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J N Ihle

Publications and source records attributed to J N Ihle.

At least 19 recordsLinked to original sources
PubMed2000

Stat5a/b contribute to interleukin 7-induced B-cell precursor expansion, but abl- and bcr/abl-induced transformation are independent of stat5.

The cytokines interleukin 7 (IL-7) and interleukin 4 (IL-4) regulate lymphoid differentiation and function and activate the transcription factor Stat5. Using mice deficient for the 2 highly related transcription factors, Stat5a and Stat5b (Stat5a/b(-/-)), we investigated the role of Stat5 for B-cell differentiation, expansion, and function. Peripheral blood B cells of Stat5-deficient mice are significantly reduced, but no proliferation defects in response to various mitogenic stimuli are found. Also, IgM and IgG1 antibody production and immunoglobulin class switching are not affected. Pre- and pro-B cells of Stat5-deficient animals were found to have reduced responses to IL-7. Pro- and pre-B cells are the target cells of the abl oncogene and numerous studies have suggested that Stat5a/b is essential for transformation by derivatives of the Abelson (abl) gene. To assess the role of Stat5a/b in transformation, we have evaluated the ability of various abl derivatives to transform cells from Stat5a/b-deficient mice in vitro or in vivo. We demonstrate that the absence of Stat5a/b is not essential for the induction of lymphoid or myeloid tumors in vivo or on the ability to transform bone marrow cells in vitro.

Animals↗
PubMed2000

A small amphipathic alpha-helical region is required for transcriptional activities and proteasome-dependent turnover of the tyrosine-phosphorylated Stat5.

Cytokines induce the tyrosine phosphorylation and associated activation of signal transducers and activators of transcription (Stat). The mechanisms by which this response is terminated are largely unknown. Among a variety of inhibitors examined, the proteasome inhibitors MG132 and lactacystin affected Stat4, Stat5 and Stat6 turnover by significantly stabilizing the tyrosine-phosphorylated form. However, these proteasome inhibitors did not affect downregulation of the tyrosine-phosphorylated Stat1, Stat2 and Stat3. With Stat5 isoforms, we have observed that tyrosine-phosphorylated carboxyl-truncated forms of Stat5 proteins were considerably more stable than phosphorylated wild-type forms of the protein. Also, the C-terminal region of Stat5 could confer proteasome-dependent downregulation to Stat1. With a series of C-terminal deletion mutants, we have defined a relatively small, potentially amphipathic alpha-helical region that is required for the rapid turnover of the phosphorylated Stat5 proteins. The region is also required for transcriptional activation, suggesting that the functions are linked. The results are consistent with a model in which the transcriptional activation domain of activated Stat5 is required for its transcriptional activity and downregulation through a proteasome-dependent pathway.

Acetylcysteine↗
PubMed2000

Antiapoptotic activity of Stat5 required during terminal stages of myeloid differentiation.

Stat5 is activated by multiple receptors of hematopoietic cytokines. To study its role during hematopoiesis, we have generated primary chicken myeloblasts expressing different dominant-negative (dn) alleles of Stat5. This caused a striking inability to generate mature cells, due to massive apoptosis during differentiation. Bcl-2 was able to rescue differentiating cells expressing dnStat5 from apoptosis, suggesting that during cytokine-dependent differentiation the main function of the protein is to ensure cell survival. Our findings with dnStat5-expressing chicken myeloblasts were confirmed with primary hematopoietic cells from Stat5a/Stat5b-deficient mice. Bone marrow cells from these animals displayed a strong increase in apoptotic cell death during GM-CSF-dependent functional maturation in vitro. The antiapoptotic protein Bcl-x was induced by GM-CSF and IL-3 in a Stat5-dependent fashion. Ectopic expression of Bcl-x rescued Stat5-deficient bone marrow cells from apoptosis, indicating that Stat5 promotes the survival of myeloid progenitor cells through its ability to induce transcription of the bcl-x gene. Finally, the recruitment of myeloid cells to inflammatory sites was found strongly impeded in Stat5-deficient mice. Taken together, our findings suggest that Stat5 may promote cytokine-dependent survival and proliferation of differentiating myeloid progenitor cells in stress or pathological situations, such as inflammation.

