Faculty Bibliography

Daudi B lymphoblastoid cells are highly sensitive to the anti-growth and anti-viral effects of interferon (IFN). Unlike many cell lines, these cells show prolonged transcription of IFN-stimulated genes following treatment with IFN-alpha. This prolonged response correlated with the continued presence of the activated transcription factor, IFN-stimulated gene factor 3 (ISGF3). Pulse-chase labeling experiments indicated that the half-life of the phosphorylation of signal transducers and activators of transcription (Stat)1 and Stat2 was short (<2 h) although the turnover of the proteins themselves was slow (>24 h), indicative of a constitutive phosphatase activity. The administration of protein-tyrosine kinase inhibitors at any time point during IFN stimulation led to rapid inhibition of the response, indicating that tyrosine kinase activity was continuously required. Catalytic activity of Jak1 and Tyk2 kinases remained elevated for prolonged periods following stimulation. Continuous presence of IFN-alpha was necessary for maintaining prolonged activation of ISGF3 and of Janus kinases, an activity that was blocked by antibodies to IFN-alpha or by cycloheximide. Conditioned medium of IFN-alpha-stimulated cells was capable of stimulating STAT activation in naive cells. Taken together, these results suggest that the response to IFN-alpha is controlled by the duration of stimulated Janus kinase activity over the background of constitutive dephosphorylation and that this response can be sustained by autocrine secretion of IFN-alpha

IFNs are antiproliferative cytokines that have growth-inhibitory effects on various normal and malignant cells. Therefore, they have been used in the treatment of certain forms of cancer, such as chronic myelogenous leukemia and hairy cell leukemia. However, there is little evidence that IFNs would be effective in the treatment of acute myelogenous leukemia, and molecular mechanisms underlying IFN unresponsiveness have not been clarified. Here we have studied the activation and induction of IFN-specific transcription factors signal transducer and activator of transcription (STAT) 1, STAT2, and p48 in all-trans-retinoic acid (ATRA)-differentiated myeloid leukemia cells using promyelocytic NB4, myeloblastic HL-60, and monoblastic U937 cells as model systems. These cells respond to ATRA by growth inhibition and differentiation. We show that in undifferentiated NB4 cells, 2',5'-oligoadenylate synthetase and MxB gene expression is not activated by IFN-alpha, possibly due to a relative lack of signaling molecules, especially p48 protein. However, during ATRA-induced differentiation, steady-state STAT1, STAT2, and especially p48 mRNA and corresponding protein levels were elevated both in NB4 and U937 cells, apparently correlating to an enhanced responsiveness of these cells to IFNs. ATRA treatment of NB4 cells sensitized them to IFN action as seen by increased IFN-gamma activation site DNA-binding activity or by efficient formation of IFN-alpha-specific ISGF3 complex and subsequent oligoadenylate synthetase and MxB gene expression. Lack of p48 expression could be one of the mechanisms of promyelocytic leukemia cell escape from growth-inhibitory effects of IFN-alpha

An international workshop on 'Cytokines, Signaling and the Jak/Stat Pathway' organized by Peter C. Heinrich of the Klinikum der RWTH Aachen under the auspices of DFG-Forschergruppe was held in Aachen, Germany, on 4-5 October 1996. In this review David Levy notes that even though some diagrams shown at the meeting displayed more arrows than there were meeting participants, remarkable progress has been made in the unraveling of the complexities of cytokine signal transduction. The central issue in the field of cytokine signaling is specificity, and this issue was addressed through experimental results from biochemical, molecular, and genetic studies of signal transduction pathways

