Transcription factor Ets-2 regulates the expression of key lymphotropic factors.

Transcription factor Ets-2 downregulates the expression of cytokine genes and HIV-1 in resting T-cells. Herein, we studied whether Ets-2 regulates the expression of lymphotropic factors (LFs) NFAT2, NF-κΒ/p65, c-Jun, c-Fos, which regulate the activation/differentiation of T-cells, and kinase CDK10, which controls Ets-2 degradation and repression activity. In silico analysis revealed Ets-2 binding sites on the promoters of NFAT2, c-Jun, c-Fos. The T-cell lines Jurkat (models T-cell signaling/activation) and H938 (contains the HIV-1-LTR) were transfected with an Ets-2 overexpressing vector, in the presence/absence of mitogens. mRNA and protein levels were assessed by qPCR and Western immunoblotting, respectively. Ets-2 overexpression in unstimulated Jurkat increased NFAT2 and c-Jun mRNA/protein, c-Fos mRNA and NF-κΒ/p65 protein, and decreased CDK10 protein. In unstimulated H938, Ets-2 upregulated NFAT2, c-Jun and CDK10 mRNA/protein and NF-κΒ/p65 protein. In stimulated Jurkat, Ets-2 increased NFAT2, c-Jun and c-Fos mRNA/protein and decreased CDK10 mRNA/protein. In stimulated H938 Ets-2 increased NFAT2, c-Jun and c-Fos protein and reduced CDK10 protein levels. Furthermore, Ets-2 overexpression modulated the expression of pro- and anti-apoptotic genes in both cell lines. Ets-2 upregulates the expression of key LFs involved in the activation of cytokine genes or HIV-1 in T-cells, either through its physical interaction with gene promoters or through its involvement in signaling pathways that directly impact their expression. The effect of Ets-2 on CDK10 expression in H938 vs Jurkat cells dictates that, additionally to Ets-2 degradation, CDK10 may facilitate Ets-2 repression activity in cells carrying the HIV-1-LTR, contributing thus to the regulation of HIV latency in virus-infected T-cells.

Transcription Factor Ets-2 Acts as a Preinduction Repressor of Interleukin-2 (IL-2) Transcription in Naive T Helper Lymphocytes.

IL-2 is the first cytokine produced when naive T helper (Th) cells are activated and differentiate into dividing pre-Th0 proliferating precursors. IL-2 expression is blocked in naive, but not activated or memory, Th cells by the transcription factor Ets-2 that binds to the antigen receptor response element (ARRE)-2 of the proximal IL-2 promoter. Ets-2 acts as an independent preinduction repressor in naive Th cells and does not interact physically with the transcription factor NFAT (nuclear factor of activated T-cells) that binds to the ARRE-2 in activated Th cells. In naive Th cells, Ets-2 mRNA expression, Ets-2 protein levels, and Ets-2 binding to ARRE-2 decrease upon cell activation followed by the concomitant expression of IL-2. Cyclosporine A stabilizes Ets-2 mRNA and protein when the cells are activated. Ets-2 silences directly constitutive or induced IL-2 expression through the ARRE-2. Conversely, Ets-2 silencing allows for constitutive IL-2 expression in unstimulated cells. Ets-2 binding to ARRE-2 in chromatin is stronger in naive compared with activated or memory Th cells; in the latter, Ets-2 participates in a change of the IL-2 promoter architecture, possibly to facilitate a quick response when the cells re-encounter antigen. We propose that Ets-2 expression and protein binding to the ARRE-2 of the IL-2 promoter are part of a strictly regulated process that results in a physiological transition of naive Th cells to Th0 cells upon antigenic stimulation. Malfunction of such a repression mechanism at the molecular level could lead to a disturbance of later events in Th cell plasticity, leading to autoimmune diseases or other pathological conditions.

HIV-1 Transcriptional Activator Tat Inhibits IL2 Expression by Preventing the Presence of Pol II on the IL2 Promoter.

