An early onset progressive motor neuron disorder in Scyl1-deficient mice is associated with mislocalization of TDP-43.

The molecular and cellular bases of motor neuron diseases (MNDs) are still poorly understood. The diseases are mostly sporadic, with ~10% of cases being familial. In most cases of familial motor neuronopathy, the disease is caused by either gain-of-adverse-effect mutations or partial loss-of-function mutations in ubiquitously expressed genes that serve essential cellular functions. Here we show that deletion of Scyl1, an evolutionarily conserved and ubiquitously expressed gene encoding the COPI-associated protein pseudokinase SCYL1, causes an early onset progressive MND with characteristic features of amyotrophic lateral sclerosis (ALS). Skeletal muscles of Scyl1(-/-) mice displayed neurogenic atrophy, fiber type switching, and disuse atrophy. Peripheral nerves showed axonal degeneration. Loss of lower motor neurons (LMNs) and large-caliber axons was conspicuous in Scyl1(-/-) animals. Signs of neuroinflammation were seen throughout the CNS, most notably in the ventral horn of the spinal cord. Neural-specific, but not skeletal muscle-specific, deletion of Scyl1 was sufficient to cause motor dysfunction, indicating that SCYL1 acts in a neural cell-autonomous manner to prevent LMN degeneration and motor functions. Remarkably, deletion of Scyl1 resulted in the mislocalization and accumulation of TDP-43 (TAR DNA-binding protein of 43 kDa) and ubiquilin 2 into cytoplasmic inclusions within LMNs, features characteristic of most familial and sporadic forms of ALS. Together, our results identify SCYL1 as a key regulator of motor neuron survival, and Scyl1(-/-) mice share pathological features with many human neurodegenerative conditions.

Chromatin condensation via the condensin II complex is required for peripheral T-cell quiescence.

Naive T cells encountering their cognate antigen become activated and acquire the ability to proliferate in response to cytokines. Stat5 is an essential component in this response. We demonstrate that Stat5 cannot access DNA in naive T cells and acquires this ability only after T-cell receptor (TCR) engagement. The transition is not associated with changes in DNA methylation or global histone modification but rather chromatin decondensation. Condensation occurs during thymocyte development and proper condensation is dependent on kleisin-ß of the condensin II complex. Our findings suggest that this unique chromatin condensation, which can affect interpretations of chromatin accessibility assays, is required for proper T-cell development and maintenance of the quiescent state. This mechanism ensures that cytokine driven proliferation can only occur in the context of TCR stimulation.

Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.

Here we identify a component of the nuclear RNA cap-binding complex (CBC), Ars2, that is important for miRNA biogenesis and critical for cell proliferation. Unlike other components of the CBC, Ars2 expression is linked to the proliferative state of the cell. Deletion of Ars2 is developmentally lethal, and deletion in adult mice led to bone marrow failure whereas parenchymal organs composed of nonproliferating cells were unaffected. Depletion of Ars2 or CBP80 from proliferating cells impaired miRNA-mediated repression and led to alterations in primary miRNA processing in the nucleus. Ars2 depletion also reduced the levels of several miRNAs, including miR-21, let-7, and miR-155, that are implicated in cellular transformation. These findings provide evidence for a role for Ars2 in RNA interference regulation during cell proliferation.

Negative regulation of Jak2 by its auto-phosphorylation at tyrosine 913 via the Epo signaling pathway.

