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Biomedical subjects

Olli Silvennoinen

Publications and source records attributed to Olli Silvennoinen.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of RNA helicase A as component of STAT6-dependent enhanceosome.

Signal transducer and activator of transcription 6 (STAT6) is a regulator of transcription for interleukin-4 (IL-4)-induced genes. The ability of STAT6 to activate transcription depends on functional interaction with other transcription factors and coactivators. We have characterized the mechanism of STAT6-mediated transcriptional activation by identifying STAT6 transcription activation domain (TAD) interacting nuclear proteins. The first of the identified proteins was coactivator protein p100, which regulates IL-4-induced transcription by connecting STAT6 with other transcriptional regulators. Here, we describe RNA helicase A (RHA) as a novel component of STAT6 transcriptosome. In vitro and in vivo experiments indicated that RHA did not directly interact with STAT6, but p100 protein was found to mediate the assembly of the ternary complex of STAT6-p100-RHA. In chromatin immunoprecipitation studies RHA together with p100 enhanced the binding of STAT6 on the human Igepsilon promoter after IL-4 stimulation. RHA enhanced the IL-4-induced transcription, and the participation of RHA in IL-4-regulated transcription was supported by RNAi experiments. Our results suggest that RHA has an important role in the assembly of STAT6 transcriptosome. As RHA is also known to interact with chromatin modifying proteins, the RHA containing protein complexes may facilitate the entry of transcriptional apparatus to the IL-4 responsive promoters.

Animals↗
PubMed2006

Ets transcription factors regulate AIRE gene promoter.

Autoimmune regulator (AIRE) directs the expression of self-antigens in thymus. Defects in AIRE gene cause an organ-specific autoimmune disease called autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED). AIRE protein is mainly expressed in thymic medullary epithelial cells, thus implying a strict control over its expression pattern. To date, only limited information is available on mechanisms responsible for the regulation of AIRE gene. Here, we show that Ets transcription factor family members Ets-1, Ets-2, and ESE-1 have positive effect on AIRE transcription. Site-directed mutagenesis and transfection studies revealed that two of the three Ets binding sites in AIRE promoter are functional and this finding has been confirmed by the electrophoretic mobility shift assay. The AIRE promoter activity could be stimulated by phorbol myristate acetate (PMA) and this activation was further enhanced by Ets transcription factors. Our results demonstrate for the first time that AIRE gene is a downstream target for the Ets family of transcription factors.

Base Sequence↗
PubMed2006

Binding of SH2-B family members within a potential negative regulatory region maintains JAK2 in an active state.

The tyrosine kinase Janus kinase 2 (JAK2) transduces signaling for the majority of known cytokine receptor family members and is constitutively activated in some cancers. Here we examine the mechanisms by which the adapter proteins SH2-Bbeta and APS regulate the activity of JAK2. We show that like SH2-Bbeta, APS binds JAK2 at multiple sites and that binding to phosphotyrosine 813 is essential for APS to increase active JAK2 and to be phosphorylated by JAK2. Binding of APS to a phosphotyrosine 813-independent site inhibits JAK2. Both APS and SH2-Bbeta increase JAK2 activity independent of their N-terminal dimerization domains. SH2-Bbeta-induced increases in JAK2 dimerization require only the SH2 domain and only one SH2-Bbeta to be bound to a JAK2 dimer. JAK2 mutations and truncations revealed that amino acids 809 to 811 in JAK2 are a critical component of a larger regulatory region within JAK2, most likely including amino acids within the JAK homology 1 (JH1) and JH2 domains and possibly the FERM domain. Together, our data suggest that SH2-Bbeta and APS do not activate JAK2 as a consequence of their own dimerization, recruitment of an activator of JAK2, or direct competition with a JAK2 inhibitor for binding to JAK2. Rather, they most likely induce or stabilize an active conformation of JAK2.

Adaptor Proteins, Signal Transducing↗
PubMed2005

SLIM trims STATs: ubiquitin E3 ligases provide insights for specificity in the regulation of cytokine signaling.

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway has evolved to serve highly specialized functions in the regulation of hematopoiesis, cell metabolism, and immune responses. The duration, strength, and specificity of cytokine signaling are controlled by several mechanisms, including the ubiquitin-proteasome pathway, which modulates the turnover of cytokine receptors and activated JAKs. The specificity of the ubiquitin pathway is achieved through various E3 ligase complexes that recognize and interact with distinct target proteins, often in a phosphorylation-dependent manner. Intriguing new information about the ubiquitin pathway came with the identification of an E3 ubiquitin ligase, SLIM, that specifically interacts with activated STAT1 and STAT4 and induces their ubiquitination and degradation. These findings, together with the evidence from paramyxoviruses about the role of ubiquitination as a highly specific STAT inhibition mechanism, highlight the role of E3 ubiquitin ligases as specificity determinants in the regulation of STAT activation, and open the field for investigation of additional E3s that target other STAT proteins.

