Promyelocytic Leukemia Zinc Finger Protein Research Articles

Transcriptional Regulatory Cascades in Runx2-Dependent Bone Development

The development of the musculoskeletal system is a complex process that involves very precise control of bone formation and growth as well as remodeling during postnatal life. Although the understanding of the transcriptional mechanisms of osteogenesis has increased considerably, the molecular regulatory basis, especially the gene regulatory network of osteogenic differentiation, is still poorly understood. This review provides the reader with an overview of the key transcription factors that govern bone formation, highlighting their function and regulation linked to Runt-related transcription factor 2 (Runx2). Runx2 as the master transcription factor of osteoblast differentiation, Twist, Msh homeobox 2 (Msx2), and promyelocytic leukemia zinc-finger protein (PLZF) acting upstream of Runx2, Osterix (Osx) acting downstream of Runx2, and activating transcription factor 4 (ATF4) and zinc-finger protein 521 (ZFP521) acting as cofactors of Runx2 are discussed, and their relevance for tissue engineering is presented. References are provided for more in-depth personal study.

Systemic Human T Cell Developmental Processes in Humanized Mice Cotransplanted With Human Fetal Thymus/Liver Tissue and Hematopoietic Stem Cells

In many humanized mouse models, there are few T cells in the engrafted human cell, whereas the number of B cells is high. We attempted to overcome this limitation and investigate whether the entire process of human T cell development arose similarly to the process in humans, as previously reported. To produce an advanced humanized mice model, we transplanted human fetal liver/thymus tissue subrenally and injected human CD34(+) stem cells intravenously into NOD/SCID/IL2Rgamma null (NSG) mice. Humanized mice transplanted with fetal thymus/liver tissues and fetal liver-derived CD34(+) stem cells (FLT+FLCD34) showed higher levels of human cells and T cells than mice transplanted with fetal liver-derived CD34(+) stem cells only (FLCD34). In the transplanted thymus tissue of FLT+FLCD34 mice, thymus seeding progenitors (TSPs), early thymic progenitors (ETPs), pre-T cells, and all the other human T cell populations were identified. In the periphery, FLT+FLCD34 mice have high levels of CD45RA(+) T cells; conversely, FLCD34 mice have higher levels of CD45RO(+) T cells. The CD45RO(+) T cells of FLCD34 mice proliferated rapidly after stimulation and exhibited innate T cells properties, expressing PLZF (promyelocytic leukemia zinc finger protein). Human T cells educated by mouse MHC II in mice without a human thymus differ from normal human T cells. On the basis of these findings, numerous T cell-tropic human diseases could be explored in our humanized mice and molecular aspects of human T cell development could be also studied extensively.

GATA2 Mutations in Pediatric Acute Myeloid Leukemia: A Study of the Japanese Childhood AML Cooperative Study Group.