Janus kinase 2 (Jak2) has a pivotal role in erythropoietin (Epo) signaling pathway, including erythrocyte differentiation and Stat5 activation. In the course of screening for critical phosphorylation of tyrosine residues in Jak2, we identified tyrosine 913 (Y(913)) as a novel and functional phosphorylation site, which negatively regulates Jak2. Phosphorylation at Y(913) rapidly occurred and was sustained for at least 120 min after Epo stimulation, in contrast to the transient phosphorylation of Y(1007/1008) in the activation loop of Jak2. Interestingly, phosphorylation defective mutation of Y(913) (Y(913)F) results in a significant enhancement of Epo-induced Jak2 activation, whereas phosphorylation mimic mutation of Y(913) (Y(913)E) completely abrogated its activation. Furthermore, Jak2 deficient fetal liver cells expressing Y(913)F mutant generated many mature erythroid BFU-E and CFU-E colonies, while Y(913)E mutant failed to reconstitute Jak2 deficiency. We also demonstrate, in Jak1, phosphorylation of Y(939), a corresponding tyrosine residue with Y(913), negatively regulated Jak1 signaling pathway. Accordingly, our results suggest that this tyrosine phosphorylation in JH1 domain may be involved in common negative regulation mechanism for Jak family.

Leukemia inhibitory factor regulates trophoblast giant cell differentiation via Janus kinase 1-signal transducer and activator of transcription 3-suppressor of cytokine signaling 3 pathway.

Suppressor of cytokine signaling 3 (SOCS3) inhibits leukemia-inhibitory factor (LIF) signaling and acts as a negative regulator. Deletion of SOCS3 causes embryonic lethality because of placental failure, and genetic reduction of LIF or the LIF receptor (LIFR) in SOCS3-deficient mice rescues placental defects and embryonic lethality; this indicates that SOCS3 is an essential inhibitor of LIFR signaling. However, the downstream signaling molecule that acts as a link between the LIFR and SOCS3 has not been identified. In this study we explored the downstream signaling of LIFR. The administration of LIF to SOCS3-heterozygous pregnant mice promotes trophoblast giant cell differentiation and accelerates placental failure in SOCS3-deficient mice. SOCS3-deficient trophoblast stem cells show enhanced and prolonged signal transducer and activator of transcription 3 (Stat3) activation by LIF stimulation. Further, in the trophoblasts of SOCS3-deficient placenta and differentiating cells from the choriocarcinoma-derived cell line Rcho-1 cells, constitutive activation of Stat3 is observed. The forced expression of SOCS3, dominant-negative Stat3, and dominant-negative Janus kinase 1 (JAK1) in Rcho-1 cells significantly suppressed the trophoblast giant cell differentiation of these cells. In addition, the number of trophoblast giant cells is significantly reduced concomitant with an increased number of precursor trophoblasts in JAK1-deficient placentas. Finally, JAK1 deficiency rescues placental defects and embryonic lethality in SOCS3-deficient mice. These results indicate that the LIFR signaling is finely coordinated by JAK1, Stat3, and SOCS3 and regulates trophoblast giant cell differentiation. In addition, these data establish that LIFR-JAK1-Stat3-SOCS3 signaling is an essential pathway for the regulation of trophoblast giant cell differentiation.

Hax1-mediated processing of HtrA2 by Parl allows survival of lymphocytes and neurons.

Cytokines affect a variety of cellular functions, including regulation of cell numbers by suppression of programmed cell death. Suppression of apoptosis requires receptor signalling through the activation of Janus kinases and the subsequent regulation of members of the B-cell lymphoma 2 (Bcl-2) family. Here we demonstrate that a Bcl-2-family-related protein, Hax1, is required to suppress apoptosis in lymphocytes and neurons. Suppression requires the interaction of Hax1 with the mitochondrial proteases Parl (presenilin-associated, rhomboid-like) and HtrA2 (high-temperature-regulated A2, also known as Omi). These interactions allow Hax1 to present HtrA2 to Parl, and thereby facilitates the processing of HtrA2 to the active protease localized in the mitochondrial intermembrane space. In mouse lymphocytes, the presence of processed HtrA2 prevents the accumulation of mitochondrial-outer-membrane-associated activated Bax, an event that initiates apoptosis. Together, the results identify a previously unknown sequence of interactions involving a Bcl-2-family-related protein and mitochondrial proteases in the ability to resist the induction of apoptosis when cytokines are limiting.

Jak2 FERM domain interaction with the erythropoietin receptor regulates Jak2 kinase activity.

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement.

