Biomedical subjects

James N Ihle

Publications and source records attributed to James N Ihle.

At least 19 recordsLinked to original sources

PubMed2007

Jak2: normal function and role in hematopoietic disorders.

Janus kinase 2 (Jak2) associates with cytokine receptors and is essential for signal transduction by mediating tyrosine phosphorylation. Kinase activity is regulated by a series of interactions beginning with the requirement to bind to specific domains in receptors, suppression of activation by the pseudokinase domain, and the requirement for phosphorylation within the activation loop. Recent studies have implicated de-regulation of Jak2 kinase activity by chromosomal translocations in hematopoietic tumors and mutations within the pseudokinase domain in a spectrum of myeloproliferative diseases.

Gene Expression Regulation, Neoplastic↗

PubMed2006

Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2.

The tyrosine kinase, Janus kinase-2 (Jak2), plays a pivotal role in signal transduction through a variety of cytokine receptors, including the receptor for erythropoietin (Epo). Although the physiological relevance of Jak2 has been definitively established, less is known about its regulation. In studies assessing the roles of sites of tyrosine phosphorylation, we identified Y(119) in the FERM (band 4.1, Ezrin, radixin and moesin) domain as a phosphorylation site. In these studies, we demonstrate that the phosphorylation of Y(119) in response to Epo downregulates Jak2 kinase activity. Using a phosphorylation mimic mutation (Y(119)E), downregulation is shown to involve dissociation of Jak2 from the receptor complex. Conversely, a Y(119)F mutant is more stably associated with the receptor complex. Thus, in cytokine responses, ligand binding induces activation of receptor associated Jak2, autophosphorylation of Y(119) in the FERM domain and the subsequent dissociation of the activated Jak2 from the receptor and degradation. This regulation occurs with the receptors for Epo, thrombopoietin and growth hormone but not with the receptor for interferon-gamma.

Amino Acid Substitution↗

PubMed2006

Two domains of the erythropoietin receptor are sufficient for Jak2 binding/activation and function.

Biochemical and genetic studies have shown that Jak2 is an essential component of EpoR signal transduction which is required for normal erythropoiesis. However, whether Jak2 is the sole direct mediator of EpoR signal transduction remains controversial. To address this issue, we have used an extensive and systematic mutational analysis across the EpoR cytoplasmic tail and transmembrane domain with the goal of determining whether mutants that negatively affected EpoR biological activity but retained Jak2 activation could be identified. Analysis of over 40 mutant receptors established that two large domains in the membrane-proximal region, which include the previously defined Box1 and Box2 domains as well as a highly conserved glycine among cytokine receptors, are required for Jak2 binding and activation and to sustain biological activity of the receptor. Importantly, none of the mutants that lost the ability to activate Jak2 retained the ability to bind Jak2, thus questioning the validity of models of receptor reorientation for Jak2 activation. Also, no correlation was made between cell surface expression of the receptor and its ability to bind Jak2, thus questioning the role of Jak2 in trafficking the receptor to the plasma membrane. Collectively, the results suggest that Jak2 is the sole direct signaling molecule downstream of EpoR required for biological activity.

Amino Acid Sequence↗

PubMed2006

A role for STAT5A/B in protection of peripheral T-lymphocytes from postactivation apoptosis: insights from gene expression profiling.

Activation of the transcription factors STAT5A and STAT5B by JAK1 and JAK3 tyrosine kinases is a key event in downstream signaling of gammac (common gamma chain)-family cytokine receptors in lymphoid cells. STAT5A/B-deficiency in mice causes, among other consequences, a reduced size and altered composition of the peripheral T-cell pool with predominance of an activated or memory-like population (CD4(+)/CD44(high)/CD62L(low)) and a proliferative deficiency following antigenic stimulation and subsequent IL-2 treatment. To further elucidate the critical function of STAT5A/B in homeostasis and activation of murine naïve peripheral T-lymphocytes, we have analyzed global gene expression of STAT5A/B-deficient versus wild-type splenic T-cells by profiling with high-density oligonucleotide arrays (Affymetrix). Comparison of (1) basal gene expression of untreated peripheral STAT5A/B-deficient and control T-cells and (2) immediate early gene induction upon in vitro stimulation of either population with IL-2 has revealed differential expression of a broad range of genes potentially contributing to the defects of STAT5A/B deficient T-cells. In the context of enhanced apoptotic rates of STAT5A/B(-/-)-T-cells in vivo and upon TCR-stimulation in culture our data suggest a role for STAT5 in post-activation survival beyond regulation of antiapoptotic Bcl-2 proteins and hence provide new insights into the nature of the late proliferative block in the T-cell compartment caused by STAT5-deficiency.

