Zsuzsanna S. Nagy

STAT6 transcription factor is a facilitator of the nuclear receptor PPARγ-regulated gene expression in macrophages and dendritic cells.

Summary Peroxisome proliferator-activated receptor γ (PPARγ) is a lipid-activated transcription factor regulating lipid metabolism and inflammatory response in macrophages and dendritic cells (DCs). These immune cells exposed to distinct inflammatory milieu show cell type specification as a result of altered gene expression. We demonstrate here a mechanism how inflammatory molecules modulate PPARγ signaling in distinct subsets of cells. Proinflammatory molecules inhibited whereas interleukin-4 (IL-4) stimulated PPARγ activity in macrophages and DCs. Furthermore, IL-4 signaling augmented PPARγ activity through an interaction between PPARγ and signal transducer and activators of transcription 6 (STAT6) on promoters of PPARγ target genes, including FABP4. Thus, STAT6 acts as a facilitating factor for PPARγ by promoting DNA binding and consequently increasing the number of regulated genes and the magnitude of responses. This interaction, underpinning cell type-specific responses, represents a unique way of controlling nuclear receptor signaling by inflammatory molecules in immune cells.

The PHB1/2 Phosphocomplex Is Required for Mitochondrial Homeostasis and Survival of Human T Cells

Many immune pathologies are the result of aberrant regulation of T lymphocytes. A functional proteomics approach utilizing two-dimensional gel electrophoresis coupled with mass spectrometry was employed to identify differentially expressed proteins in response to T cell activation. Two members of the prohibitin family of proteins, Phb1 and Phb2, were determined to be up-regulated 4–5-fold upon activation of primary human T cells. Furthermore, their expression was dependent upon CD3 and CD28 signaling pathways that synergistically led to the up-regulation (13–15-fold) of Phb1 and Phb2 mRNA levels as early as 48 h after activation. Additionally, orthophosphate labeling coupled with phosphoamino acid analysis identified Phb1 to be serine and Phb2 serine and tyrosine phosphorylated. Tyrosine phosphorylation of Phb2 was mapped to Tyr248 using mass spectrometry and confirmed by mutagenesis and phosphospecific antibodies. In contrast to previous reports of Phb1 and Phb2 being nuclear localized, subcellular fractionation, immunofluorescent, and electron microscopy revealed both proteins to localize to the mitochondrial inner membrane of human T cells. Accordingly, small interfering RNA-mediated knockdown of Phbs in Kit225 cells resulted in disruption of mitochondrial membrane potential. Additionally, Phb1 and Phb2 protein levels were up-regulated 2.5-fold during cytokine deprivation-mediated apoptosis of Kit225 cells, suggesting this complex plays a protective role in human T cells. Taken together, Phb1 and Phb2 are novel phosphoproteins up-regulated during T cell activation that function to maintain mitochondrial integrity and thus represent previously unrecognized therapeutic targets for regulating T cell activation, differentiation, viability, and function.

STAT3 : An Important Regulator of Multiple Cytokine Functions

Maintaining T cell homeostasis is critical for normal immune response. Three sequential signals activate T cells, with signal 3 delivered by multiple cytokines that regulate cell proliferation, differentiation, and survival/death. Cytokines binding to their receptors engages two key molecular families, namely, Janus tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats). Among Stats, Stat3 is involved in the generation of T helper 17 (Th17) cells, regulation of dendritic cells, and acute inflammatory response. These aspects of Stat3 function are important for transplantation. We discuss Stat3s role in innate and adaptive immunity as well as its potential for therapeutic intervention.

Interleukin-2 family cytokines stimulate phosphorylation of the Pro-Ser-Pro motif of Stat5 transcription factors in human T cells: resistance to suppression of multiple serine kinase pathways.