AbstractAbstract 2536 BackgroundAcute myeloid leukemia (AML) is a complex disease caused by mutations, epigenetic modifications, and deregulated expression of genes leading to increased proliferation and decreased differentiation of hematopoietic progenitor cells. Although many important molecular markers have already been discovered in AML, no known prognosis-associated cytogenetic aberrations or mutations were detectable in a subset of AML patients. In this regard, recent reports of somatic mutations affecting the C-terminal zinc finger (ZF) 2 of GATA2 are intriguing, because these GATA2 mutations were associated with the progression of chronic myeloid leukemia, whereas hereditary ZF2 of GATA2 mutations predispose to AML and myelodysplastic syndrome. GATA2 mutations were also reported as a predisposing gene of the monocytopenia, mycobacterial infection (MonoMAC) syndrome and the dendritic cell, monoctye, B and NK lymphoid deficiency (DCML) syndrome. GATA2 belongs to a family of zinc finger transcription factors, and is important for hematopoietic stem cell proliferation and normal megakaryocytic development. These findings prompted us to search for possible GATA2 mutations in pediatric AML. MethodsTo explore the frequency and clinical impact of GATA2 mutations, we examined 157 Japanese pediatric AML patients, including 13 with FAB-M3 and 10 with Down syndrome (DS) who were treated on different treatment protocol, by PCR following direct sequencing. As GATA2 mutations thus far reported almost exclusively involved exons 4–6 that encode zinc finger 1 and 2 domain, we confined our analysis to these exons. ResultsGATA2 missense mutations were found in 7 out of 157 patients (4.5%). Notably, All of GATA2 mutations were located in ZF2 in this study, although almost all of GATA2 mutations in adult AML were located in ZF1. Wild type GATA2 were found in 3 of 3 AML patients with GATA2 mutation. The other 4 patients had no history of Mono MAC syndrome, suggesting that these mutations were acquired. The zinc finger region of GATA2 is required for binding to promyelocytic leukemia zinc finger (PLZF) protein can interact with GATA2 and can modify its transactivation capacity. Interestingly, 2 GATA2 mutations were found in FAB-M3 in this study, GATA2 mutations also may be associated with acute promyelocytic leukemia. Clinical and molecular features between patients with and without GATA2 mutations were not significantly different in the clinical parameters (WBC, age, sex, etc.), and the outcome of GATA2 mutation positive patients was not poor when compared to GATA2 mutation negative patients: Two of the 7 patients received allogeneic-stem cell transplantation (Allo-SCT) and one of them died of gastrointestinal hemorrhage after SCT. The other 5 patients who did not receive the SCT were still alive. ConclusionGATA2 is a new predisposition gene for pediatric AML and shows functional changes caused by mutations within a highly conserved threonine repeat located in ZF2. Interestingly, MonoMAC and DCML syndromes were not observed in de novo AML patients with GATA2 mutations. Although further investigation is needed, our results indicated that GATA2 mutations were associated with a favorable outcome in pediatric AML. Most of the patients with GATA2 mutations have been classified into an intermediate risk group in our study, however, their favorable outcome suggests that less aggressive treatment strategy without SCT might be appropriate for AML patients carrying GATA2 mutations. Disclosures:No relevant conflicts of interest to declare.

The (pro)renin receptor ((P)RR) can act as a repressor of Wnt signalling

The (pro)renin receptor ((P)RR) and Wnt signalling are both involved in different diseases ranging from cardiac and renal end-organ damage to cancer. (P)RR function involves signalling via the transcription factor promyelocytic leukemia zinc finger protein (PLZF) as well as the furin-mediated generation of vacuolar proton-translocating ATPase (V-ATPase)-associated and soluble (P)RR isoforms. Recently, the (P)RR was described as adaptor protein of Wnt (co)receptors. The aim of this study was to analyse the contribution of these distinct (P)RR functions to Wnt signalling.Using Tcf/Lef reporter gene systems in HEK293T and HepG2 cells and quantification of endogenous axin2 mRNA and protein levels in HEK293T cells we were able to demonstrate that full-length (P)RR acts as a repressor of Wnt signalling in a system preactivated either by Wnt3a stimulation or by constitutively active β-catenin. These repressive effects are mediated by Dvl but are independent of the mutation status of β-catenin. Furthermore, the V-ATPase complex, but not PLZF translocation or renin enzymatic activity, is necessary for the induction of Tcf/Lef-responsive genes by Wnt3a.Our data indicate interference of (P)RR and Wnt cascades, a fact that has to be considered concerning pathophysiology of cardio-renal and oncological entities as well as in drug development programs targeting (P)RR or Wnt pathways.