Animals↗

PubMed2006

Trophoblast stem cells rescue placental defect in SOCS3-deficient mice.

Stem cells have important clinical and experimental potentials. Trophoblast stem (TS) cells possess the ability to differentiate into trophoblast subtypes in vitro and contribute to the trophoblast lineage in vivo. Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of cytokine signaling. Targeted disruption of SOCS3 revealed embryonic lethality on E12.5; it was caused by placental defect with enhanced leukemia inhibitory factor receptor signaling. A complementation of the wild-type (WT) placenta by using tetraploid rescue technique showed that the embryonic lethality in SOCS3-deficient embryo was due to the placental defect. Here we demonstrate that TS cells supplementation rescues placental defect in SOCS3-deficient embryos. In the rescued placenta, TS cells were integrated into the placental structure, and a substantial structural improvement was observed in the labyrinthine layer that was disrupted in the SOCS3-deficient placenta. Importantly, by supplying TS cells, living SOCS3-deficient embryos were detected at term. These results indicate a functional contribution of TS cells in the placenta and their potential application.

Animals↗

PubMed2005

Role of erythropoietin receptor signaling in Friend virus-induced erythroblastosis and polycythemia.

Friend virus is an acutely oncogenic retrovirus that causes erythroblastosis and polycythemia in mice. Previous studies suggested that the Friend virus oncoprotein, gp55, constitutively activates the erythropoietin receptor (EPOR), causing uncontrolled erythroid proliferation. Those studies showed that gp55 confers growth factor independence on an interleukin-3 (IL-3)-dependent cell line (Ba/F3) when the EPOR is coexpressed. Subsequently, we showed that a truncated form of the stem-cell kinase receptor (sf-STK) is required for susceptibility to Friend disease. Given the requirement for sf-STK, we sought to establish the in vivo significance of gp55-mediated activation of the EPOR. We found that the cytoplasmic tyrosine residues of the EPOR, and signal transducer and activator of transcription-5 (STAT5), which acts through these sites, are not required for Friend virus-induced erythroblastosis. The EPOR itself was required for the development of erythroblastosis but not for gp55-mediated erythroid proliferation. Interestingly, the murine EPOR, which is required for gp55-mediated Ba/F3-cell proliferation, was dispensable for erythroblastosis in vivo. Finally, gp55-mediated activation of the EPOR and STAT5 are required for Friend virus-induced polycythemia. These results suggest that Friend virus activates both sf-STK and the EPOR to cause deregulated erythroid proliferation and differentiation.

Animals↗

PubMed2005

Stat5 tetramer formation is associated with leukemogenesis.

Activation of Stat5 is frequently found in leukemias. To study the mechanism and role of Stat5 activation, we introduced a constitutively activated Stat5a mutant, cS5F, into murine bone marrow (BM) cells. BM transplantation with cS5F-transfected cells caused development of multilineage leukemias in lethally irradiated wild-type or nonirradiated Rag2(-/-) mice. The leukemic cells showed strongly enhanced levels of cS5F tetramers but unchanged cS5F dimer levels in a DNA binding assay. Moreover, Stat5a mutants engineered to form only dimers, but not tetramers, failed to induce leukemias. In addition, Stat5 tetramers were found to accumulate in excess compared to dimers in various human leukemias. These data suggest that Stat5 tetramers are associated with leukemogenesis.

Animals↗

PubMed2005

Evi-1 expression in Xenopus.