HIV-1 infection leads to a gradual loss of T helper cells, chronic immune activation, and eventual immune system breakdown. HIV-1 causes deregulation of the expression of IL-2, a cytokine important for T helper cell growth and survival, which is downregulated in HIV-1 patients. The present study addresses the regulation of IL2 expression via HIV-1 Tat transcriptional activator. We used J-LAT cells, a T cell line that serves as a latency model for studies of HIV-1 expression in T cells, and as controls a T cell line lacking HIV-1 elements and a T cell line with a stably integrated copy of the HIV-1-LTR promoter. We show that endogenously expressed Tat inhibits IL2 transcription in J-Lat cells via its presence in the ARRE-1/2 elements of the IL2 promoter and that the inhibition of IL2 expression is mediated by Tat inhibiting Pol II activity at the IL2 promoter, which is mediated by preventing the presence of Pol II at the ARRE-1/2 elements. Overall, Tat is present at the IL2 promoter, apart from its cognate HIV-1 LTR target. This supports our current knowledge of how HIV-1 affects the host transcriptional machinery and reflects the potential of Tat to disrupt transcriptional regulation of host genes to manipulate cell responses.

Different immunosuppressive combinations on T-cell regulation in renal transplant recipients.

BACKGROUND/AIMS: Recent studies indicate that regulatory T-cells (Tregs) promote transplant tolerance. We studied Treg levels in 39 stable renal transplant recipients to determine the sizes of the Treg populations and the effects of treatment regimens thereof. METHODS: All patients (19 with good graft function and 20 with chronic allograft nephropathy) received induction therapy (basiliximab) and were on triple immunosuppressive regimens with calcineurin inhibitors (cyclosporine or tacrolimus), mycophenolate mofetil (MMF) or everolimus and steroids. Twenty healthy subjects served as controls. Whole blood samples were stained with anti-CD4, CD25, CD127, and FoxP3 antibodies and analyzed by flow cytometry to determine CD4+CD25(high)FoxP3+/- and CD4+ CD25(high)CD127(-/low) Treg levels. RESULTS: All patients had significantly reduced CD4+CD25(high)FoxP3+/- but no CD4+ CD25(high)CD127(-/low) Treg levels compared to controls. Renal allograft function did not correlate with Treg levels. Statistically significant correlations between CD4+CD25(high)Foxp3+ Tregs and tacrolimus levels and CD4+CD25(high)Foxp3- Tregs and HLA-DR mismatching were detected. Patients receiving MMF had significantly higher CD4+CD25(high)Foxp3+ Tregs compared to patients on everolimus who were also receiving lower doses of calcineurin inhibitors. CONCLUSION: Overall, immunosuppression lowers CD4+CD25(high)FoxP3+/- Treg levels significantly in the periphery in renal transplant recipients. In addition, different immunosuppressive regimens have different impacts on CD4+CD25(high)FoxP3+ Tregs, a fact that may influence long-term allograft survival.

Expression patterns of leptin receptor (OB-R) isoforms and direct in vitro effects of recombinant leptin on OB-R, leptin expression and cytokine secretion by human hematopoietic malignant cells.

Several studies have implicated leptin in the pathophysiology of neoplasias. We investigated the direct effect of leptin on malignant hematopoietic tissue that included: primary acute myeloid leukemia (AML) cells, leukemic cell lines and bone marrow biopsies from multiple myeloma (MM) patients. PBMC, T-cells, B-cells and monocytes from healthy subjects served as controls. We defined the patterns of OB-R isoform expression in AML cells and leukemic cell lines in comparison to control cells by RT-PCR. rLeptin upregulated the expression of OB-R and endogenous leptin in AML blasts and certain cell lines but not in control cells. Cytometric Bead Array analysis of pro- and anti-inflammatory cytokines showed that rleptin upregulates IL-6 secretion by AML cells, various cytokines by the leukemic cell lines tested and IL-10 secretion by control PBMC, contributed by monocytes. Western immunoblotting revealed that the effect of rleptin was independent of JAK-2/phospho-JAK-2 protein levels. Finally, MM biopsies stained positive for leptin and, to a lesser extend, OB-R. Immunoreactivity was confined mostly to the nucleus of the myeloma cells. Normal myelocytes, promyelocytes and megakaryocytes stained weakly positive, and erythroid cells were constantly negative. We propose that the leptin/OB-R system is strongly and directly involved in supporting the growth of hematopoietic malignancies.