Development of promyelocytic leukemia zinc finger-expressing innate CD4 T cells requires stronger T-cell receptor signals than conventional CD4 T cells

MHC class II-expressing thymocytes and thymic epithelial cells can mediate CD4 T-cell selection resulting in functionally distinct thymocyte-selected CD4 (T-CD4) and epithelial-selected CD4 (E-CD4) T cells, respectively. However, little is known about how T-cell receptor (TCR) signaling influences the development of these two CD4 T-cell subsets. To study TCR signaling for T-CD4 T-cell development, we used a GFP reporter system of Nur77 in which GFP intensity directly correlates with TCR signaling strength. T-CD4 T cells expressed higher levels of GFP than E-CD4 T cells, suggesting that T-CD4 T cells received stronger TCR signaling than E-CD4 T cells during selection. Elimination of Ras GTPase-activating protein enhanced E-CD4 but decreased T-CD4 T-cell selection efficiency, suggesting a shift to negative selection. Conversely, the absence of IL-2-inducible T-cell kinase that causes poor E-CD4 T-cell selection due to insufficient TCR signaling improved T-CD4 T-cell generation, consistent with rescue from negative selection. Strong TCR signaling during T-CD4 T-cell development correlates with the expression of the transcription factor promyelocytic leukemia zinc finger protein. However, although modulation of the signaling strength affected the efficiency of T-CD4 T-cell development during positive and negative selection, the signaling strength is not as important for the effector function of T-CD4 T cells. These findings indicate that innate T-CD4 T cells, together with invariant natural killer T cells and γδ T cells, receive strong TCR signals during their development and that signaling requirements for the development and the effector functions are distinct.

Expression of the promyelocytic leukemia zinc-finger in T-lymphoblastic lymphoma and leukemia has strong implications for their cellular origin and greater association with initial bone marrow involvement

The promyelocytic leukemia zinc-finger (PLZF) is essential for the development of innate T cells (as represented by natural killer T cells) for acquisition of their unique innate immune properties. We evaluated the PLZF protein expression in a variety of immature and mature lymphoid malignancies. PLZF was preferentially expressed in T-lymphoblastic lymphoma/acute lymphoblastic leukemia (T-LBL/ALL) in 50% of the 54 cases. Among 51 cases of peripheral T-cell lymphoma not otherwise specified, only one (2%) expressed PLZF. One mycosis fungoides case expressed PLZF in lymph node involved by tumor. Otherwise, PLZF was not detected in any other type of lymphoma. In T-LBL/ALL, PLZF expression was more common in CD4/CD8 double-negative (67%) or CD8 single-positive subtypes (73%) than in CD4/CD8 double-positive (13%) and CD4 single-positive subtypes (0%) (P=0.001). Importantly, PLZF and CD1a expression were mutually exclusive in T-LBL/ALL (P=0.001). This was also the case for T-cell receptor βF1 expression (P=0.000). Most (96%) of the PLZF-positive T-LBL/ALL cases showed initial bone marrow involvement compared with 39% of PLZF-negative cases (P=0.000). Based on these findings, we suggest that T-LBL/ALLs that express PLZF arise from early immature double-negative thymocytes when the T-cell receptor β chain has not yet expressed or innate T-cell precursors, and strongly imply bone marrow involvement.

Germline stem cell lineage tracing by free‐floating immunofluorescent assay of mouse seminiferous tubule

Whole-mount immunohistochemistry (whole-mount IH) of the seminiferous tubule is widely used to investigate the self-renewal and differentiation of spermatogonial stem cells (SSCs). Examination of the length of spermatogonial cysts is critical for tracing SSCs lineage by using Whole-mount IH. However, it is difficult for antibody molecules to penetrate into the depth of seminiferous epithelium because its thickness and the tight peritubular myoid and basement membrane outside. Here, we developed a free-floating immunofluorescent procedure of mouse seminiferous tubules using regular incubation time and normal antibody concentration. Microscopic results showed that undifferentiated spermatogonia were positively labeled by promyelocytic leukemia zinc finger protein, E-cadherin, and glial cell line-derived neurotrophic factor family receptor alpha 1, respectively. Spermatogonial cysts in varied length were revealed clearly and spermatogonia subpopulations including A(single) (A(s)), A(paired) (A(pr)), and A(aligned) (A(al)) were distinguished in lower background images. This method provides us an alternate simple way to trace the lineage of individual SSCs and show their three-dimensional locations and distributions within their niches anatomically in next step.