Adaptor Proteins, Signal Transducing↗
PubMed2005

SUMO-1 conjugation selectively modulates STAT1-mediated gene responses.

Signal transducers and activators of transcription 1 (STAT1) is a critical mediator of interferon (IFN)-induced gene responses. Recently, STAT1 was found to become modified by small ubiquitin-like modifier 1 (SUMO-1) conjugation at Lys703 through the SUMO E3 ligase function of protein inhibitors of activated STAT (PIAS) proteins. However, the physiologic function of sumoylation in STAT1 is still unclear. Here, we show that mutations in the SUMO attachment site in STAT1 result in increased transcriptional activity in a fashion that is selective among IFN-gamma target genes. The sumoylation-defective STAT1 mutant displayed increased induction of guanylate-binding protein 1 (GBP1) and transporters associated with antigen presentation 1 (TAP1) transcription but not interferon regulatory factor 1 (IRF1) transcription. Moreover, the sumoylation-defective mutant STAT1-KR showed a prolonged DNA-binding activity and nuclear localization in response to IFN-gamma stimulation. These results suggest that sumoylation has a defined negative regulatory effect on selective STAT1-mediated transcription responses.

Animals↗
PubMed2005

The transcriptional co-activator protein p100 recruits histone acetyltransferase activity to STAT6 and mediates interaction between the CREB-binding protein and STAT6.

STAT6 is a critical regulator of transcription for interleukin-4 (IL-4)-induced genes. Activation of gene expression involves recruitment of coactivator proteins that function as bridging factors connecting sequence-specific transcription factors to the basal transcription machinery, and as chromatin-modifying enzymes. Coactivator proteins CBP/p300 have been implicated in regulation of transcription in all STATs. CBP is also required for STAT6-mediated gene activation, but the underlying molecular mechanisms are still elusive. In this study we investigated the mechanisms by which STAT6 recruits CBP and chromatin-modifying activities to the promoter. Our results indicate that while STAT1-interacted directly with CBP, the interaction between STAT6 and CBP was found to be mediated through p100 protein, a coactivator protein that has previously been shown to stimulate the transcription of IL-4-induced genes. The staphylococcal nuclease-like (SN)-domains of p100 directly interacted with amino acids 1099-1758 of CBP, while p100 did not associate with SRC-1, another coactivator of STAT6. p100 was found to recruit histone acetyltransferase (HAT) activity to STAT6 in vivo. Chromatin immunoprecipitation studies demonstrated that p100 increases the STAT6-p100-CBP ternary complex formation in the human Igepsilon promoter. p100 also increased the amount of acetylated histone H4 at the Igepsilon promoter, and siRNAs directed against p100 effectively inhibited Igepsilon reporter gene expression. Our results suggest that p100 has an important role in the assembly of STAT6 transcriptosome, and that p100 stimulates IL-4-dependent transcription by mediating interaction between STAT6 and CBP and recruiting chromatin modifying activities to STAT6-responsive promoters.

Acetyltransferases↗
PubMed2005

Monocyte anergy is present in patients with severe acute pancreatitis and is significantly alleviated by granulocyte-macrophage colony-stimulating factor and interferon-gamma in vitro.