The Evi-1 gene was first identified as a site for viral integration in murine myeloid leukemia. Evi-1 is a zinc finger transcription factor that has been implicated in the development of myeloid neoplasia. In humans, disruption of the Evi-1 locus, by chromosomal rearrangements, is associated with myeloid leukemia and myelodyplastic syndromes. Here, we report the cloning and developmental pattern of expression of Xenopus Evi-1. xEvi-1 is expressed during oogenesis and during embryonic development. In situ hydridization reveals that xEvi-1 has a dynamic expression profile during early embryonic development. Expression of Evi-1 is detected by in situ hybridization in the pronephric tissue, the brain and in neural crest derivatives of the head and neck.

Amino Acid Sequence↗

PubMed2004

Determination of the transphosphorylation sites of Jak2 kinase.

Janus kinases are the key enzymes involved in the initial transmission of signals in response to type I and II cytokines. Activation of the signal begins with the transphosphorylation of Jak kinases. Substrates that give rise to downstream events are recruited to the receptor complex in part by interactions with phosphorylated tyrosines. The identity of many of the phosphotyrosines responsible for recruitment has been elucidated as being receptor-based tyrosines. The ability of Jaks to recruit substrates through their own phosphotyrosines has been demonstrated for tyrosines in the kinase activation loop. Recent studies demonstrate that other tyrosines have implications in regulatory roles of Jak kinase activity. In this study, baculovirus-produced Jak2 was utilized to demonstrate that transphosphorylation of Jak kinases occurs on multiple residues throughout the protein. We demonstrate that among the tyrosines phosphorylated, those in the kinase domain occur as expected, but many other sites are also phosphorylated. The tyrosines conserved in the Jak family are the object of this study, although many of them are phosphorylated, many are not. This result suggests that conservation of tyrosines is perhaps as important in maintaining structure of the Jak family. Additionally, non-Jak family conserved tyrosines are phosphorylated suggesting that the individual Jaks ability to phosphorylated specific tyrosines may influence signals emitting from activated Jaks.

Amino Acid Sequence↗

PubMed2004

Re-examination of the role of suppressor of cytokine signaling 1 (SOCS1) in the regulation of toll-like receptor signaling.

Suppressor of cytokine signaling 1 (SOCS1) is an obligate negative regulator of cytokine signaling and most importantly in vivo, signaling via the interferon-gamma (IFN-gamma) receptor. SOCS1, via its Src homology 2 domain, binds to phosphotyrosine residues in its targets, reducing the amplitude of signaling from cytokine receptors. SOCS1 is also implicated in blocking Toll-like receptor (TLR) signaling in macrophages activated by TLR agonists such as lipopolysaccharide (LPS), thus regulating multiple steps in the activation of innate immune responses. To rigorously test this, we isolated macrophages from Socs1-/- mice on multiple genetic backgrounds. We found no evidence that SOCS1 blocked TLR-activated pathways, endotoxin tolerance, or nitric oxide production. However, Socs1-/-;IFN-gamma-/- mice were extremely susceptible to LPS challenge, confirming previous findings. Because LPS induces IFN-beta production from macrophages, we tested whether SOCS1 regulates IFN-alpha/beta receptor signaling. We find that SOCS1 is required to inhibit IFN-alpha/beta receptor signaling in vitro. Furthermore, the absence of a single allele encoding TYK2, a JAK (Janus kinase) family member essential IFN-alpha/beta receptor signaling, rescued Socs1-/- mice from early lethality, even in the presence of IFN-gamma. We conclude that previous reports linking SOCS1 to TLR signaling are most likely due to effects on IFN-alpha/beta receptor signaling.

Animals↗

PubMed2004

Regulation of progesterone levels during pregnancy and parturition by signal transducer and activator of transcription 5 and 20alpha-hydroxysteroid dehydrogenase.

The two highly related signal transducers and activators of transcription (Stats), Stat5a and Stat5b, are major mediators of prolactin signaling in both the mammary gland and in the ovary. Deficiencies in Stat5b, or in both Stat5a and Stat5b, result in loss of pregnancy during midgestation and are correlated with an increase in ovarian 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) and a decrease in serum progesterone, which normally declines only immediately before parturition. To determine the relative contribution of 20alpha-HSD to progesterone metabolism and Stat5 function during pregnancy and parturition, we created a 20alpha-HSD-deficient strain of mice by gene disruption. Mice deficient for 20alpha-HSD sustain high progesterone levels and display a delay in parturition of several days demonstrating that 20alpha-HSD regulates parturition downstream of the prostaglandin F2alpha receptor in an essential and nonredundant manner. Moreover, 20alpha-HSD deficiency partially corrected the abortion of pregnancies associated with Stat5b deficiency, supporting the concept that prolactin activation of Stat5b is important in suppressing 20alpha-HSD gene expression and thereby allowing the maintenance of progesterone levels that are required to sustain pregnancy.