Promyelocytic Leukemia Zinc Finger Protein Activates GATA4 Transcription and Mediates Cardiac Hypertrophic Signaling from Angiotensin II Receptor 2

BackgroundPressure overload and prolonged angiotensin II (Ang II) infusion elicit cardiac hypertrophy in Ang II receptor 1 (AT1) null mouse, whereas Ang II receptor 2 (AT2) gene deletion abolishes the hypertrophic response. The roles and signals of the cardiac AT2 receptor still remain unsettled. Promyelocytic leukemia zinc finger protein (PLZF) was shown to bind to the AT2 receptor and transmit the hypertrophic signal. Using PLZF knockout mice we directed our studies on the function of PLZF concerning the cardiac specific transcription factor GATA4, and GATA4 targets.Methodology and Principal FindingsPLZF knockout and age-matched wild-type (WT) mice were treated with Ang II, infused at a rate of 4.2 ng·kg−1·min−1 for 3 weeks. Ang II elevated systolic blood pressure to comparable levels in PLZF knockout and WT mice (140 mmHg). WT mice developed prominent cardiac hypertrophy and fibrosis after Ang II infusion. In contrast, there was no obvious cardiac hypertrophy or fibrosis in PLZF knockout mice. An AT2 receptor blocker given to Ang II-infused wild type mice prevented hypertrophy, verifying the role of AT2 receptor for cardiac hypertrophy. Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that PLZF bound to the GATA4 gene regulatory region. A Luciferase assay verified that PLZF up-regulated GATA4 gene expression and the absence of PLZF expression in vivo produced a corresponding repression of GATA4 protein.ConclusionsPLZF is an important AT2 receptor binding protein in mediating Ang II induced cardiac hypertrophy through an AT2 receptor-dependent signal pathway. The angiotensin II-AT2-PLZF-GATA4 signal may further augment Ang II induced pathological effects on cardiomyocytes.

Far-Infrared Therapy Induces the Nuclear Translocation of PLZF Which Inhibits VEGF-Induced Proliferation in Human Umbilical Vein Endothelial Cells

Many studies suggest that far-infrared (FIR) therapy can reduce the frequency of some vascular-related diseases. The non-thermal effect of FIR was recently found to play a role in the long-term protective effect on vascular function, but its molecular mechanism is still unknown. In the present study, we evaluated the biological effect of FIR on vascular endothelial growth factor (VEGF)-induced proliferation in human umbilical vein endothelial cells (HUVECs). We found that FIR ranging 3∼10 µm significantly inhibited VEGF-induced proliferation in HUVECs. According to intensity and time course analyses, the inhibitory effect of FIR peaked at an effective intensity of 0.13 mW/cm2 at 30 min. On the other hand, a thermal effect did not inhibit VEGF-induced proliferation in HUVECs. FIR exposure also inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinases in HUVECs. FIR exposure further induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO generation in VEGF-treated HUVECs. Both VEGF-induced NO and reactive oxygen species generation was involved in the inhibitory effect of FIR. Nitrotyrosine formation significantly increased in HUVECs treated with VEGF and FIR together. Inhibition of phosphoinositide 3-kinase (PI3K) by wortmannin abolished the FIR-induced phosphorylation of eNOS and Akt in HUVECs. FIR exposure upregulated the expression of PI3K p85 at the transcriptional level. We further found that FIR exposure induced the nuclear translocation of promyelocytic leukemia zinc finger protein (PLZF) in HUVECs. This induction was independent of a thermal effect. The small interfering RNA transfection of PLZF blocked FIR-increased PI3K levels and the inhibitory effect of FIR. These data suggest that FIR induces the nuclear translocation of PLZF which inhibits VEGF-induced proliferation in HUVECs.