Alleles↗
PubMed2000

Phospholipase Cgamma2 is essential in the functions of B cell and several Fc receptors.

Many receptors activate phospholipase Cgamma1 or -gamma2. To assess the role of PLCgamma2, we derived enzyme-deficient mice. The mice are viable but have decreased mature B cells, a block in pro-B cell differentiation, and B1 B cell deficiency. IgM receptor-induced Ca2+ flux and proliferation to B cell mitogens are absent. IgM, IgG2a, and IgG3 levels are reduced, and T cell-independent antibody production is absent. The similarity to Btk- or Blnk-deficient mice demonstrates that PLCgamma2 is downstream in Btk/Blnk signaling. FcRgamma signaling is also defective, resulting in a loss of collagen-induced platelet aggregation, mast cell FcepsilonR function, and NK cell FcgammaRIII and 2B4 function. The results define a signal transduction pathway broadly utilized by immunoglobulin superfamily receptors.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Reconstitution of early lymphoid proliferation and immune function in Jak3-deficient mice by interleukin-3.

Expansion of early lymphoid progenitors requires interleukin-7 (IL-7), which functions through gamma(c)-mediated receptor activation of Jak3. Jak3 deficiency is a cause of severe combined immunodeficiency (SCID) in humans and mice. IL-3 activates many of the same signaling pathways as IL-7, such as Stat5, but achieves this effect through the activation of Jak2 rather than Jak3. We hypothesized that expansion of an IL-7-responsive precursor population through a Jak3-independent pathway using IL-3 may stimulate early lymphoid progenitors and restore lymphopoiesis in Jak3(-/-) mice. Newborn Jak3(-/-) mice that were injected with IL-3 demonstrated thymic enlargement, a 2- to 20-fold increase in thymocyte numbers, and up to a 10-fold expansion in the number of CD4(+), CD8(+), and B220(+)/IgM(+) splenic lymphocytes, consistent with an effect upon an early lymphoid progenitor population. In contrast to control mice, IL-3-treated Jak3(-/-) mice challenged with the allogeneic major histocompatibility complex (MHC) class I-bearing tumor P815 developed a specific CD8-dependent cytotoxic T lymphocyte (CTL) response. IL-3-treated mice also mounted influenza-specific CTL responses and survival was prolonged. The beneficial effects of IL-3 are proposed to be produced by stimulation of a lymphoid precursor population of IL-7Ralpha(+)/IL-3Ralpha(+) cells that we identified in wild-type bone marrow. In vitro, we show that an early IL-7R(+) lymphoid progenitor population expresses IL-3R and proliferates in response to IL-3 and that IL-3 activates Stat5 comparably to IL-7. Clinically, IL-3 may therefore be useful treatment for X-linked and Jak3-deficient SCID patients who lack bone marrow donors.

Animals↗
PubMed1999

SOCS1 deficiency causes a lymphocyte-dependent perinatal lethality.

SOCS1 is an SH2-containing protein that is primarily expressed in thymocytes in a cytokine- and T cell receptor-independent manner. SOCS1 deletion causes perinatal lethality with death by 2-3 weeks. During this period thymic changes include a loss of cellularity and a switch from predominantly CD4+ CD8+ to single positive cells. Peripheral T cells express activation antigens and proliferate to IL-2 in the absence of anti-CD3. In addition, IFNgamma is present in the serum. Reconstitution of the lymphoid lineage of JAK3-deficient mice with SOCS1-deficient stem cells recapitulates the lethality and T cell alterations. Introducing a RAG2 or IFNgamma deficiency eliminates lethality. The results demonstrate that lymphocytes are critical to SOCS1-associated perinatal lethality and implicate SOCS1 in lymphocyte differentiation or regulation.

Age Factors↗
PubMed1999

SOCS3 is essential in the regulation of fetal liver erythropoiesis.

