The ubiquitin-conjugating enzyme UBE2O modulates c-Maf stability and induces myeloma cell apoptosis.

BACKGROUND: UBE2O is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown. METHODS: Mass spectrometry was applied to identify c-Maf ubiquitination-associated proteins. Immunoprecipitation was applied for c-Maf and UBE2O interaction. Immunoblotting was used for Maf protein stability. Luciferase assay was used for c-Maf transcriptional activity. Lentiviral infections were applied for UBE2O function in multiple myeloma (MM) cells. Flow cytometry and nude mice xenografts were applied for MM cell apoptosis and tumor growth assay, respectively. RESULTS: UBE2O was found to interact with c-Maf, a critical transcription factor in MM, by the affinity purification/tandem mass spectrometry assay and co-immunoprecipitation assays. Subsequent studies showed that UBE2O mediated c-Maf polyubiquitination and degradation. Moreover, UBE2O downregulated the transcriptional activity of c-Maf and the expression of cyclin D2, a typical gene modulated by c-Maf. DNA microarray revealed that UBE2O was expressed in normal bone marrow cells but downregulated in MGUS, smoldering MM and MM cells, which was confirmed by RT-PCR in primary MM cells, suggesting its potential role in myeloma pathophysiology. When UBE2O was restored, c-Maf protein in MM cells was significantly decreased and MM cells underwent apoptosis. Furthermore, the human MM xenograft in nude mice showed that re-expression of UBE2O delayed the growth of myeloma xenografts in nude mice in association with c-Maf downregulation and activation of the apoptotic pathway. CONCLUSIONS: UBE2O mediates c-Maf polyubiquitination and degradation, induces MM cell apoptosis, and suppresses myeloma tumor growth, which provides a novel insight in understanding myelomagenesis and UBE2O biology.

Allergenicity potential of red kidney bean (Phaseolus vulgaris L.) proteins in orally treated BALB/c mice and passively sensitized RBL-2H3 cells.

Red kidney bean (Phaseolus vulgaris L.) is one the most commonly consumed legumes that requires an in depth understanding of its allergenicity. Therefore, the aim of this study was to explore the allergenicity of red kidney bean proteins following oral exposure in BALB/c mice and elucidate the levels of Th1/Th2 transcription factors induced by red kidney bean proteins in rat basophilic leukemia cells (RBL-2H3 cells) passively sensitized with the sera of red kidney bean sensitized mice. Red kidney bean proteins showed enhanced levels of total and specific IgE, anaphylactic symptoms, thymic stromal lymphopoietin (TSLP) and peritoneal albumin over control. Enhanced release of ß-hexosaminidase along with up regulated expressions of GATA-3, STAT-6, T-bet, c-MAF and NFAT were observed in the RBL-2H3 cells exposed with red kidney bean proteins when compared to that of the controls. Taken together, exposure of red kidney bean proteins may cause allergic symptoms in mice and the ambivalent effect on Th2/Th1 transcription factors in RBL-2H3 cells.

Prognostic impact of tumour-infiltrating Th2 and regulatory T cells in classical Hodgkin lymphoma.

Classical Hodgkin Lymphoma (cHL) is morphologically characterized by a small number of tumour cells, Hodgkin and Reed-Sternberg (HRS) cells, surrounded by numerous tumour-infiltrating lymphocytes (TIL). The functional role of these TIL is still controversial. While generally considered to represent an anti-tumour immune response, TIL in cHL might result from the profoundly deregulated immunity of cHL patients. Eighty-seven cases of cHL were available to evaluate the prognostical significance of tumour-infiltrating cytotoxic T lymphocytes (CTL), T helper 1 (Th1) cells, T helper 2 (Th2) cells and regulatory T cells (Treg). We confirm that in cHL the microenvironment is dominated by Th2 cells and Treg and show that large numbers of Th2 cells are associated with significantly improved disease-free survival (p = 0.021) and event-free survival (p = 0.012). Furthermore, a high ratio of Treg over Th2 cells resulted in a significantly shortened disease-free survival (p = 0.025). These observations suggest that Treg may exert inhibitory effects on anti-tumour immune responses mediated through Th2 cells and that Th2 cells may be more important for effective anti-tumour immunity than anticipated.

c-Maf expression in angioimmunoblastic T-cell lymphoma.