The Promyelocytic Leukemia Zinc Finger Protein: Two Decades of Molecular Oncology

The promyelocytic leukemia zinc finger (PLZF) protein, also known as Zbtb16 or Zfp145, was first identified in a patient with acute promyelocytic leukemia, where a reciprocal chromosomal translocation t(11;17)(q23;q21) resulted in a fusion with the RARA gene encoding retinoic acid receptor alpha. The wild-type Zbtb16 gene encodes a transcription factor that belongs to the POK (POZ and Krüppel) family of transcriptional repressors. In addition to nine Krüppel-type sequence-specific zinc fingers, which make it a member of the Krüppel-like zinc finger protein family, the PLZF protein contains an N-terminal BTB/POZ domain and RD2 domain. PLZF has been shown to be involved in major developmental and biological processes, such as spermatogenesis, hind limb formation, hematopoiesis, and immune regulation. PLZF is localized mainly in the nucleus where it exerts its transcriptional repression function, and many post-translational modifications affect this ability and also have an impact on its cytoplasmic/nuclear dissociation. PLZF achieves its transcriptional regulation by binding to many secondary molecules to form large multi-protein complexes that bind to the regulatory elements in the promoter region of the target genes. These complexes are also capable of physically interacting with its target proteins. Recently, PLZF has become implicated in carcinogenesis as a tumor suppressor gene, since it regulates the cell cycle and apoptosis in many cell types. This review will examine the major advances in our knowledge of PLZF biological activities that augment its value as a therapeutic target, particularly in cancer and immunological diseases.

MHC Class II-Restricted Interaction between Thymocytes Plays an Essential Role in the Production of Innate CD8+ T Cells

We have recently shown that MHC class II-dependent thymocyte-thymocyte (T-T) interaction successfully generates CD4(+) T cells (T-T CD4(+) T cells), and that T-T CD4(+) T cells expressing promyelocytic leukemia zinc finger protein (PLZF) show an innate property both in mice and humans. In this article, we report that the thymic T-T interaction is essential for the conversion of CD8(+) T cells into innate phenotype in the physiological condition. CD8(+) T cells developed in the presence of PLZF(+) CD4(+) T cells showed marked upregulation of eomesodermin (Eomes), activation/memory phenotype, and rapid production of IFN-γ on ex vivo stimulation. Their development was highly dependent on the PLZF expression in T-T CD4(+) T cells and the IL-4 secreted by PLZF(+) T-T CD4(+) T cells. The same events may take place in humans, as a substantial number of Eomes expressing innate CD8(+) T cells were found in human fetal thymi and spleens. It suggests that PLZF(+) T-T CD4(+) T cells in combination with Eomes(+) CD8(+) T cells might actively participate in the innate immune response against various pathogens, particularly in human perinatal period.

A Corticosteroid-Responsive Transcription Factor, Promyelocytic Leukemia Zinc Finger Protein, Mediates Protection of the Cochlea from Acoustic Trauma

Animals can be induced to resist cochlear damage associated with acoustic trauma by exposure to a variety of "conditioning" stimuli, including restraint stress, moderate level sound, heat stress, hypoxia, and corticosteroids. Here we identify in mice a corticosteroid-responsive transcription factor, PLZF (promyelocytic leukemia zinc finger protein), which mediates conditioned protection of the cochlea from acoustic trauma. PLZF mRNA levels in the cochlea are increased following conditioning stimuli, including restraint stress, dexamethasone administration, and moderate-to-high level acoustic stimulation. Heterozygous mutant (luxoid.Zbtb16(LU)/J) mice deficient in PLZF have hearing and responses to acoustic trauma similar to their wild type littermates but are unable to generate conditioning-induced protection from acoustic trauma. PLZF immunoreactivity is present in the spiral ganglion, lateral wall of the cochlea, and organ of Corti, all targets for acoustic trauma. PLZF is also present in the brain and PLZF mRNA in brain is elevated following conditioning stimuli. The identification of a transcription factor that mediates conditioned protection from trauma provides a tool for understanding the protective action of corticosteroids, which are widely used in treating acute hearing loss, and has relevance to understanding the role of corticosteroids in trauma protection.