Transcriptional responses to interferon (IFN) are mediated by tyrosine phosphorylation and nuclear translocation of transcription factors of the signal transducer and activator of transcription (Stat) family. The Stat1 protein is required for all transcriptional responses to IFN (both type I and type II). Responses to type I IFN (alpha and beta) also require Stat2 and the IFN regulatory factor family protein p48, which form a heterotrimeric transcription complex with Stat1 termed ISGF3. Stat1 homodimers formed in response to IFN-gamma treatment can also interact with p48 and function as transcriptional activators. We now show that Stat2 is capable of forming a stable homodimer that interacts with p48, can be recruited to DNA, and can activate transcription, raising a question of why Stat1 is required. Analysis of the transcriptional competence, affinity, and specificity of Stat2-p48 complexes compared with other Stat protein-containing transcription factor complexes suggests distinct roles for each component. Although Stat2 is a potent transactivator, it does not interact stably with DNA in complex with p48 alone. Adding Stat1 increases the affinity and alters the sequence selectivity of p48-DNA interactions by contacting a half-site of its palindromic recognition motif adjacent to a p48 interaction sequence. Thus, ISGF3 assembly involves p48 functioning as an adaptor protein to recruit Stat1 and Stat2 to an IFN-alpha-stimulated response element, Stat2 contributes a potent transactivation domain but is unable to directly contact DNA, while Stat1 stabilizes the heteromeric complex by contacting DNA directly

Interferon (IFN) induces gene expression by phosphorylating latent transcription factors of the STAT family. Two different STAT multimeric complexes that bind distinct enhancer elements are activated by IFN alpha and IFN gamma, dictated by the DNA-binding protein ISGF3 gamma p48. This protein, a member of the IFN regulatory factor (IFR) family, acts as an adaptor protein to redirect STAT multimers from their intrinsic palindromic sequence specificity to interactions with a composite element composed of an IRF site juxtaposed with a STAT half-site. Sequence similarity within the IRF family suggests that other members could serve as adaptor proteins for transcriptional activators. Recent evidence suggests that PIP (LSIRF) sequesters the Ets protein PU.1 at a composite DNA element lends support to this adaptor hypothesis

Oncastatin M (OSM) is one member of the leukemia inhibitory factor/interleukin-6 family of cytokines that has been shown to be a growth regulatory molecule. In osteoblastic cultures, OSM causes marked phenotypic changes and the enhanced secretion of interleukin-6. In this study, we have shown that stimulation of murine and human osteoblastic cultures and a human osteosarcoma cell line with OSM resulted in the tyrosine phosphorylation of a number of cellular proteins including members of both the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) family of signaling proteins. The JAKs, a family of intracellular kinases, and the STATs, a family of transcription factors, have both previously been shown to be tyrosine phosphorylated and activated in response to various cytokines, interferons, and growth factors in cells of non-skeletal origin. Using three different sources of cells of the osteoblast lineage, we demonstrate that OSM induces a rapid but transient tyrosine phosphorylation of the three JAK family members tested, JAK1, JAK2 and Tyk2. In addition, two members of the STAT family, Stat1alpha and Stat3, are tyrosine phosphorylated in osteoblastic cells in culture in response to OSM. OSM activation of this pathway in cells of the osteoblast lineage will result in the transcription of specific genes that ultimately may be associated with osteoblast function

The STAT1 transcription factor is activated in response to many cytokines and growth factors. To study the requirement for STAT1 in vivo, we disrupted the Stat1 gene in embryonic stem (ES) cells and in mice. Stat1(-1-)ES cells were unresponsive to interferon (IFN), but retained responsiveness to leukemia inhibitory factor (LIF) and remained LIF dependent for undifferentiated growth. Stat1(-1-1) animals were born at normal frequencies and displayed no gross developmental defects. However, these animals failed to thrive and were extremely susceptible to viral disease. Cells and tissues from Stat1(-1-) mice were unresponsive to IFN, but remained responsive to all other cytokines tested. Thus, STAT1 appears to be specific for IFN pathways that are essential for viability in the face of otherwise innocuous pathogens