OBJECTIVES: Severe acute pancreatitis (AP) is frequently associated with immune suppression, which increases the risk of infections, organ failure, and death. Our aims were to measure monocyte function (ie, HLA-DR expression and tumor necrosis factor-alpha [TNF-alpha] production as markers of immune suppression) in patients with severe AP and to determine whether treatment of blood samples with granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interferon-gamma (IFN-gamma) corrected the functional defects of monocytes in vitro. METHODS: The study consisted of 28 patients with severe AP who were treated at intensive care unit and in whom the proportion of HLA-DR-positive monocytes in the circulation was less than 70%, and 28 matched control subjects who were selected from healthy laboratory personnel. HLA-DR density was determined by whole blood flow cytometry. Monocyte TNF-alpha production in response to bacterial lipopolysaccharides (LPSs) was studied in a whole blood assay. Aliquots of blood were supplemented with IFN-gamma (all 28 patients), GM-CSF (the last 24 patients), or both (the last 12 patients). RESULTS: The median proportion of HLA-DR-positive monocytes was 45% in patients (range, 18%-73%) and was 98% in controls (range, 86%-100%; P < 0.001). TNF-alpha levels in response to LPSs were lower in patients (545 pg/mL; range, 84-1990 pg/mL) than in controls (1415 pg/mL; range, 660-5490 pg/mL; P < 0.001). The proportion of HLA-DR-positive cells correlated positively with TNF-alpha levels (r = 0.56; P < 0.01). Both GM-CSF and IFN-gamma increased HLA-DR expression of monocytes in patients (98%; range, 74%-100% for GM-CSF; 99%; range, 86%-100% for IFN-gamma; both P < 0.001). The combination restored monocyte HLA-DR expression (99%; range, 96%-100%; P = 0.002). Compared with basal levels, GM-CSF increased TNF-alpha production of monocytes both in blood samples from patients (median, 1320 pg/mL; range, 35-8015 pg/mL) and controls (median, 3450 pg/mL; range, 1040-9835 pg/mL; both P < 0.001). IFN-gamma increased TNF-alpha production by monocytes in patients (683 pg/mL; range, 186-2705 pg/mL; P < 0.05) but not in controls (1658 pg/mL; range, 765-4755 pg/mL; P = 0.31). With the combination of GM-CSF and IFN-gamma, the TNF-alpha levels of monocytes in patients (3185 pg/mL; range, 545-8280 pg/mL) and in controls (2800 pg/mL; range, 1080-6860 pg/mL) were comparable. CONCLUSIONS: The proportion of HLA-DR-positive monocytes correlates with TNF-alpha production, and they both reflect the degree of immune suppression. The low proportion of HLA-DR-positive monocytes in AP can be reversed in vitro by GM-CSF and/or IFN-gamma. The GM-CSF and IFN-gamma treatments also increase LPS-induced TNF-alpha production. By the combination of GM-CSF and IFN-gamma, but not by either agent alone, LPS-induced TNF-alpha production of monocytes was equally high in patients and in controls.

Acute Disease↗
PubMed2004

The Janus kinases (Jaks).

The Janus kinase (Jak) family is one of ten recognized families of non-receptor tyrosine kinases. Mammals have four members of this family, Jak1, Jak2, Jak3 and Tyrosine kinase 2 (Tyk2). Birds, fish and insects also have Jaks. Each protein has a kinase domain and a catalytically inactive pseudo-kinase domain, and they each bind cytokine receptors through amino-terminal FERM (Band-4.1, ezrin, radixin, moesin) domains. Upon binding of cytokines to their receptors, Jaks are activated and phosphorylate the receptors, creating docking sites for signaling molecules, especially members of the signal transducer and activator of transcription (Stat) family. Mutations of the Drosophila Jak (Hopscotch) have revealed developmental defects, and constitutive activation of Jaks in flies and humans is associated with leukemia-like syndromes. Through the generation of Jak-deficient cell lines and gene-targeted mice, the essential, nonredundant functions of Jaks in cytokine signaling have been established. Importantly, deficiency of Jak3 is the basis of human autosomal recessive severe combined immunodeficiency (SCID); accordingly, a selective Jak3 inhibitor has been developed, forming a new class of immunosuppressive drugs.

Animals↗
PubMed2004

Pim-1 kinase inhibits STAT5-dependent transcription via its interactions with SOCS1 and SOCS3.

Signal transducer and activator of transcription 5 (STAT5) plays a critical role in cytokine-induced survival of hematopoietic cells. One of the STAT5 target genes is pim-1, which encodes an oncogenic serine/threonine kinase. Here we demonstrate that Pim-1 inhibits STAT5-dependent transcription in cells responsive to interleukin-3, prolactin, or erythropoietin. Ectopic expression of Pim-1 in cytokine-dependent FDCP1 myeloid cells results in reduced tyrosine phosphorylation and DNA binding of STAT5, indicating that Pim-1 interferes already with the initial steps of STAT5 activation. However, the Pim-1 kinase does not directly phosphorylate or bind to STAT5. By contrast, Pim-1 interacts with suppressor of cytokine signaling 1 (SOCS1) and SOCS3 and potentiates their inhibitory effects on STAT5, most likely via phosphorylation-mediated stabilization of the SOCS proteins. Thus, both Pim and SOCS family proteins may be components of a negative feedback mechanism that allows STAT5 to attenuate its own activity.

Animals↗
PubMed2004

STATs as critical mediators of signal transduction and transcription: lessons learned from STAT5.