The Promyelocytic Leukemia Zinc Finger (PLZF) Protein Exerts Neuroprotective Effects in Neuronal Cells and is Dysregulated in Experimental Stroke

Stroke is one of the major medical burdens in industrialized countries. Animal experiments indicate that blockade of the angiotensin AT1 receptor (AT1R) improves neurological outcome after cerebral ischemia. These protective effects are partially mediated by the angiotensin AT2 receptor (AT2R). The transcription factor promyelocytic leukemia zinc finger (PLZF) was identified as a direct adapter protein of the AT2R. Furthermore, our group was able to demonstrate that PLZF also directly binds and mediates the effects of the human (pro)renin receptor [(P)RR] which is involved in brain development. Therefore, we hypothesized that PLZF is involved in neuroprotection. Here we show that PLZF and its receptors (P)RR and AT2R exhibited an ubiquitous expression pattern in different brain regions. Furthermore, stable PLZF overexpression in human neuronal cells was able to mediate neuroprotection in a glutamate toxicity model in vitro. Consistently, PLZF mRNA and protein were downregulated on the ipsilateral side in a stroke model in vivo, whereas the neurodetrimental PLZF target genes cyclin A2 and BID were upregulated under this condition. Further analyses indicated that the neuroprotective AT2R is upregulated upon stable PLZF overexpression in cultured neuronal cells. Finally, reporter gene assays demonstrated the functionality of (P)RR promoter polymorphisms regarding basal and PLZF-induced activity.

Identification of Regulatory Foxp3+ Invariant NKT Cells Induced by TGF-β

Invariant NKT (iNKT) cells were shown to prevent the onset of experimental autoimmune encephalomyelitis in mice following administration of their specific TCR agonist alpha-galactosylceramide. We found that this protection was associated with the emergence of a Foxp3(+) iNKT cell population in cervical lymph nodes. We demonstrate that the differentiation of these cells is critically dependent on TGF-beta in both mice and humans. Moreover, in vivo generation of Foxp3(+) iNKT cells was observed in the TGF-beta-rich environment of the murine gut. Foxp3(+) iNKT cells displayed a phenotype similar to that of Foxp3(+) regulatory T cells, and they suppress through a contact-dependent, glucocorticoid-induced TNFR-mediated mechanism. Nevertheless, Foxp3(+) iNKT cells retain distinctive NKT cell characteristics, such as promyelocytic leukemia zinc finger protein expression and preferential homing to the liver following adoptive transfer, where they stably maintained Foxp3 expression. Our data thus unveil an unexpected capacity of iNKT cells to acquire regulatory functions that may contribute to the establishment of immunological tolerance.

Novel approach to gene expression profiling in Sézary syndrome

Microarray hybridization studies in Sézary syndrome (SS) have compared T lymphocytes from patients with cutaneous T-cell lymphoma with those of normal controls; a major limitation of this design is that significant inherent genetic variability of lymphocyte populations between individuals may produce differences in gene expression unrelated to disease state. The objective of this study was to minimize the heterogeneity of information derived from whole-genome expression analysis and to identify specific genetic differences between highly purified malignant and nonmalignant (control) T cells from the same patient with SS. Peripheral blood mononuclear cells were obtained from a patient with SS, stained with anti-T-cell receptor Vb (TCR-Vb) antibodies, and sorted by multiparameter flow cytometry. Malignant cells expressed the dominant TCR-Vb; control T cells lacked the dominant TCR-Vb but were otherwise phenotypically identical (CD3+CD4+CD45RO+). These cell populations were compared using the Illumina Inc. Sentrix Human-6 expression BeadChip system. Transcriptome analysis using the J5 test, which was selected for data analysis based on an efficiency analysis of competing statistical methods, showed differential expression of 44 genes between the malignant and nonmalignant cell subsets. Promyelocytic leukaemia zinc finger protein (ZBTB16) was the most profoundly upregulated gene in the malignant cell population, while interferon regulatory factor 3 (IRF3) and interferon-induced protein 35 (IFI35), which are important elements of the cellular response to viral infection, were significantly downregulated. The results of this study suggest the feasibility of this novel comparative approach to genomic profiling in SS. Using this method, we identified several differentially expressed genes and pathways not previously described in SS. While these findings require validation in larger studies, they may be important in SS pathogenesis.

