Paired Box Gene 5 Research Articles

Astragalus polysaccharides ameliorates experimental colitis by regulating memory B cells metabolism

It is well-established that the reduced Memory B cells (MBCs) play an important role in the pathogenesis of ulcerative colitis (UC), rendering them a potential therapeutic target for UC intervention. Astragalus polysaccharide (APS), a primary active constituent derived from the classic traditional Chinese medicine Astragalus membranaceus (AM), has been used for centuries in the treatment of UC in both human and animal subjects due to its renowned immunomodulatory properties. However, it is unknown whether APS can regulate MBCs to alleviate experimental colitis. In the present investigation, the murine colitis was successfully induced using dextran sulphate sodium (DSS) and subsequently treated with APS for a duration of 7 days. APS exhibited significant efficacy in reducing the disease activity index (DAI), colonic weight index, the index of colonic weight/colonic length. Furthermore, APS mitigated colonic pathological injuries, restored the colonic length, elevated the immunoglobulin A (IgA), transforming growth factor-β1 (TGF-β1) and interleukin (IL)-10 levels, while concurrently suppressing IgG, IgM, IL-6, tumor necrosis factor alpha (TNF-α) levels. Crucially, the quantities of MBCs, IgA+MBCs and forkhead box P3 (Foxp3+) MBCs were notably increased along with a concurrent decrease in IgG1+MBCs, IG2a+MBCs, IgG2b+MBCs after APS administration in colitis mice. Additionally, the Mitotracker red expressions of MBCs and their subgroups demonstrated a significantly up-regulation. Meanwhile, the transcriptomics analysis identified mitochondrial metabolism as the predominant and pivotal mechanism underlying APS-mediated mitigation of DSS-induced colitis. Key differentially expressed genes, including B-cell linker (BLNK), aldehyde dehydrogenase 1A1 (ALDH1A1), B-cell lymphoma 6 (BCL-6), B-lymphocyte-induced maturation protein 1 (Blimp-1), paired box gene 5 (PAX5), purinergic 2 × 7 receptor (P2X7R), B Cell activation factor (BAFF), B Cell activation factor receptor (BAFFR), CD40, nuclear factor kappa-B (NF-κB), IL-6 and so on were implicated in this process. These mRNA expressions were validated through quantitative polymerase chain reaction (qPCR) and immunohistochemistry. These findings revealed that APS effectively restored MBCs and their balance to ameliorate DSS-induced colitis, which was potentially realized via promoting mitochondrial metabolism to maintain MBCs activation.

EBF1, PAX5, and MYC: regulation on B cell development and association with hematologic neoplasms.

During lymphocyte development, a diverse repertoire of lymphocyte antigen receptors is produced to battle against pathogens, which is the basis of adaptive immunity. The diversity of the lymphocyte antigen receptors arises primarily from recombination-activated gene (RAG) protein-mediated V(D)J rearrangement in early lymphocytes. Furthermore, transcription factors (TFs), such as early B cell factor 1 (EBF1), paired box gene 5 (PAX5), and proto-oncogene myelocytomatosis oncogene (MYC), play critical roles in regulating recombination and maintaining normal B cell development. Therefore, the aberrant expression of these TFs may lead to hematologic neoplasms.

PAX5 activates telomerase activity and proliferation in keloid fibroblasts by transcriptional regulation of SND1, thus promoting keloid growth in burn-injured skin.

Staphylococcal nuclease domain-containing 1 (SND1) that functioned as an oncogene in a variety of tumors was upregulated in burn-injured skin tissues, and this study aims to investigate the effect of SND1 on keloid and elucidate the underlying mechanism. Keloid fibroblasts (KFs) and normal skin fibroblasts (NFs) were isolated from the keloid tissues and adjacent normal skin tissues of keloid patients. The SND1 expression was assessed in keloid tissues and KFs with Western blot assay. Gain- and loss-of-function experiments were performed to investigate the role of SND1 in proliferation, colony formation, telomerase activity, expression of fibrogenic genes and production of pro-inflammatory factors in KFs. Chromatin immunoprecipitation (CHIP) and Dual-luciferase reporter gene assays were used to verify the interaction of Paired-box gene 5 (PAX5) on SND1 promoter. Then, a series of rescue experiments were performed to verify the effects of SND1 overexpression on PAX5 knockdown-mediated KF functions. Finally, the role of SND1 in keloid formation in vivo was validated in mice with keloid implantation. SND1 was upregulated in keloid tissues and KFs. SND1 positively regulated proliferation, colony formation, telomerase activity, production of pro-inflammatory factors and expression of fibrogenic genes. PAX5 directly bound to the SND1 promoter to transcriptionally regulate SND1 expression and positively regulated SND1-mediated KF functions via the ERK/JNK pathway. In vivo assay further demonstrated that SND1 displayed a positive effect on keloid formation. SND1 transcriptionally regulated by PAX5 promotes keloid formation through activating telomerase activity via the ERK/JNK signaling pathways, which provides a promising therapeutic target for clinical treatment of burned skin keloid.