SOCS3 (CIS3/JAB2) is an SH2-containing protein that binds to the activation loop of Janus kinases, inhibiting kinase activity, and thereby suppressing cytokine signaling. During embryonic development, SOCS3 is highly expressed in erythroid lineage cells and is Epo independent. Transgene-mediated expression blocks fetal erythropoiesis, resulting in embryonic lethality. SOCS3 deletion results in an embryonic lethality at 12-16 days associated with marked erythrocytosis. Moreover, the in vitro proliferative capacity of progenitors is greatly increased. SOCS3-deficient fetal liver stem cells can reconstitute hematopoiesis in lethally irradiated adults, indicating that its absence does not disturb bone marrow erythropoiesis. Reconstitution of lymphoid lineages in JAK3-deficient mice also occurs normally. The results demonstrate that SOCS3 is critical in negatively regulating fetal liver hematopoiesis.

Animals↗
PubMed1999

The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop.

The Janus family of protein tyrosine kinases (JAKs) regulate cellular processes involved in cell growth, differentiation and transformation through their association with cytokine receptors. However, compared with other kinases, little is known about cellular regulators of the JAKs. We have recently identified a JAK-binding protein (JAB) that inhibits JAK signaling in cells. In the studies presented here we demonstrate that JAB specifically binds to the tyrosine residue (Y1007) in the activation loop of JAK2, whose phosphorylation is required for activation of kinase activity. Binding to the phosphorylated activation loop requires the JAB SH2 domain and an additional N-terminal 12 amino acids (extended SH2 subdomain) containing two residues (Ile68 and Leu75) that are conserved in JAB-related proteins. An additional N-terminal 12-amino-acid region (kinase inhibitory region) of JAB also contributes to high-affinity binding to the JAK2 tyrosine kinase domain and is required for inhibition of JAK2 signaling and kinase activity. Our studies define a novel type of regulation of tyrosine kinases and might provide a basis for the design of specific tyrosine kinase inhibitors.

Amino Acid Sequence↗
PubMed1999

Stat5 is required for IL-2-induced cell cycle progression of peripheral T cells.

Many cytokines activate two highly homologous Stat proteins, 5a and 5b. Mice deficient in both genes lack all growth hormone and prolactin functions but retain functions associated with cytokines such as erythropoietin. Here, we demonstrate that, while lymphoid development is normal, Stat5a/b mutant peripheral T cells are profoundly deficient in proliferation and fail to undergo cell cycle progression or to express genes controlling cell cycle progression. In addition, the mice lack NK cells, develop splenomegaly, and have T cells with an activated phenotype, phenotypes seen in IL-2 receptor beta chain-deficient mice. These phenotypes are not seen in mice lacking Stat5a or Stat5b alone. The results demonstrate that the Stat5 proteins, redundantly, are essential mediators of IL-2 signaling in T cells.

Animals↗
PubMed1999

Stat5 activation is uniquely associated with cytokine signaling in peripheral T cells.

The activation and subsequent proliferation of peripheral T cells requires the engagement of the T cell and a cytokine receptor, typically the IL-2 or IL-4 receptors. Critical to understanding the regulation of peripheral T cells is the knowledge of the unique contributions of each receptor to full T cell activation and cell cycle progression. Mice deficient in Stat5a and Stat5b have demonstrated the essential role that these highly related proteins play in cell cycle progression following peripheral T cell activation. Here we demonstrate that activation of the Stat5 proteins by tyrosine phosphorylation is uniquely contributed by cytokine receptor signaling and specifically does not occur through the T cell receptor complex.

Animals↗
PubMed1998

Signaling by the cytokine receptor superfamily.

A variety of cytokines that regulate functions of multiple lineages share the utilization of receptors that are structurally and functionally related and are referred to as the cytokine receptor superfamily. These receptors associate with one or more of the four mammalian Janus kinases (Jaks) and ligand-induced receptor aggregation results in their activation. Critical roles for Jak3 and Jak2 are demonstrated by the phenotypes of mice that lack each gene. Among the substrates of the Jaks are one or more of the seven members of the signal transducers and activators of transcription (Stats). Each Stat family member plays a critical role in the biological functions of specific cytokines as demonstrated by the phenotype of mice lacking one or more of these genes.