20-alpha-Hydroxysteroid Dehydrogenase↗

PubMed2004

Regulation of ZAP-70 activation and TCR signaling by two related proteins, Sts-1 and Sts-2.

T cells play a central role in the recognition and elimination of foreign pathogens. Signals through the T cell receptor (TCR) control the extent and duration of the T cell response. To ensure that T cells are not inappropriately activated, signaling pathways downstream of the TCR are subject to multiple levels of positive and negative regulation. Herein, we describe two related proteins, Sts-1 and Sts-2, that negatively regulate TCR signaling. T cells from mice lacking Sts-1 and Sts-2 are hyperresponsive to TCR stimulation. The phenotype is accompanied by increased Zap-70 phosphorylation and activation, including its ubiquitinylated forms. Additionally, hyperactivation of signaling proteins downstream of the TCR, a marked increase in cytokine production by Sts1/2(-/-) T cells, and increased susceptibility to autoimmunity in a mouse model of multiple sclerosis is observed. Therefore, Sts-1 and Sts-2 are critical regulators of the signaling pathways that regulate T cell activation.

Animals↗

PubMed2004

The centrosomal, putative tumor suppressor protein TACC2 is dispensable for normal development, and deficiency does not lead to cancer.

TACC2 is a member of the transforming acidic coiled-coil-containing protein family and is associated with the centrosome-spindle apparatus during cell cycling. In vivo, the TACC2 gene is expressed in various splice forms predominantly in postmitotic tissues, including heart, muscle, kidney, and brain. Studies of human breast cancer samples and cell lines suggest a putative role of TACC2 as a tumor suppressor protein. To analyze the physiological role of TACC2, we generated mice lacking TACC2. TACC2-deficient mice are viable, develop normally, are fertile, and lack phenotypic changes compared to wild-type mice. Furthermore, TACC2 deficiency does not lead to an increased incidence of tumor development. Finally, in TACC2-deficient embryonic fibroblasts, proliferation and cell cycle progression as well as centrosome numbers are comparable to those in wild-type cells. Therefore, TACC2 is not required, nonredundantly, for mouse development and normal cell proliferation and is not a tumor suppressor protein.

Animals↗

PubMed2004

Absence of an essential role for thymic stromal lymphopoietin receptor in murine B-cell development.

The murine cytokine thymic stromal lymphopoietin (TSLP) supports the development of B220+ IgM+ immature B cells and induces thymocyte proliferation in vitro. Human TSLP, by contrast, activates CD11c+ dendritic cells, but not B or T cells. Recent studies have demonstrated that the receptor for TSLP consists of a heterodimer of the interleukin 7 (IL-7) alpha chain and a novel protein that resembles the hematopoietic cytokine receptor common gamma chain. We examined signal transduction by the gamma-like chains using chimeric receptor proteins. The cytoplasmic domain of the human, but not of the murine, gamma-like chain, activates Jak2 and Stat5 and supports the proliferation of hematopoietic cell lines. In order to assess the role of the murine gamma-like chain in vivo, we generated gamma-like chain-deficient mice. Receptor-deficient mice are unresponsive to TSLP but exhibit no obvious phenotypic defects. In particular, hematopoietic cell development appeared normal. B-cell development, including the IgM+ compartment, was unaffected by loss of the TSLP pathway, as were T lymphopoiesis and lymphocyte proliferation in vitro. Cytokine receptors that utilize the common gamma chain signal through the lymphocyte-specific kinase Jak3. Mice deficient in Jak3 exhibit a SCID phenotype but harbor a residual B220+ splenic lymphocyte population. We demonstrate here that this residual lymphocyte population is lost in mice lacking both the gamma-like chain and Jak3.