Upregulation of miR-1254 promotes Hepatocellular Carcinoma Cell Proliferation, Migration, and Invasion via Inactivation of the Hippo-YAP signaling pathway by decreasing PAX5.

Increasing evidence suggests that microRNAs (miRNAs) affect the progression of hepatocellular carcinoma (HCC). However, the exact function and mechanism of miR-1254 in HCC remains unclear. This study explored the effects of miR-1254 on the biological behavior of HCC cells and determined the underlying mechanism. RT-qPCR was used to detect the expression of miR-1254. Gain- or loss-of-function assays determined if miR-1254 affected the biological function of HCC cells in vitro. Dual luciferase reporter assays confirmed the target gene of miR-1254. Tumor xenografts in mice were used to explore the effects of miR-1254 on tumorigenesis and metastasis of HCC. miR-1254 was upregulated in HCC tissues and cell lines and linked to larger tumor size, aggressive vascular invasion and higher Edmondson grade. Lentiviral-based overexpression and knockdown experiments indicated that miR-1254 promoted proliferation, migration, invasion, and the epithelial-mesenchymal transition of HCC cells. The paired box gene 5 (PAX5) was downregulated in HCC tissues, negatively correlated with miR-1254 expression, and confirmed to be a direct target of miR-1254. Restoration of PAX5 reversed the effects of miR-1254 on the biological behavior of HCC cells. Advanced mechanism studies suggested that PAX5 might mediate miR-1254 by regulating the Hippo signaling pathway. Tumor xenografts in mice confirmed that miR-1254 promoted tumorigenesis and metastasis, and led to poor survival. In conclusion, miR-1254 promoted proliferation, migration, and invasion of HCC cells via decreasing Hippo signaling through targeting PAX5 in vitro and in vivo. This miRNA might be a therapeutic target for HCC.

Transactivation of PTGS2 by PAX5 signaling potentiates cisplatin resistance in muscle-invasive bladder cancer cells

Cisplatin (CDDP)-based systematic chemotherapy remains the mainstay of treatment for muscle-invasive bladder cancer (MIBC). However, acquired resistance to CDDP, a multifactorial process governed by an array of signals acting at different levels, is the major problem in BC treatment. Here, we report for the first time that, expression of Paired-box gene 5 (PAX5), a B-cell essential transcription factor, was significantly induced in CDDP-resistant BC tissues and in experimentally-induced CDDP-resistant BC cells. Inhibition of PAX5 expression by shRNA treatment effectively improved CDDP sensitivity in BC cells, whereas overexpression of PAX5 potentiated CDDP resistance through supporting BC cell survival. Mechanistically, using luciferase reporter and chromatin immunoprecipitation assays, we identified prostaglandin-endoperoxide synthase 2 (PTGS2, also called COX2), a potent enzyme responsible for prostanoids formation and inflammatory response, as the direct down-stream target of PAX5. PAX5 exerted its oncogenic function during the pathogenesis of CDDP resistance via stimulation of PTGS2 transcription. These observations collectively suggest that dysregulation of PAX5/PTGS2 cascade plays a causal role in the induction of CDDP resistance and gene silencing approaches targeting this pathway may therefore provide a novel therapeutic strategy for overcoming CDDP resistance in BC.