Animals↗
PubMed1998

Interleukin-4 and -13 induce upregulation of the murine macrophage 12/15-lipoxygenase activity: evidence for the involvement of transcription factor STAT6.

When human monocytes or alveolar macrophages are cultured in the presence of interleukin (IL)-4 or IL-13, the expression of the reticulocyte-type 15-lipoxygenase is induced. In mice a 15-lipoxygenase is not expressed, but a leukocyte-type 12-lipoxygenase is present in peritoneal macrophages. To investigate whether both lipoxygenase isoforms exhibit a similar regulatory response toward cytokine stimulation, we studied the regulation of the leukocyte-type 12-lipoxygenase of murine peritoneal macrophages by interleukins and found that the activity of this enzyme is upregulated in a dose-dependent manner when the cells were cultured in the presence of the IL-4 or IL-13 but not by IL-10. When peripheral murine monocytes that do not express the lipoxygenase were treated with IL-4 expression of 12/15-lipoxygenase mRNA was induced, suggesting pretranslational control mechanisms. In contrast, no upregulation of the lipoxygenase activity was observed when the macrophages were prepared from homozygous STAT6-deficient mice. Peritoneal macrophages of transgenic mice that systemically overexpress IL-4 exhibited a threefold to fourfold higher 12-lipoxygenase activity than cells prepared from control animals. A similar upregulation of 12-lipoxygenase activity was detected in heart, spleen, and lung of the transgenic animals. Moreover, a strong induction of the enzyme was observed in red cells during experimental anemia in mice. The data presented here indicate that (1) the 12-lipoxygenase activity of murine macrophages is upregulated in vitro and in vivo by IL-4 and/or IL-13, (2) this upregulation requires expression of the transcription factor STAT6, and (3) the constitutive expression of the enzyme appears to be STAT6 independent. The cytokine-dependent upregulation of the murine macrophage 12-lipoxygenase and its induction during experimental anemia suggests its close relatedness with the human reticulocyte-type 15-lipoxygenase despite their differences in the positional specificity of arachidonic acid oxygenation.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1998

Selective regulation of Bcl-XL by a Jak kinase-dependent pathway is bypassed in murine hematopoietic malignancies.

Bcl-2 family proteins are key regulators of apoptosis and function as cell death antagonists (e.g., Bcl-2, Bcl-XL, and Mcl-1) or agonists (e.g., Bax, Bad, and Bak). Here we report that among the Bcl-2 family of proteins tested (Bcl-2, Bcl-XL, Mcl-1, Bax, Bad, and Bak), Bcl-XL was unique in that its protein levels were tightly regulated by hemopoietins in both immortal and primary myeloid progenitors. Investigating signaling pathways utilized by cytokine receptors established that the regulation of Bcl-XL protein levels is mediated by the Jak kinase pathway and is independent of other signaling effectors including STATs, PI-3' kinase, and Ras. Moreover, we provide the first direct evidence that Bcl-X is altered in cancer, because bcl-X expression was activated selectively by retroviral insertions in murine myeloid and T-cell hemopoietic malignancies. Tumors harboring bcl-X insertions had altered bcl-X RNAs, expressed elevated levels of Bcl-XL protein, and lacked the requirements for cytokines normally essential for cell survival. Finally, overexpression of Bcl-XL effectively protected IL-3-dependent myeloid cells from apoptosis following removal of trophic factors. Therefore, Bcl-XL functions as a key cytokine regulated anti-apoptotic protein in myelopoiesis and contributes to leukemia cell survival.

Animals↗
PubMed1998

Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses.

A variety of cytokines mediate the activation of Janus protein tyrosine kinases (Jaks). The Jaks then phosphorylate cellular substrates, including members of the signal transducers and activators of transcription (Stat) family of transcription factors. Among the Stats, the two highly related proteins, Stat5a and Stat5b, are activated by a variety of cytokines. To assess the role of the Stat5 proteins, mutant mice were derived that have the genes deleted individually or together. The phenotypes of the mice demonstrate an essential, and often redundant, role for the two Stat5 proteins in a spectrum of physiological responses associated with growth hormone and prolactin. Conversely, the responses to a variety of cytokines that activate the Stat5 proteins, including erythropoietin, are largely unaffected.