Signal transducer and activator of transcription (Stat)5a and Stat5b are critical for normal immune function. Progression of T cells through G1‐S phase of cell cycle requires T cell receptor (TCR)‐ and/or cytokine‐inducible tyrosine phosphorylation of Stat5a/b. Stat5a/b may also, in a cell‐dependent manner, be constitutively or cytokine‐inducibly phosphorylated on a Pro‐Ser‐Pro (PSP) motif located within the transcriptional activation domain. Phosphorylation of the PSP motif is needed for maximal transcriptional activation by Stat5, at least in certain promoter contexts. The basal and cytokine‐inducible serine phosphorylation state of Stat5a/b has not been determined in T cells. Using primary human T cells and T lymphocytic cell lines coupled with novel phospho‐specific antibodies to this conserved phosphoserine motif in Stat5a or Stat5b, we report that: Stat5a and Stat5b were unphosphorylated on the PSP motif under basal conditions and became markedly phosphorylated in response to several T cell growth factor stimuli, including interleukin (IL)‐2, ‐7, ‐9, and ‐15 and phorbol ester 12‐myristate 13‐acetate but not TCR engagement; inducible Stat5a/b serine phosphorylation differed quantitatively and temporally; and Stat5a/b serine phosphorylation was, in contrast to inducible Stat3 serine phosphorylation, insensitive to inhibitors of mitogen‐activated protein kinase, phosphatidylinositol‐3 kinase, and mammalian target of rapamycin or deletion of Raf‐A, ‐B, or ‐C by antisense oligonucleotides. We conclude that IL‐2 family cytokines tightly control Stat5 serine phosphorylation through a kinase distinct from the Stat3 serine kinase.

STAT5 regulation of BCL10 parallels constitutive NFκB activation in lymphoid tumor cells

BackgroundSignal Transducer and Activator of Transcription 5 A and B (STAT5) are key survival factors in cells of the lymphoid lineage. Identification of novel, tissue-specific STAT5 regulated genes would advance the ability to combat diseases due to aberrant STAT5 signaling. In the present work a library of human STAT5 bound genomic elements was created and validated.ResultsOf several STAT5 responsive genomic regulatory elements identified, one was located within the first intron of the human BCL10 gene. Chromatin immuno-precipitation reactions confirmed constitutive in vivo STAT5 binding to this intronic fragment in various human lymphoid tumor cell lines. Interestingly, non-phosphorylated STAT5 was found in the nuclei of Kit225 and YT cells in the absence of cytokine stimulation that paralleled constitutive NFκB activation. Inhibition of the hyperactive JAK3/STAT5 pathway in MT-2 cells via the Mannich-base, NC1153, diminished the constitutive in vivo occupancy of BCL10-SBR by STAT5, reduced NFκB activity and BCL10 protein expression in a dose dependent manner. Moreover, depletion of STAT5 via selective antisense oligonucleotide treatment similarly resulted in decreased BCL10 mRNA and protein expression, cellular viability and impaired NFκB activity independent of IL-2.ConclusionThese results suggest that the NFκB regulator BCL10 is an IL-2-independent STAT5 target gene. These findings proffer a model in which un-activated STAT5 can regulate pathways critical for lymphoid cell survival and inhibitors that disrupt STAT5 function independent of tyrosine phosphorylation may be therapeutically effective in treating certain leukemias/lymphomas.

Specific Inhibition of Stat5a/b Promotes Apoptosis of IL-2-Responsive Primary and Tumor-Derived Lymphoid Cells