Smooth muscle α-actin is a direct target of PLZF: effects on the cytoskeleton and on susceptibility to oncogenic transformation

Changes in cell morphology and rearrangements of the actin cytoskeleton are common features accompanying cell transformation induced by various oncogenes. In this study, we show that promyelocytic leukemia zinc finger protein (PLZF) binds to the promoter of smooth muscle α-actin, reducing mRNA and protein levels encoded by this gene and resulting in a reorganization of the actin cytoskeleton. In cultures of chicken embryo fibroblasts (CEF), this effect on α-actin expression is correlated with a change in cellular phenotype from spindle shaped to polygonal and flattened. This morphological change is dependent on Ras function. The polygonal, flattened CEF show a high degree of resistance to the transforming activity of several oncoproteins. Our results support the conclusion that reorganization of the actin cytoskeleton plays an important role in tumor suppression by PLZF.

Effects of glial cell line-derived neurotrophic factor on the proliferation and differentiation of PLZF and c-Kit of spermatogonial stem cells of rats in vitro

Objective To investigate the effects of glial cell line-derived neurotrophic factor(GDNF)on the gene transcriptions and expressions of promyelocytic leukaemia zinc finger(PLZF)and c-Kit in spermatogonial stem cells(SSC).Methods SSC was cultured in DMEM plus different concentrations of GDNF(0,10,50,100 ng/ml).Quantitative reverse transcription PCR was used to detect the mRNA of PLZF and c-Kit.The expressions of PLZF and c-Kit protein were detected by Western blot assay.Results With the control group and 10ng/ml of GDNF group cell growth has no obvious influence,but with 50 ng/ml of GDNF group and 100 ng/ml of GDNF group cell growth was enhanced significantly.In the control group,the PLZF mRNA was 0.28±0.13 and c-Kit mRNA was 0.65±0.21.In the 10 ng/ml of GDNF group,PLZF mRNA was 0.27±0.14 and c-Kit tuRNA was 0.62±0.19.Compared with the control group,10 ng/ml of GDNF group had not influence on PLZF and c-Kit mRNA.In the 50 ng/ml of GDNF group PLZF mRNA was 0.64±0.28 and c-Kit mRNA was 0.34±0.15.Compared with the control group,50 ng/ml of GDNF enhanced the transcription of PLZF mRNA(P＜0.05),decreased the transeription of c-Kit mRNA (P＜0.05).In the 100 ng/ml of GDNF gmup,PLZF mRNA was 0.68±0.27 and c-Kit mRNA was 0.28±0.18.In the 100 ng/ml of GDNF group,there was no difference compared with 50 ng/ml of GDNF.In the control group,PLZF protein was 0.34±0.13 and c-Kit protein was 0.72±0.27.In 10 ng/ml of GDNF group,PLZF protein was 0.38±0.18 and c-Kit protein was 0.69±0.26.Compared with the control group,10 ng/ml of GDNF had no influence on PLZF and c-Kit protein.In 50 ng/ml of GDNF group,PLZF protein was 0.68±0.26 and c-Kit protein was 0.35±0.15.50 ng/ml of GDNF enhanced the expression of PLZF protein(P＜0.05),decreased the expression of c-Kit protein(P＜0.05).In 100 ng/ml of GDNF group PLZF protein was 0.70±0.27 and c-Kit protein Was 0.32±0.11,100 ng/ml of GDNF had no influence on PLZF and c-Kit protein compared with 50 ng/ml of GDNF.Conclusions Higher concentration of GDNF could enhance the transcription and expression of PLZF,and decrease the transcription and expression of c-Kit in SSC.GDNF has possible role to regulate and control proliferation and differentiation of SSC. Key words: Spermatogonia; Glial cell line-derived neurotrophic factor; Promyelocytic leukaemia zinc finger; Proto-oncogene proteins; c-kit