Construction of an M1GS Ribozyme for Targeted and Rapid mRNA Cleavage; Application on the Ets-2 Oncogene.

BACKGROUND: RNase P-mediated cleavage of target RNAs has been proposed as a promising tool for gene silencing. Ets-2 proto-oncogene controls the expression of a wide variety of genes involved in cancer and immunity. OBJECTIVE: Construction of a functional RNase P-based ribozyme (M1GS303) that targets Ets-2 mRNA. METHODS: The accessible sites for targeting of Ets-2 mRNA were identified by footprinting analysis. M1GS303 ribozyme was constructed by cloning. The activity of the ribozyme in the presence or absence of spiramysin in E. coli cells and human cell lines was quantified by RT-PCR. The efficiency of the ribozyme in silencing the endogenous expression of Ets-2 in human cell lines was examined by RT-PCR, western blot and immunofluorescence analysis. RESULTS: In E. coli cells co-transformed with plasmids bearing M1GS303 and the ets-2 target gene, Ets-2 mRNA was decreased by 93% 12h after IPTG induction in the absence, and after 4h in the presence of spiramycin. Ets-2 was rapidly downregulated in the human embryonic kidney cell line HEK293 and the T-cell line Jurkat transfected with an M1GS303 plasmid; the silencing effect of M1GS303 was considerably faster when the cells were cultured with spiramycin. In Jurkat cells, Ets-2-downregulation resulted in upregulation of the expression of IL-2, IL-4 and IFN-α cytokine genes that have Ets-2 binding sites on their promoters, whereas it had no effect on the expression of the IL-10 gene that lacks Ets-2 binding sites on its promoter. CONCLUSIONS: M1GS303 ribozyme cleaves effectively Ets-2 mRNA in bacteria and mammalian cells, and its activity is enhanced by spiramycin. Downregulation of ets-2 gene in the T-cell line Jurkat upregulates IL-2, IL-4 and IFN-α cytokine genes. M1GS technology may be a better alternative to conventional gene-interference therapies and the delineation of the effects of gene silencing in various pathologies.

Ets-2 Acts As a Transcriptional Repressor of the Human Immunodeficiency Virus Type 1 through Binding to a Repressor-Activator Target Sequence of 5'-LTR.

HIV-1 is transcriptionally active in activated T helper (Th)-cells and inactive in naive or resting memory Th-cells. Ets-2 is a preinduction transcriptional repressor of the IL-2 gene in naive Th-cells and a candidate transcriptional repressor of HIV-1 in the same cells, because the -279 to -250 upstream region of HIV-1-LTR [repressor-activator target sequence (RATS)], that participates in HIV-1-LTR transcriptional silencing, encompasses the AAGGAG Ets-2 binding site. In this proof of concept study, we investigated whether Ets-2 represses the expression of HIV-1. To assess whether Ets-2 can repress HIV-1 transcriptional activation acting through RATS, we transfected Jurkat cells with an Ets-2 overexpression plasmid (pCDNA3-ets-2) or Ets-2 silencing plasmids (ets-2-shRNA) and, as target genes, plasmids carrying the whole HIV-1-LTR sequence (HIV-1-LTR-CAT) or two copies of the RATS sequence (2× RATS-CAT) or a point mutation in the Ets-2 binding site (2× mutantRATS-CAT) or CMV-CAT (control). Ets-2 overexpression resulted in a significant reduction of HIV-1-LTR-CAT and 2× RATS-CAT activities in stimulated cells, but not of the 2× mutantRATS-CAT or CMV-CAT. Ets-2 silencing led to increased activities of HIV-1-LTR-CAT and 2× RATS-CAT in unstimulated cells, but had no effect on the activities of 2× mutantRATS-CAT and CMV-CAT. To assess Ets-2 binding to HIV-1-LTR-RATS in naive Th-cells, we isolated naive Th-cell nuclear proteins and passed them through an Ets-2 antibody column; electrophoretic mobility shift assays were performed using an RATS probe mixed with consecutive protein eluates. Ets-2 bound to the HIV-1-LTR-RATS in a dose-dependent manner. To assess Ets-2 binding to RATS in vivo, Jurkat cells were transfected with 2× RATS-CAT and stained for the Ets-2 protein and the RATS sequence by combining immunofluorescence and fluorescence in situ hybridization techniques. In unstimulated cells, Ets-2 bound to RATS, whereas no binding was observed in stimulated cells. To test for RATS specificity, the same experiments were performed with 2× mutantRATS-CAT, and no binding of Ets-2 was observed. The results were corroborated by chromatin immunoprecipitation assays performed with the same cells. Our results show that Ets-2 is a transcriptional repressor of HIV-1. Repression of HIV-LTR-RATS mediated by Ets-2 may account for the low-level transcription and replication of HIV-1 in naive Th-cells, and contribute to the viral latency and maintenance of viral reservoirs in patients, despite long-term therapy.