Animals↗

PubMed2003

SOCS3 regulates the plasticity of gp130 signaling.

Suppressor of cytokine signaling (SOCS) proteins are feedback inhibitors of the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathway. SOCS3 is upregulated by several signals in macrophages and has been implicated as a regulator of various signaling pathways. Here we show that phosphorylation of STAT3 is prolonged in mouse Socs3-deficient macrophages after stimulation with interleukin-6 (IL-6) but not IL-10, indicating that SOCS3 specifically affects signaling mediated by IL-6 and gp130. IL-6 induces a wider transcriptional response in Socs3-deficient macrophages than in wild-type cells; this response is dominated by interferon (IFN)-regulated genes owing to an excess of STAT1 phosphorylation. Thus, SOCS3 functions to control the quality of the response to IL-6 and prevents the activation of an IFN-induced program of gene expression.

Animals↗

PubMed2003

Jak1 deficiency leads to enhanced Abelson-induced B-cell tumor formation.

The Janus kinase Jak1 has been implicated in tumor formation by the Abelson oncogene. In this study we show that loss of Jak1 does not affect in vitro transformation by v-abl as defined by the ability to induce cytokine-independent B-cell colony formation or establishment of B-cell lines. However, Jak1-deficient, v-abl-transformed cell lines were more tumorgenic than wild-type cells when transplanted subcutaneously into severe combined immunodeficient (SCID) mice or injected intravenously into nude mice. Jak1 deficiency was associated with a loss in the ability of interferon-gamma (IFN-gamma)to induce growth arrest and/or apoptosis of v-abl-transformed pre-B cells or tumor growth in SCID mice. Moreover, IFN-gamma mRNA could be detected in growing tumors, and tumor cells explanted from SCID mice had lost the ability to respond to IFN-gamma in 9 of 20 cases, whereas the response to interferon-alpha (IFN-alpha) remained intact. Importantly, a similar increase in tumorgenicity was observed when IFN-gamma-deficient cells were injected into SCID mice, identifying the tumor cell itself as the main source of IFN-gamma. These findings demonstrate that Jak1, rather than promoting tumorgenesis as previously proposed, is critical in mediating an intrinsic IFN-gamma-dependent tumor surveillance.

Abelson murine leukemia virus↗

PubMed2003

SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation.

Suppressor of cytokine signaling 3 (SOCS3) binds cytokine receptors and thereby suppresses cytokine signaling. Deletion of SOCS3 causes an embryonic lethality that is rescued by a tetraploid rescue approach, demonstrating an essential role in placental development and a non-essential role in embryo development. Rescued SOCS3-deficient mice show a perinatal lethality with cardiac hypertrophy. SOCS3-deficient placentas have reduced spongiotrophoblasts and increased trophoblast secondary giant cells. Enforced expression of SOCS3 in a trophoblast stem cell line (Rcho-1) suppresses giant cell differentiation. Conversely, SOCS3-deficient trophoblast stem cells differentiate more readily to giant cells in culture, demonstrating that SOCS3 negatively regulates trophoblast giant cell differentiation. Leukemia inhibitory factor (LIF) promotes giant cell differentiation in vitro, and LIF receptor (LIFR) deficiency results in loss of giant cell differentiation in vivo. Finally, LIFR deficiency rescues the SOCS3-deficient placental defect and embryonic lethality. The results establish SOCS3 as an essential regulator of LIFR signaling in trophoblast differentiation.

Animals↗

PubMed2003

Puma is an essential mediator of p53-dependent and -independent apoptotic pathways.

Puma encodes a BH3-only protein that is induced by the p53 tumor suppressor and other apoptotic stimuli. To assess its physiological role in apoptosis, we generated Puma knockout mice by gene targeting. Here we report that Puma is essential for hematopoietic cell death triggered by ionizing radiation (IR), deregulated c-Myc expression, and cytokine withdrawal. Puma is also required for IR-induced death throughout the developing nervous system and accounts for nearly all of the apoptotic activity attributed to p53 under these conditions. These findings establish Puma as a principal mediator of cell death in response to diverse apoptotic signals, implicating Puma as a likely tumor suppressor.

Animals↗