PAX5α and PAX5β mRNA expression in breast Cancer: Relation to serum P53 and MMP2

Background: Many studies evaluated the role of paired box gene 5 (PAX5) in breast cancer. However, few investigated PAX5α and PAX5β isoforms individually. Objective: The aim of the present study is to evaluate mRNA expression of PAX5α and PAX5β in breast cancer and assessing their underlying pathological roles through investigating their correlations to matrix metalloproteinase2 (MMP2), p53 and cancer antigen 15-3 (CA15-3). Methods: Evaluation of PAX5α and PAX5β RNA expression in breast tissue samples was done by real-time PCR, serum MMP2 and p53 by ELISA and CA15-3 by chemiluminescent immunoassay in 55 infiltrating ductal carcinoma patients, 25 fibroadenoma patients and 15 control females who underwent plastic breast surgery. Results: PAX5α RNA was reduced while PAX5β RNA was elevated in breast cancer patients compared to other groups (p<0.01). The best cutoff value of PAX5α was ⩽1.15 with 78.2% sensitivity and 90% specificity and for PAX5β it was ⩾2.01 with 83.6% sensitivity and 82.5% specificity. The RNA expression of PAX5β was higher in advanced grades and stages (p<0.01), and significantly correlated to p53 (r: 0.51, p<0.01), and CA15-3 (r: 0.45, p<0.01). Moreover, PAX5α was negatively correlated to PAX5β (r: −0.42, p<0.01), p53 (r: −0.53, p<0.01) and MMP2 (r: −0.22, p<0.05). Conclusions: The downregulation of PAX5α with upregulation of PAX5β RNA expressions could highlight the possible oncogenic role of PAX5 as a transcriptional factor in tumorigenesis and progression of breast cancer.

The complex translocation (9;14;14) involving IGH and CEBPE genes suggests a new subgroup in B-lineage acute lymphoblastic leukemia.

Many subtypes of acute lymphoblastic leukemia (ALL) are associated with specific chromosomal rearrangements. The complex translocation t(9;14;14), a variant of the translocation (14;14)(q11;q32), is a rare but recurrent chromosomal abnormality involving the immunoglobulin heavy-chain (IGH) and CCAAT enhancer-binding protein (CEBPE) genes in B-lineage ALL (B-ALL) and may represent a new B-ALL subgroup. We report here the case of a 5-year-old girl with B-ALL, positive for CD19, CD38 and HLA-DR. A direct technique and G-banding were used for chromosomal analysis and fluorescentin situ hybridization (FISH) with BAC probes was used to investigate a possible rearrangement of the IGH andCEBPE genes. The karyotype exhibit the chromosomal aberration 46,XX,del(9)(p21),t(14;14)(q11;q32). FISH with dual-color break-apartIGH-specific and CEPBE-specific bacterial artificial chromosome (BAC) probes showed a complex t(9;14;14) associated with a deletion of cyclin-dependent kinase inhibitor 2A (CDKN2A) and paired box gene 5 (PAX5) at 9p21-13 and duplication of the fusion gene IGH-CEBPE.

Identification of two promoters for human D-amino acid oxidase gene: implication for the differential promoter regulation mediated by PAX5/PAX2.

D-amino acid oxidase (DAO) is a flavoenzyme that metabolizes d-amino acids. Until now, the DAO expression mechanism is still unclear. Our assessment of human DAO (hDAO) promoter activity using luciferase reporter system indicated the proximal upstream region of exon1 (-237/+1) has promoter activity (P1). Interestingly, we identified an alternative promoter in the proximal upstream region of exon2 (+4,126/+4,929) (P2). This alternative promoter has stronger activity than that of P1. Our results also revealed a negative regulatory segment (+1,163/+1,940) in intron1; that would act in concert with P1 and P2. Bioinformatics analyses elucidated the conservation of transcription factor PAX5 family binding sites among species. These sites (-60/-31) and (+4,464/+4,493), locate in P1 and P2 of hDAO, respectively. Gel shift assays demonstrated P1 contains a site (-60/-31) for PAX5 binding while P2 has three sites for both paired box gene 2 (PAX2) and paired box gene 5 (PAX5) binding. The dual roles of PAX5 family in regulating hDAO transcription by modulating promoter activity of P1 and activating promoter activity of P2 were implicated based on the site-directed mutagenesis experiment. Altogether, our data suggested the differential regulation of hDAO expression by two promoters whose activities may be modulated by the binding of PAX2 and PAX5.

Positive intergenic feedback circuitry, involving EBF1 and FOXO1, orchestrates B-cell fate

Recent studies have identified a number of transcriptional regulators, including E2A, early B-cell factor 1 (EBF1), FOXO1, and paired box gene 5 (PAX5), that promote early B-cell development. However, how this ensemble of regulators mechanistically promotes B-cell fate remains poorly understood. Here we demonstrate that B-cell development in FOXO1-deficient mice is arrested in the common lymphoid progenitor (CLP) LY6D(+) cell stage. We demonstrate that this phenotype closely resembles the arrest in B-cell development observed in EBF1-deficient mice. Consistent with these observations, we find that the transcription signatures of FOXO1- and EBF1-deficient LY6D(+) progenitors are strikingly similar, indicating a common set of target genes. Furthermore, we found that depletion of EBF1 expression in LY6D(+) CLPs severely affects FOXO1 mRNA abundance, whereas depletion of FOXO1 activity in LY6D(+) CLPs ablates EBF1 transcript levels. We generated a global regulatory network from EBF1 and FOXO1 genome-wide transcription factor occupancy and transcription signatures derived from EBF1- and FOXO1-deficient CLPs. This analysis reveals that EBF1 and FOXO1 act in a positive feedback circuitry to promote and stabilize specification to the B-cell lineage.