Stat5a/b exhibits 96% homology and are required for normal immune function. The present studies examined Stat5a/b function in lymphoid cells by specific and simultaneous disruption of both proteins using novel phosphorothioate-2′-O-methoxyethyl antisense oligodeoxynucleotides (asODN). Efficient delivery was confirmed by the presence of fluorescent TAMRA-labeled ODN in ≥55 and 95% in human primary and tumor cell lines, respectively. Acute asODN administration reduced levels of Stat5a (90%) in 6 h, whereas Stat5b required nearly 48 h to attain the same inhibition, suggesting that the apparent turnover rate for Stat5a was 8-fold higher than that for Stat5b. Expression of the closely related Stat3 protein was unchanged after asODN treatment, however. Molecular ablation of Stat5a/b promoted apoptotic cell death in a significant population of primary PHA-activated T cells (72%) and lymphoid tumor cell line (e.g., YT; 74%) within 24 h, as assessed by 1) visualization of karyolytic nuclear degeneration and other generalized cytoarchitectural alterations, 2) enzymatic detection of TdT-positive DNA degradation, and 3) automated cytometric detection of annexin V translocation. Contrary to findings from Stat5a/b-null mice, cell cycle progression did not appear to be significantly affected. Interestingly, IL-2-insensitive and unprimed T cells and Jurkat cells remained mostly unaffected. Finally, evidence is provided that the cytotoxicity associated with Stat5a/b ablation may derive from activation of caspase-8, an initiator protease that contributes to apoptotic cell commitment. We propose that in lymphoid cells competent to activate Stat5a and Stat5b, both proteins preferentially mediate an antiapoptotic survival influence.

Regulation of T cell homeostasis by JAKs and STATs.

Regulation of T cell homeostasis is critical for maintaining normal immune function. An imbalance in T cell proliferation can result in disorders ranging from cancer and autoimmunity to immunodeficiencies. Full activation of T cells requires three sequential signals, where signal 3, which is delivered by multiple cytokines, regulates proliferation, differentiation, and survival/death. Signaling from cytokines through their receptors is primarily delivered by two molecular families, namely Janus tyrosine kinases (JAKs) and signal transducers and activators of transcription (STATs). Invaluable knowledge about JAKs and STATs has arisen from studies of mice made genetically deficient in these molecules, analyses of tumor models, and studies of expression patterns by proteomics/genomics, which all have begun to define the role of JAKs and STATs in survival versus apoptosis. These findings also have suggested ways in which JAKs and STATs may be manipulated for therapeutic intervention in lymphoid-derived diseases. This review seeks to focus on the role of JAK tyrosine kinases and STAT transcription factors in mediating the lymphocyte life cycle and how they might be manipulated for therapeutic applications.

Protein Phosphatase 2A Regulates Interleukin-2 Receptor Complex Formation and JAK3/STAT5 Activation

Reversible protein phosphorylation plays a key role in interleukin-2 (IL-2) receptor-mediated activation of Janus tyrosine kinase 3 (JAK3) and signal transducer and activator of transcription 5 (STAT5) in lymphocytes. Although the mechanisms governing IL-2-induced tyrosine phosphorylation and activation of JAK3/STAT5 have been extensively studied, the role of serine/threonine phosphorylation in controlling these effectors remains to be elucidated. Using phosphoamino acid analysis, JAK3 and STAT5 were determined to be serine and tyrosine-phosphorylated in response to IL-2 stimulation of the human natural killer-like cell line, YT. IL-2 stimulation also induced serine/threonine phosphorylation of IL-2Rβ, but not IL-2Rγ. To investigate the regulation of serine/threonine phosphorylation in IL-2 signaling, the roles of protein phosphatase 1 (PP1) and 2A (PP2A) were examined. Inhibition of phosphatase activity by calyculin A treatment of YT cells resulted in a significant induction of serine phosphorylation of JAK3 and STAT5, and serine/threonine phosphorylation of IL-2Rβ. Moreover, inhibition of PP2A, but not PP1, diminished IL-2-induced tyrosine phosphorylation of IL-2Rβ, JAK3, and STAT5, and abolished STAT5 DNA binding activity. Serine/threonine phosphorylation of IL-2Rβ by a staurosporine-sensitive kinase also blocked its association with JAK3 and IL-2Rγ in YT cells. Taken together, these data indicate that serine/threonine phosphorylation negatively regulates IL-2 signaling at multiple levels, including receptor complex formation and JAK3/STAT5 activation, and that this regulation is counteracted by PP2A. These findings also suggest that PP2A may serve as a therapeutic target for modulating JAK3/STAT5 activation in human disease.