Inhibition of angiogenesis by the BTB domain of promyelocytic leukemia zinc finger protein

Promyelocytic leukemia zinc finger is a negative regulator of cell cycle progression. In this study, we showed that PLZF inhibits endothelial cell angiogenesis using a human umbilical vein endothelial cell system. We also focused on characterizing the specific function of the BTB domain of PLZF as a novel apoptotic and anti-angiogenic protein via deletion mapping analysis. The BTB domain directly inhibited tube formation, as well as the biological functions of angiostatic activity in vivo, and reduced the expression of p-Akt and p-eNOS, which play a significant role in angiogenesis when stimulated by VEGF. These results strongly suggest that the BTB domain could potentially modulate the apoptotic and anti-angiogenic effects of PLZF.

THY1 is a conserved marker of undifferentiated spermatogonia in the pre-pubertal bull testis

The undifferentiated spermatogonial population consists of stem and progenitor germ cells which function to provide the foundation for spermatogenesis. The stem cell component, termed spermatogonial stem cells (SSCs), is capable of self-renewal and differentiation. These unique attributes have made them a target for novel technologies to enhance reproductive function in males. With bulls, culture and transplantation of SSCs have the potential to enhance efficiency of cattle production and provide a novel avenue to generate transgenic animals. Isolation of SSCs is an essential component for the development of these techniques. In rodents and non-human primates, undifferentiated spermatogonia and SSCs express the surface marker THY1. The hypothesis tested in this study was that THY1 is a conserved marker of the undifferentiated spermatogonial population in bulls. Flow cytometric analyses showed that the THY1+ cell fraction comprises a rare sub-population in testes of pre-pubertal bulls. Immunocytochemical analyses of the isolated THY1+ fraction for expression of VASA showed that this cell population is comprised mostly of germ cells. Additionally, expression of the undifferentiated spermatogonial specific transcription factor promyelocytic leukemia zinc finger (PLZF, ZBTB16) protein was found to be enriched in the isolated THY1+ testis cell fraction. Lastly, xenogeneic transplantation of bull testis cells into seminiferous tubules of immunodeficient mice resulted in greater than sixfold more colonies from isolated THY1+ cells compared to the unselected total testis cell population indicating SSC enrichment. Collectively, these results demonstrate that THY1 is a marker of undifferentiated spermatogonia in testes of pre-pubertal bulls, and isolation of THY1+ cells results in their enrichment from the total testis cell population.

Human endogenous retrovirus protein Rec interacts with the testicular zinc-finger protein and androgen receptor

More than 2000 human endogenous retrovirus (HERV) sequences are present in the human genome, yet only a few are intact and able to produce proteins. The normal functions of these, if any, are unknown, but some HERV proteins have been implicated in cancers, in particular germ-cell cancers. For instance, it has been documented that (i) patients with germ-cell tumours frequently produce antibodies against HERV proteins; (ii) transgenic mice expressing HERV-K (HML-2) rec are prone to testicular carcinoma in situ; and (iii) Rec can bind and suppress a guardian of germline stem-cell pluripotency, the promyelocytic leukaemia zinc-finger protein (PLZF). This study identified the PLZF-related testicular zinc-finger protein (TZFP) as a binding partner of HERV-K (HML-2) Rec. Interactions occurred via the N- and C-terminal domains of Rec and the C-terminal DNA-binding zinc-finger domain of TZFP (aa 375-450). Not much is known about the function of TZFP. The protein is expressed predominantly in the testis, where it functions as a transcriptional repressor that is active during specific stages of spermatogenesis. The most intensely studied function of TZFP is that of a co-repressor of the activated androgen receptor (AR). Here, it was shown that Rec can form a trimeric complex with TZFP and AR, and can relieve the TZFP-mediated repression of AR-induced transactivation. In addition, Rec was able to overcome the direct transcriptional repression by TZFP of the c-myc gene promoter in reporter assays. Thus, HERV-K (HML-2) Rec may function as an oncoprotein by de-repressing oncogenic transcription factors such as AR.