Establishment of an orthotopic transplantation tumor model of hepatocellular carcinoma in mice.

To improve the outcome of orthotopic transplantation in a mouse model, we used an absorbable gelatin sponge (AGS) in nude mice to establish an orthotopic implantation tumor model. MHCC-97L hepatocellular carcinoma (HCC) cells stably expressing the luciferase gene were injected into the subcutaneous region of nude mice. One week later, the ectopic tumors were harvested and transplanted into the left liver lobe of nude mice. The AGS was used to establish the nude mouse orthotopic implantation tumor model. The tumor suppressor gene, paired box gene 5 (PAX5), which is a tumor suppressor in HCC, was transfected into HCC cells to validate the model. Tumor growth was measured by bioluminescence imaging technology. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and histopathology were used to confirm the tumorigenicity of the implanted tumor from the MHCC-97L cell line. We successfully developed an orthotopic transplantation tumor model in nude mice with the use of an AGS. The success rate of tumor transplantation was improved from 60% in the control group to 100% in the experimental group using AGS. The detection of fluorescent signals showed that tumors grew in all live nude mice. The mice were divided into 3 groups: AGS-, AGS+/PAX5- and AGS+/PAX5+. Tumor size was significantly smaller in PAX5 transfected nude mice compared to control mice (P < 0.0001). These fluorescent signal results were consistent with observations made during surgery. Pathologic examination further confirmed that the tissues from the ectopic tumor were HCC. Results from RT-PCR proved that the HCC originated from MHCC-97L cells. Using an AGS is a convenient and efficient way of establishing an indirect orthotopic liver transplantation tumor model with a high success rate.

Epigenetic inactivation of paired box gene 5, a novel tumor suppressor gene, through direct upregulation of p53 is associated with prognosis in gastric cancer patients

Using genome-wide methylation screening, we identified that paired box gene 5 (PAX5) is involved in human cancer development. However, the function of PAX5 in gastric cancer (GC) development is largely unclear. We analyzed its epigenetic inactivation, biological functions and clinical application in GC. PAX5 was silenced in seven out of eight GC cell lines. A significant downregulation was also detected in paired gastric tumors compared with adjacent non-cancerous tissues. The downregulation of PAX5 was closely linked to the promoter hypermethylation status and could be restored with demethylation treatment. Ectopic expression of PAX5 in silenced GC cell lines (AGS and BGC823) inhibited colony formation and cell viability, arrested cell cycle, induced apoptosis, suppressed cell migration and invasion and repressed tumorigenicity in nude mice. Consistent with the induction of apoptosis by PAX5 in vitro, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining showed significantly enhanced apoptotic cells in PAX5-expressed tumors compared with the vector control tumors. On the other hand, knockdown of PAX5 by PAX5-short hairpin RNA increased the cell viability and proliferation. The anti-tumorigenic function of PAX5 was revealed to be mediated by upregulating downstream targets of tumor protein 53 (p53), p21, BCL2-associated X protein, metastasis suppressor 1 and tissue inhibitors of metalloproteinase 1, and downregulating BCL2, cyclin D1, mesenchymal-epithelial transition factor (MET) and matrix metalloproteinase 1. Immunoprecipitation assay demonstrated that PAX5 directly bound to the promoters of p53 and MET. Moreover, PAX5 hypermethylation was detected in 77% (144 of 187) of primary GCs compared with 10.5% (2/19) of normal gastric tissues (P<0.0001). GC patients with PAX5 methylation had a significant poor survival compared with the unmethylated cases as demonstrated by Cox regression model and log-rank test. In conclusion, PAX5 is a novel functional tumor suppressor in gastric carcinogenesis. Detection of methylated PAX5 can be utilized as an independent prognostic factor in GC.