Signal transducers and activators of transcription (Stats) comprise a family of seven transcription factors that are activated by a variety of cytokines, hormones and growth factors. Stats are activated through tyrosine phosphorylation, mainly by Jak kinases, that lead to their dimerization, nuclear translocation and regulation of target gene expression. Stat5 was originally identified as a transcription factor that regulates the beta-casein gene in response to prolactin (PRL), but Stat5 is activated also by several other cytokines and growth factors. The molecular mechanisms that underlie Stat5-mediated transcription involve interactions and cooperation with sequence specific transcription factors and transcriptional coregulators. Our studies identified p100 protein as a coactivator for Stat5, and suggest the existence of a positive regulatory loop in PRL-induced transcription, where PRL stabilizes p100 protein, which in turn can cooperate with Stat5 in transcriptional activation. Suppressors of cytokine signaling (SOCS) proteins are important negative regulators of Stats. A target gene for Stat5, the serine/threonine kinase Pim-1, was found to cooperate with SOCS-1 and SOCS-3 to inhibit Stat5 activity suggesting that Pim-1 together with SOCS-1 and SOCS-3 are components of a negative feedback mechanism that allows Stat5 to regulate its own activation.

Animals↗
PubMed2004

Molecular basis of Stat1 and PU.1 cooperation in cytokine-induced Fcgamma receptor I promoter activation.

The high-affinity receptor for IgG (FcgammaRI) is a myeloid cell-specific and IFN-gamma-induced gene, and thereby serves as a paradigm for cytokine-induced cell type-specific gene responses. The expression of FcgammaRI is regulated by PU.1 and Stat1 transcription factors. We established an experimental model to analyze the individual functions of Stat1 and PU.1 in cytokine-induced transcription of the natural FcgammaRI promoter in U3A cells lacking both factors. PU.1 was required for both the basal activity and for the IFN-gamma-induced FcgammaRI promoter activation, while Stat1 alone could not initiate transcription. In contrast, in the context of a heterologous promoter, PU.1 inhibited the Stat1-mediated transcription. Systematic analysis of Stat1 and PU.1 mutants and FcgammaRI promoter elements revealed that activation of the promoter required the DNA binding, and the transactivation functions of both Stat1 and PU.1. PU.1 and Stat1 bound the promoter elements independently, and no physical interaction between the proteins was observed. The requirement of PU.1 for FcgammaRI promoter activity was supported by demonstration of in vitro interaction between PU.1 and components of the basal transcription machinery TBP and RNA polymerase II. Deletion of the acidic transactivation domain of PU.1 greatly diminished both the FcgammaRI promoter activity as well as the interaction with RNA polymerase II. In contrast, Stat1 did not interact with TBP or RNA polymerase II. These results define functional cooperativity between PU.1 and Stat1 in FcgammaRI promoter activation where PU.1 serves as an amplifier and bridging factor with the basal transcription machinery.

Animals↗
PubMed2003

PIAS proteins promote SUMO-1 conjugation to STAT1.

Signal transducer and activator of transcription 1 (STAT1) is a critical mediator of interferon-gamma (IFN-gamma)-induced transcription that is regulated through posttranslational modifications and through transacting proteins such as protein inhibitor of activated STAT1 (PIAS1). PIAS proteins have been shown to function as E3-type small ubiquitin-like modifier (SUMO) ligases, and sumoylation has been identified as a modulatory mechanism for several transcription factors. Here we show that STAT1 is subject to SUMO-1 modification, and sumoylation occurs in vivo and in vitro at a single, evolutionary conserved amino acid residue Lys703. Members of the PIAS family of proteins were found to strongly stimulate sumoylation of STAT1. Furthermore, activation of STAT1 by IFN-gamma or pervanadate induced SUMO-1 conjugation. Mutation of Lys703 in STAT1 resulted in increased IFN-gamma-mediated transactivation, suggesting a negative regulatory function for sumoylation. These results indicate that STAT1 is covalently modified by SUMO-1 in cytokine signaling and that PIAS proteins promote SUMO-1 conjugation to STAT1.

DNA-Binding Proteins↗
PubMed2003

Tudor and nuclease-like domains containing protein p100 function as coactivators for signal transducer and activator of transcription 5.

Signal transducer and activator of transcription 5 (Stat5) plays a critical role in prolactin (PRL)-induced transcription of several milk protein genes. Stat5-mediated gene regulation is modulated by cooperation of Stat5 with cell type- and promoter-specific transcription factors as well as by interaction with transcriptional coregulators. Recently, the expression of a tudor and staphylococcal nuclease-like domains containing protein p100 was found to be increased in mammary epithelial cells during lactation in response to lactogenic hormones. p100 was initially identified as a transcriptional coactivator of the Epstein-Barr virus nuclear antigen 2. In this study we investigated the potential role of p100 in PRL-induced Stat5-mediated transcriptional activation. PRL stimulation increased the p100 protein levels in HC11 mouse mammary epithelial cells. p100 did not affect the early activation events of Stat5, but p100 enhanced the Stat5-dependent transcriptional activation in HC11 cells. p100 associated with Stat5 both in vivo and in vitro, and the interaction was mediated by both the tudor and staphylococcal nuclease-like domains of p100. Together these results suggest that p100 functions as a transcriptional coactivator for Stat5-dependent gene regulation and the existence of a positive regulatory loop in PRL-induced transcription, in which PRL stabilizes p100 protein, which in turn can cooperate with Stat5 in transcriptional activation.

