in our assays. We also specifically tested truncations of the LMP1 promoter element and showed that the -204 to +40 region had increased levels of activation compared with a larger region, -512 to +40, which contains two recombination signal-binding protein J kappa binding sites. The smaller, -204 to +40 promoter region contains specific binding sites for the Ets family transcription factor PU.1, transcription activating factor/cyclic AMP response element, and Sp1, all of which are known to function as activators of transcription. Our data therefore suggest a potential role for LANA in regulation of the major EBV latent promoters in KSHV- and EBV-coinfected cells. Furthermore, LANA may be able to activate transcription of viral and cellular promoters in the absence of EBNA2, potentially through association with transcription factors bound to their cognate sequences within the -204 to +40 region. This regulation of viral gene expression is critical for persistence of these DNA tumor viruses and most likely involved in mediating the oncogenic process in these coinfected cells.

Enhanced Expression and Autoimmunity of Recombination Signal Binding Protein-jκ in Human Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is a major cause of heart failure in younger individuals. Its prognosis is poor with 40–50% of patients dying within 2 years after diagnosis. Although the etiology of DCM is poorly understood, there is increasing evidence that DCM may represent an autoimmune disease in a significant subset of patients. In order to identify candidate antigens in DCM, we applied a molecular strategy which combines recombinant expression cloning and autoimmunological screening procedures. A left ventricle from a male DCM patient was explanted at heart transplantation and a human DCM left ventricular cDNA-expression library was constructed. 2 × 106 clones were immunologically screened with serum collected from the same patient prior transplantation. Subsequent rounds of screening and purification allowed isolation of a positive clone which was sequenced and identified as Recombination Signal Binding Protein-jκ (RBP-jκ). RBP-jκ is an already identified transcription factor, e.g., involved in Epstein-Barr-virus-induced immortalization processes. Radioactively labeled RBP-jκ protein was synthesized via in vitro translation using the isolated RBP-jκ cDNA. This RBP-jκ protein was used for immunoprecipitation reactions to screen sera of healthy controls and patients suffering of DCM for the presence of RBP-jκ autoantibodies. Analysis revealed that only 31% (n = 16) of healthy but 70.6% of DCM patients (n = 17) carry an autoantibody against RBP-jκ. Patients suffering from ischemic cardiomyopathy showed a prevalence of 22% of RBP-jκ autoantibodies. Western analysis with an monoclonal antibody raised against RBP-jκ showed that RBP-jκ was overexpressed to 488 ± 140% in DCM hearts compared to non-failing controls (n = 8). Autologous immunological screening of a cDNA expression library is a powerful and novel technology to gain insights into the etiology of human idiopathic DCM. Human DCM displays an autoimmune response against RBP-jκ and an overexpression of RBP-jκ. Since RBP-jκ is involved in cellular immortalization and exerts antiapoptotic effects, the increased RBP-jκ autoantibody level during DCM may inhibit this growth-regulating feature of RBP-jκ. In this setting, enhanced myocardial RBP-jκ expression could represent a compensatory but ineffective response to counteract the increased rate of apoptosis in DCM. Furthermore, RBP-jκ may be a useful diagnostic marker for DCM.

NF-kappaB2 is a putative target gene of activated Notch-1 via RBP-Jkappa.

NF-kappaB2 (p100/p52), a member of the NF-kappaB/Rel family of transcription factors, is involved in the regulation of a variety of genes important for immune function. Previously, we have shown that the NF-kappaB2 gene is regulated in a positive and a negative manner. Two kappaB elements within the NF-kappaB2 promoter mediate tumor necrosis factor alpha-inducible transactivation. In addition, we have shown that there exists a transcriptional repression in the absence of NF-kappaB. To identify a DNA binding activity responsible for this transcriptional repression, we have partially purified a nuclear complex, named Rep-kappaB. Here we further analyze this putative repressive binding activity. Detailed examination of Rep-kappaB-DNA interaction revealed the sequence requirements for binding to be almost identical to those of recombination signal binding protein Jkappa (RBP-Jkappa), the mammalian homolog of the protein encoded by Drosophila suppressor of hairless [Su(H)]. In addition, in electromobility shift assays, Rep-kappaB binding activity is recognized by an antibody directed against RBP-Jkappa. By performing transient-transfection assays, we show that human RBP-Jkappa represses basal as well as RelA (p65)-stimulated NF-kappaB2 promoter activity. Studies in Drosophila melanogaster have shown that Su(H) is implicated in the Notch signaling pathway regulating cell fate decisions. In transient-transfection assays we show that truncated Notch-1 strongly induces NF-kappaB2 promoter activity. In summary, our data clearly demonstrate that Rep-kappaB is closely related or identical to RBP-Jkappa. RBP-Jkappa is a strong transcriptional repressor of NF-kappaB2. Moreover, this repression can be overcome by activated Notch-1, suggesting that NF-kappaB2 is a novel putative Notch target gene.