The oncogene c-Maf was recently found to be overexpressed in approximately 50% of multiple myeloma cases, and a role for c-Maf in promoting cyclin D2 expression has been postulated. We previously examined c-Maf expression in various T-cell lymphomas by reverse-transcription polymerase chain reaction and found extremely elevated c-Maf levels in angioimmunoblastic T-cell lymphoma (AILT). In this study, we examined T-cell lymphomas for c-Maf and cyclin expression immunohistochemically. Of 93 cases of T-cell lymphomas we investigated in the current study, c-Maf expression was seen in 23 out of 31 cases of AILT, 3 out of 11 of adult T-cell leukemia/lymphoma, 4 out of 19 of peripheral T-cell lymphoma, unspecified [PTCL(U)], and 0 out of 11 cases of mycosis fungoides, 0 out of 11 of anaplastic large cell lymphoma, and 1 out of 10 of extranodal NK/T-cell lymphoma, nasal type. Double immunostaining in AILT revealed that the majority of c-Maf-positive cells were also positive for CD43 (MT1), CD45RO (UCHL-1), and CD4 but were negative for CD20 (L26). Additionally, cyclins D1 and D2, which stimulate cell cycle progression, were overexpressed in a large number of the c-Maf-positive AILT samples. Quantitative reverse-transcription polymerase chain reaction analysis also showed that c-Maf was overexpressed in 8/31 cases of AILT, 0/19 cases of PTCL(U), 0/11 cases of anaplastic large cell lymphoma, 0/10 cases of extranodal NK/T-cell lymphoma, nasal type, and 2/8 cases of multiple myeloma, presenting significant difference between AILT and PTCL(U) (P=0.016, chi test). These findings strongly suggest that CD4-positive neoplastic T cells in AILT show c-Maf expression and provide new insight into the pathogenesis of AILT suggesting c-Maf to be a useful diagnostic marker for AILT.

Osteopontin dysregulation and lytic bone lesions in multiple myeloma.

Osteopontin (OPN), a secreted phosphoprotein involved in immune regulation and bone homeostasis, is a major component of bone, the natural habitat of long-lived plasma cells and multiple myeloma (MM). We show that only some MM cell lines and primary patient samples express OPN at high levels. High OPN expression inversely correlates with bone disease. When we subdivide MM into molecular subtypes, OPN is significantly upregulated in patients with maf translocations, particularly in the fraction lacking bone disease. OPN is produced in osteolytic lesions: we propose that MM-derived OPN plays a critical role in bone disease by protecting bone from destruction.

Potential roles of large mafs in cell lineages and developing pancreas.

OBJECTIVES: Maf is a family of transcription factor proteins characterized by a typical bZip structure, and mafA, a member of the large-maf family, is a strong transactivator of insulin in cell lines. The present study investigated the expression profiles of the large-maf family proteins in porcine pancreatic tissue and in primary culture cells. METHODS: Immunohistochemical staining was performed to localize each maf protein. Messenger RNA expression was quantitated by real-time polymerase chain reaction, and protein expression was assessed by Western blotting. RESULTS: Islet formation was not as clear in newborn pancreatic tissue as in adult pancreatic tissue. MafA- and c-maf-positive cells were more diffusely localized in pancreatic tissue with fewer mafB-positive cell clusters scattered throughout. By contrast, islet formation was clearer, and positive staining for mafA and c-maf tended to be more prominent in the islets of adult pancreatic tissue. Messenger RNA and protein expressions were consistent with the immunohistochemical findings. MafA, mafB, and c-maf coexpressed with insulin-positive cells, and c-maf coexpressed with glucagon-positive cells in adult porcine pancreas based on the results of a double-staining study. CONCLUSIONS: Large mafs were identified in normal porcine and human pancreas, and the expression levels and localizations of the large mafs in newborn and adult pancreatic tissues differed. Mafs may play important roles in establishing endocrine function during pancreatic cell differentiation.