Characterization of a family of novel cysteine- serine-rich nuclear proteins (CSRNP).

Gene array analysis has been widely used to identify genes induced during T cell activation. Our studies identified an immediate early gene that is strongly induced in response to IL-2 in mouse T cells which we named cysteine- serine-rich nuclear protein-1 (CSRNP-1). The human ortholog was previously identified as an AXIN1 induced gene (AXUD1). The protein does not contain sequence defined domains or motifs annotated in public databases, however the gene is a member of a family of three mammalian genes that share conserved regions, including cysteine- and serine-rich regions and a basic domain, they encode nuclear proteins, possess transcriptional activation domain and bind the sequence AGAGTG. Consequently we propose the nomenclature of CSRNP-1, -2 and -3 for the family. To elucidate the physiological functions of CSRNP-1, -2 and -3, we generated mice deficient for each of these genes by homologous recombination in embryonic stem cells. Although the CSRNP proteins have the hallmark of transcription factors and CSRNP-1 expression is highly induced by IL-2, deletion of the individual genes had no obvious consequences on normal mouse development, hematopoiesis or T cell functions. However, combined deficiencies cause partial neonatal lethality suggesting that the genes have redundant functions.

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.

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.

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.

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.

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.

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.

Divergent mechanisms utilized by SOCS3 to mediate interleukin-10 inhibition of tumor necrosis factor alpha and nitric oxide production by macrophages.

The cytokine interleukin-10 (IL-10) potently inhibits macrophage function through activation of the transcription factor STAT3. The expression of SOCS3 (suppressor of cytokine signaling-3) has been shown to be induced by IL-10 in a STAT3-dependent manner. However, the relevance of SOCS3 expression to the anti-inflammatory effect of IL-10 on macrophages has been controversial. Through kinetic analysis of the requirement for SOCS3 in IL-10 inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNFalpha) transcription and translation, SOCS3 was found to be necessary for TNFalpha expression during the early phase, but not the late phase of IL-10 action. SOCS3 was essential for IL-10 inhibition of LPS-stimulated production of iNOS (inducible nitric-oxide synthase) protein and nitric oxide (NO). To determine the domains of SOCS3 protein important in mediating these effects, SOCS3-/- macrophages were reconstituted with SOCS3 mutated for the SH2, KIR, SOCS box domains, and tyrosines 204 (Tyr204) and 221 (Tyr221). The SH2 domain, SOCS box, and both Tyr204 and Tyr221 were required for IL-10 inhibition of TNFalpha mRNA and protein expression, but interestingly the KIR domain was necessary only for IL-10 inhibition of TNFalpha protein expression. In contrast, Tyr204 and Tyr221 were the only structural features of SOCS3 that were necessary in mediating IL-10 inhibition of iNOS protein expression and NO production. These data define SOCS3 as an important mediator of IL-10 inhibition of macrophage activation and that SOCS3 interferes with distinct LPS-stimulated signal transduction events through differing mechanisms.

Regulation of interleukin 7-dependent immunoglobulin heavy-chain variable gene rearrangements by transcription factor STAT5.

Rearrangement of immunoglobulin heavy-chain variable (V(H)) gene segments has been suggested to be regulated by interleukin 7 signaling in pro-B cells. However, the genetic evidence for this recombination pathway has been challenged. Furthermore, no molecular components that directly control V(H) gene rearrangement have been elucidated. Using mice deficient in the interleukin 7-activated transcription factor STAT5, we demonstrate here that STAT5 regulated germline transcription, histone acetylation and DNA recombination of distal V(H) gene segments. STAT5 associated with V(H) gene segments in vivo and was recruited as a coactivator with the transcription factor Oct-1. STAT5 did not affect the nuclear repositioning or compaction of the immunoglobulin heavy-chain locus. Therefore, STAT5 functions at a distinct step in regulating distal V(H) recombination in relation to the transcription factor Pax5 and histone methyltransferase Ezh2.

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.

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.

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.

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.