FOXP1 and PAX5 are rare but recurrent translocations partners in acute lymphoblastic leukemia

Here, we report the case of a 57-year-old man, who was diagnosed with B-cell acute lymphoblastic leukemia (B-ALL). His diagnostic workup identified a translocation t(3;9)(p13;p13). This is the fifth case reported to date that involved the forkhead box P1 gene (FOXP1) and paired box gene 5 (PAX5). The PAX5-FOXP1 translocation is a nonrandom aberration, which is recurrent in both childhood and in adult B-ALL, and may contribute to leukemogenesis by blocking differentiation of hematopoietic cells into mature B-cells.

PAX5 (paired box gene 5)

Review on PAX5 (paired box gene 5), with data on DNA, on the protein encoded, and where the gene is implicated.

B cell activator PAX5 promotes lymphomagenesis through stimulation of B cell receptor signaling

The presumed involvement of paired box gene 5 (PAX5) in B-lymphomagenesis is based largely on the discovery of Pax5-specific translocations and somatic hypermutations in non-Hodgkin lymphomas. Yet mechanistically, the contribution of Pax5 to neoplastic growth remains undeciphered. Here we used 2 Myc-induced mouse B lymphoma cell lines, Myc5-M5 and Myc5-M12, which spontaneously silence Pax5. Reconstitution of these cells with Pax5-tamoxifen receptor fusion protein (Pax5ER(TAM)) increased neoplastic growth in a hormone-dependent manner. Conversely, expression of dominant-negative Pax5 in murine lymphomas and Pax5 knockdown in human lymphomas negatively affected cell expansion. Expression profiling revealed that Pax5 was required to maintain mRNA levels of several crucial components of B cell receptor (BCR) signaling, including CD79a, a protein with the immunoreceptor tyrosine-based activation motif (ITAM). In contrast, expression of 2 known ITAM antagonists, CD22 and PIR-B, was suppressed. The key role of BCR/ITAM signaling in Pax5-dependent lymphomagenesis was corroborated in Syk, an ITAM-associated tyrosine kinase. Moreover, we observed consistent expression of phosphorylated BLNK, an activated BCR adaptor protein, in human B cell lymphomas. Thus, stimulation of neoplastic growth by Pax5 occurs through BCR and is sensitive to genetic and pharmacological inhibitors of this pathway.

IRF4 negatively regulates proliferation of germinal center B cell-derived Burkitt's lymphoma cell lines and induces differentiation toward plasma cells

To investigate the roles of interferon regulatory factor 4 (IRF4) in B-cell development, we established germinal center B cell-derived Burkitt's lymphoma cell lines that exogenously express IRF4. Daudi-IRF4 expressed IRF4 in the presence of doxycycline (inducible expression), and Raji-IRF4 constitutively expressed an IRF4-estrogen receptor chimeric protein, which was activated by 4-hydroxytamoxifen. Expression or activation of IRF4 resulted in growth inhibition accompanied by accumulation of cells in G0/G1. Upregulation of the plasma cell markers CD38 and CD138 and downregulation of the germinal center cell marker B-cell lymphoma 6 (BCL6) were also observed. Furthermore, mRNAs for BCL6 and paired box gene 5 (PAX5) were decreased and those for B-lymphocyte-induced maturation protein-1 (BLIMP1)/PR domain containing 1 (PRDM1) and X-box binding protein 1 (XBP1) were increased, which corresponds to the characteristic changes in transcription factor expression in B cells differentiating toward plasma cells. Impairment in proliferation and differentiation toward plasma cells induced by IRF4 were not inhibited by enforced expression of BCL6. These results suggest that IRF4 inhibits cell cycle progression of germinal center B cell-derived Burkitt's lymphoma cells and induces terminal differentiation toward plasma cells through mechanisms independent of BCL6 downregulation.

Effects of Growth Hormone on the Differentiation of Mouse B-Lymphoid Precursors

Growth hormone (GH) has been known to enhance immune responses directly or through insulin-like growth factor-I (IGF-I). The present study aimed to clarify the roles of GH in the differentiation of B-lineage precursors. In short-term bone marrow cultures, which contained stem cells and early B-lineage cells, GH (10 μg/L) treatment for one day decreased the percentages of stem cells (0.5-fold) and increased those of B-lineage cells (1.4-fold). Furthermore, GH changed the expressions of transcription factors for B cell progenitors differentiation such as paired box gene-5 (Pax-5), immunoglobulin-associated-α (Ig-α)/CD79a, Ig-β/CD79b, and IGF-I. Thus, a physiological concentration of GH stimulated the differentiation of B-lymphoid precursors from bone marrow stem cells. Since mRNAs of both GH and GH receptor were present in stem cells and B-cell precursors in bone marrow, GH may modulate B-lymphoid precursors development in an autocrine or paracrine manner in bone marrows.