Animals↗
PubMed1998

Jak2 is essential for signaling through a variety of cytokine receptors.

A variety of cytokines activate receptor-associated members of the Janus family of protein tyrosine kinases (Jaks). To assess the role of Jak2, we have derived Jak2-deficient mice. The mutation causes an embryonic lethality due to the absence of definitive erythropoiesis. Fetal liver myeloid progenitors, although present based on the expression of lineage specific markers, fail to respond to erythropoietin, thrombopoietin, interleukin-3 (IL-3), or granulocyte/macrophage colony-stimulating factor. In contrast, the response to granulocyte specific colony-stimulating factor is unaffected. Jak2-deficient fibroblasts failed to respond to interferon gamma (IFNgamma), although the responses to IFNalpha/beta and IL-6 were unaffected. Lastly, reconstitution experiments demonstrate that Jak2 is not required for the generation of lymphoid progenitors, their amplification, or functional differentiation. Therefore, Jak2 plays a critical, nonredundant role in the function of a specific group of cytokines receptors.

Animals↗
PubMed1998

Cytokine rescue of p53-dependent apoptosis and cell cycle arrest is mediated by distinct Jak kinase signaling pathways.

Exposure of hematopoietic progenitors to gamma-irradiation (IR) induces p53-dependent apoptosis and a p53-independent G2/M cell cycle arrest. These responses to DNA-damage can be inhibited by treatment with cytokine growth factors. Here we report that gamma-IR-induced apoptosis and cell cycle arrest are suppressed by specific cytokines (e.g., erythropoietin and interleukin-3) and that activation of the Jak kinase is necessary and sufficient for these effects. Using myleoid cells expressing a series of erythropoietin receptor (EpoR) mutants, we have demonstrated that Jak kinase-dependent signals initiated from the membrane proximal domain of EpoR were sufficient to prevent IR-induced apoptotic cell death, but failed to prevent cell cycle arrest. Cell survival by Epo did not require activation of other known signaling pathways including PI-3 kinase, PLC-gamma, Ras or Stats. Signaling targets of Jak kinase pathways included members of the Bcl-2 family of anti-apoptotic proteins, and enforced expression of Bcl-2 or Bcl-xL was as effective as cytokine treatment in blocking IR-induced apoptosis but did not prevent growth arrest. A distinct signal derived from a membrane distal domain of EpoR is required to overcome growth arrest associated with DNA damage. These findings functionally link the Jak signaling pathway to suppression of p53-mediated cell death by cytokines and demonstrate that the apoptotic and growth arrest responses to DNA damage in hematopoietic cells are modulated by distinct, cytokine specific signal transduction pathways.

Animals↗
PubMed1998

Requirement for Jak3 in mature T cells: its role in regulation of T cell homeostasis.

The tyrosine kinase Jak3 plays a key role in transducing signals from the IL-2, -4, -7, -9, and -15 receptors. Mice lacking Jak3 exhibit a profound, early block in both B and T cell development. To examine the mechanisms whereby Jak3 influences T cell function, we have reconstituted thymic development in Jak3-/- animals by introducing a Jak3 transgene in which expression was driven by the lck proximal promoter. Thymic reconstitution required Jak3 kinase activity, as catalytically inactive Jak3 did not restore early thymic development. Furthermore, the thymus-restricted expression pattern of the transgene allowed us to assess the requirement for Jak3 in peripheral T cells. In these mice, loss of Jak3 expression was associated with a failure to proliferate in response to antigen receptor crosslinking, the accumulation of T cells manifesting an activated cell surface phenotype, and an increased CD4/CD8 ratio among peripheral T cells, all of which are characteristics that were observed in Jak3-/- animals. Finally, we present data which suggest that peripheral T cells proliferate more rapidly in vivo and also undergo apoptosis more rapidly, upon loss of Jak3. Hence Jak3 exerts effects on mature peripheral T lymphocytes, as well as on thymocytes, resulting in the proper maintenance of circulating, quiescent cells.

Animals↗