The tyrosine kinase JAK1 and the transcription factors STAT1 and STAT3 are phosphorylated in response to epidermal growth factor (EGF) and other growth factors. We have used EGF receptor-transfected cell lines defective in individual JAKs to assess the roles of these kinases in STAT activation and signal transduction in response to EGF. Although JAK1 is phosphorylated in response to EGF, it is not required for STAT activation or for induction of the c-fos gene. STAT activation in JAK2- and TYK2-defective cells is also normal, and the tyrosine phosphorylation of these two kinases does not increase upon EGF stimulation in wild-type or JAK1-negative cells. In cells transfected with a kinase-negative mutant EGF receptor, there is no STAT activation in response to EGF and c-fos is not induced, showing that the kinase activity of the receptor is required, directly or indirectly, for these two responses. The data do not support a role for any of the three JAK family members tested in STAT activation and are consistent with a JAK-independent pathway in which the intrinsic kinase domain of the EGF receptor is crucial. Furthermore, data from transient transfection experiments in HeLa cells, using c-fos promoters lacking the STAT regulatory element c-sis-inducible element, indicate that this element may play only a minor role in the induction of c-fos by EGF in these cells

The T cell activation is initiated by interaction of specific Ags with TCR, followed by activation of intracellular biochemical events leading to activation of several genes. The activation of signal transducer and activator of transcription (STAT) proteins in a primary TCR-mediated activation of T cells have been explored. In purified human peripheral blood T cells, nuclear STAT proteins were activated approximately 3 h after activation by cross-linked anti-CD3 Abs. These STAT proteins were detected by using the IFN-gamma-activated sequence (GAS) and related oligonucleotides as probes in electrophoretic mobility shift assay. Analysis of the nuclear extracts with anti-STAT Abs indicated that they contained STAT-3 and additional proteins crossreactive with the STAT family. The induction of STAT activity was inhibited completely by pretreatment with either cycloheximide or cyclosporin A, thus indicating that the induction was due to a secondary factor produced by the activated T cells. As neutralizing anti-IL-6 Abs effectively down-regulated the early induction of STAT proteins and as exogenously added IL-6 rapidly activated DNA binding similar to TCR-mediated bindings, it can be concluded that IL-6 is the factor responsible for the activation of STAT proteins in a primary T cell response

Thrombopoietin (TPO) is a newly cloned cytokine which is the major regulator of circulating platelet levels, acting on both proliferation and differentiation of megakaryocytes. We have investigated the ability of TPO to activate the JAK/STAT pathway in megakaryocytic cell lines. We used either the granulocyte-macrophage colony-stimulating factor (GM-CSF)- and/or erythropoietin (EPO)-dependent UT7 cell line in which the murine TPO receptor (mumpl) had been transfected (mumpl-UT7 transfectants) or the MO7E and DAMI cells which express endogenous human TPO receptors. We demonstrated that TPO activates the kinase JAK2 and a STAT5-like transcriptional factor but not STAT1, STAT2, STAT3 or STAT4, in a very rapid and transient manner. In order to better ascertain the specificity of the activation of STAT5-related factor by TPO, we investigated the effect of other cytokines/growth factors. Both GM-CSF and EPO activated the STAT5-like factor. In contrast, neither interferon (IFN)-gamma nor the mitogenic stem cell factor (SCF) activated STAT5, although IFN-gamma did activate STAT1 in those cells. The hematopoietic DNA binding activity related to STAT5 was identified as a p97 tyrosine-phosphorylated protein band which exhibited identical gel mobility to the mammary STAT5. Because v-mpl, a truncated form of the TPO receptor c-mpl, was shown to be oncogenic, we tested the activity of v-mpl on STAT5 and found STAT5 constitutively activated in two different v-mpl-expressing cells, the transiently transfected Cos7 cells and the stable v-mpl-UT7 transfectants. Overall, our data indicate that STAT5 is widely expressed in hematopoietic cells and activated by a number of cytokines, including TPO, GM-CSF and EPO, but not by IFN-gamma or SCF