Growth hormone attenuates the transcriptional activity of Runx2 by facilitating its physical association with Stat3beta.

UNLABELLED: We document that GH controls osteoblast function by modulating the biological activity of the osteospecific transcription factor Runx2. Evidence is provided for a physical interaction between Runx2 and Stat3beta, which is enhanced by GH and downregulates the transcriptional properties of this key osteogenic regulator. INTRODUCTION: Growth hormone (GH) signals to bone either through insulin-like growth factor-1 or directly by influencing the function of osteoblasts, the bone-forming cells. This study aimed at exploring the molecular events that underlie the direct biological action of GH on osteoblastic cells, and specifically, the effects that it might exert on the function of the bone-specific transcriptional regulator Runx2. MATERIALS AND METHODS: The GH-responsive human osteoblastic cell line Saos-2 was used as our experimental system. Western blot analyses were used to monitor the presence of several parameters known to be affected by GH in these cells (i.e., downregulation of GH receptor, induction of STATs, and extracellular signal-regulated kinase [ERK] mitogen-activated protein kinase [MAPK] pathways). Electrophoretic mobility shift assays were used to assess Runx2 and Stat3 binding activity on an osteoblast-specific element (OSE2) after GH treatment. A combination of yeast two-hybrid and co-immunoprecipitation assays were performed to test for the existence of a physical Runx2.Stat3beta association. Finally, co-transfection experiments were used to investigate the interplay of the two transcription factors on the activity of a p6OSE2-Luc promoter after GH stimulation. RESULTS: We show that GH signaling through Stat3/ERK MAPK potentiates the DNA binding activity of Runx2 but, at the same time, restrains its transcriptional potential. Moreover, a novel physical interaction of Runx2 with transcription factor Stat3beta, which is enhanced by GH stimulation, was documented both in vitro and in vivo. Importantly, this interaction impairs the transcriptional activity of Runx2 without affecting its DNA binding capacity. CONCLUSION: Our data provide the first evidence that GH modulates the transcriptional function of Runx2 in osteoblastic cells by promoting its inhibitory interaction with Stat3beta. Shedding light on such mechanisms will contribute to a better understanding of GH effects on skeletal homeostasis that may impact on decisions at the clinical level, especially in diseases affecting bone quantity and quality (e.g., osteoporosis).

The bone-specific transcriptional regulator Cbfa1 is a target of mechanical signals in osteoblastic cells.

A primary goal of bone research is to understand the mechanism(s) by which mechanical forces dictate the cellular and metabolic activities of osteoblasts, the bone-forming cells. Several studies indicate that osteblastic cells respond to physical loading by transducing signals that alter gene expression patterns. Accumulated data have documented the fundamental role of the osteoblast-specific transcription factor Cbfa1 (core-binding factor) in osteoblast differentiation and function. Here, we demonstrate that low level mechanical deformation (stretching) of human osteoblastic cells directly up-regulates the expression and DNA binding activity of Cbfa1. This effect seems to be fine tuned by stretch-triggered induction of distinct mitogen-activated protein kinase cascades. Our novel finding that activated extracellular signal-regulated kinase mitogen-activated protein kinase physically interacts and phosphorylates endogenous Cbfa1 in vivo (ultimately potentiating this transcription factor) provides a molecular link between mechanostressing and stimulation of osteoblast differentiation. Elucidation of the specific modifiers and cofactors that operate in this mechanotranscription circuitry will contribute to a better understanding of mechanical load-induced bone formation which may set the basis for nonpharmacological intervention in bone loss pathologies.