Animals↗
PubMed2003

PU.1 is required for transcriptional activation of the Stat6 response element in the Igepsilon promoter.

Signal transducer and activator of transcription 6 (Stat6) has a crucial role in regulation of IL-4-induced gene responses. Stat6-binding sites are present in the promoters of both ubiquitously and cell-type-specifically expressed genes. The promoter regions of IL-4-inducible genes contain cis-acting elements for several transcription factors that act in concert with Stat6 and are also likely to modulate lineage-specific gene expression. We have observed that the Stat6 response element from the B-cell-specific Igepsilon promoter is readily activated upon IL-4 stimulation in B cells but not in non-hematopoietic cells. A minimal low-affinity PU.1-core-binding sequence (5'-AGAA-3') was identified within the Stat6 DNA-binding site in the Igepsilon promoter. Ectopic expression of the myeloid- and B-cell-specific transcription factor PU.1 restored the IL-4-inducibility of the Igepsilon-Stat6 response element in HepG2 cells, and the induction required an intact PU.1-binding sequence. Both the transactivation and the DNA-binding domains of PU.1 were required for induction of Stat6-mediated transcription. The co-operation between PU.1 and Stat6 in transactivation of the Igepsilon gene represents a molecular mechanism for the fine-tuning of cell-type-restricted expression of IL-4-induced gene responses.

Binding Sites↗
PubMed2003

Autoinhibition of Jak2 tyrosine kinase is dependent on specific regions in its pseudokinase domain.

Jak tyrosine kinases have a unique domain structure containing a kinase domain (JH1) adjacent to a catalytically inactive pseudokinase domain (JH2). JH2 is crucial for inhibition of basal Jak activity, but the mechanism of this regulation has remained elusive. We show that JH2 negatively regulated Jak2 in bacterial cells, indicating that regulation is an intrinsic property of Jak2. JH2 suppressed basal Jak2 activity by lowering the V(max) of Jak2, whereas JH2 did not affect the K(m) of Jak2 for a peptide substrate. Three inhibitory regions (IR1-3) within JH2 were identified. IR3 (residues 758-807), at the C terminus of JH2, directly inhibited JH1, suggesting an inhibitory interaction between IR3 and JH1. Molecular modeling of JH2 showed that IR3 could form a stable alpha-helical fold, supporting that IR3 could independently inhibit JH1. IR2 (725-757) in the C-terminal lobe of JH2, and IR1 (619-670), extending from the N-terminal to the C-terminal lobe, enhanced IR3-mediated inhibition of JH1. Disruption of IR3 either by mutations or a small deletion increased basal Jak2 activity, but abolished interferon-gamma-inducible signaling. Together, the results provide evidence for autoinhibition of a Jak family kinase and identify JH2 regions important for autoregulation of Jak2.

Amino Acid Sequence↗
PubMed2003

M-CSF induced differentiation of myeloid precursor cells involves activation of PKC-delta and expression of Pkare.

Macrophage-colony stimulating factor (M-CSF) regulates proliferation and differentiation of cells belonging to the monocytic lineage. We investigated the mechanisms of M-CSF differentiation signaling in follicular dendritic cell-P1 cells and analyzed the catalytic activation of different protein kinase C (PKC) isoforms. M-CSF induced rapid catalytic activation of PKC-delta and membrane translocation of the tyrosine phosphorylated form of PKC-delta. Mutation of tyrosine 807 in the M-CSF receptor (Fms) abrogates cell differentiation but not a proliferative response to M-CSF, and FmsY807F failed to activate PKC-delta. We also investigated the downstream signaling pathways from PKC-delta. A cyclic adenosine monophosphate-regulated Ser/Thr kinase gene, protein kinase X (PRKX), has been associated with macrophage differentiation in human cells. We found that M-CSF and PKC-delta induced the expression of the PRKX murine homologue: PKA-related gene. Taken together, our results indicate that PKC-delta functions as a critical mediator of M-CSF-induced differentiation signaling.

Amidohydrolases↗