Signal transducer and activator of transcription 5b (Stat5b) serine 193 is a novel cytokine induced phospho-regulatory site that is constitutively activated in primary hematopoietic malignancies

Background: Stat5 transcription factors regulate genes required for cell growth, survival, and differentiation. Results: A novel cytokine-induced Stat5b phospho-regulatory site was identified and found to be constitutively activated in hematopoietic cancers. Conclusion: This newly defined phosphorylation site acts to regulate Stat5b DNA binding and transcriptional activity. Significance: Therapeutic strategies that disrupt Stat5 serine phosphorylation may be important for controlling hematopoietic diseases such as cancer. Signal transducer and activator of transcription 5b (Stat5b) is a critical node in the signaling network downstream of external (cytokines or growth factors) or internal (oncogenic tyrosine kinases) stimuli. Maximum transcriptional activation of Stat5b requires both tyrosine and serine phosphorylation. Although the mechanisms governing tyrosine phosphorylation and activation of Stat5b have been extensively studied, the role of serine phosphorylation remains to be fully elucidated. Using mass spectrometry and phospho-specific antibodies, we identified Ser-193 as a novel site of cytokine-induced phosphorylation within human Stat5b. Stat5b Ser(P)-193 was detected in activated primary human peripheral blood mononuclear cells or lymphoid cell lines in response to several γ common (γc) cytokines, including interleukin (IL)-2, IL-7, IL-9, and IL-15. Kinetic and spatial analysis indicated that Stat5b Ser-193 phosphorylation was rapid and transient and occurred in the cytoplasmic compartment of the cell prior to Stat5b translocation to the nucleus. Moreover, inducible Stat5b Ser-193 phosphorylation was sensitive to inhibitors of mammalian target of rapamycin (mTOR), whereas inhibition of protein phosphatase 2A (PP2A) induced phosphorylation of Ser-193. Reconstitution assays in HEK293 cells in conjunction with site-directed mutagenesis, EMSA, and reporter assays indicated that Ser(P)-193 is required for maximal Stat5b transcriptional activity. Indeed, Stat5b Ser-193 was found constitutively phosphorylated in several lymphoid tumor cell lines as well as primary leukemia and lymphoma patient tumor cells. Taken together, IL-2 family cytokines tightly control Stat5b Ser-193 phosphorylation through a rapamycin-sensitive mechanism. Furthermore, constitutive Ser-193 phosphorylation is associated with Stat5b proto-oncogenic activity and therefore may serve as a novel therapeutic target for treating hematopoietic malignancies.

Inhibition of TASK-3 (KCNK9) channel biosynthesis changes cell morphology and decreases both DNA content and mitochondrial function of melanoma cells maintained in cell culture.

TASK-3 channel overexpression was shown to facilitate the survival of malignantly transformed cells, possibly by providing greater hypoxia tolerance through a still unknown mechanism. Although it has been suggested previously that TASK-3 channels are expressed in the mitochondrial membranes, their role here remains elusive. In this study, a transient transfection of TASK-3 knockdown melanoma cell cultures was produced to show the significance of TASK-3 expression. Reduction of the TASK-3 protein biosynthesis induced characteristic changes in cell morphology, reduced the amount of DNA and decreased metabolic activity and mitochondrial function of melanoma cells when compared with control. These findings indicate that TASK-3 channel expression and function is indispensable for the proliferation and/or survival of the melanoma cells, as they seem to contribute to their mitochondrial functions. The significance is that, in this study, we have shown that TASK-3 channels are expressed in the mitochondria of melanoma malignum cells, and they are essential for maintaining cellular integrity and viability. The TASK-3 knockdown melanoma cell line had altered morphology, reduced DNA content, decreased metabolic activity and impaired mitochondrial function. These data indicate that TASK-3 channels are functionally present in the mitochondria of the melanoma cells, and their function is essential for the survival of these cells, thus TASK-3 channels may be the possible targets of future anticancer therapy.