scholarly journals In Vivo Targeting of the Growth Hormone Receptor (GHR) Box1 Sequence Demonstrates that the GHR Does Not Signal Exclusively through JAK2

2010 ◽  
Vol 24 (1) ◽  
pp. 204-217 ◽  
Author(s):  
Johanna L. Barclay ◽  
Linda M. Kerr ◽  
Leela Arthur ◽  
Jennifer E. Rowland ◽  
Caroline N. Nelson ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Growth Hormone Receptor ◽  
Target Genes ◽  
Postnatal Growth ◽  
Signal Transducers ◽  
Phenotypic Differences ◽  
Protein Levels ◽  
Igf Binding ◽  
Transcript Profiles

Abstract GH is generally believed to signal exclusively through Janus tyrosine kinases (JAK), particularly JAK2, leading to activation of signal transducers and activators of transcription (STAT), ERK and phosphatidylinositol 3-kinase pathways, resulting in transcriptional regulation of target genes. Here we report the creation of targeted knock-in mice wherein the Box1 motif required for JAK2 activation by the GH receptor (GHR) has been disabled by four Pro/Ala mutations. These mice are unable to activate hepatic JAK2, STAT3, STAT5, or Akt in response to GH injection but can activate Src and ERK1/2. Their phenotype is identical to that of the GHR−/− mouse, emphasizing the key role of JAK2 in postnatal growth and the minimization of obesity in older males. In particular, they show dysregulation of the IGF-I/IGF-binding protein axis at transcript and protein levels and decreased bone length. Because no gross phenotypic differences were evident between GHR−/− and Box1 mutants, we undertook transcript profiling in liver from 4-month-old males. We compared their transcript profiles with our 391-GHR truncated mice, which activate JAK2, ERK1/2, and STAT3 in response to GH but not STAT5a/b. This has allowed us for the first time to identify in vivo Src/ERK-regulated transcripts, JAK2-regulated transcripts, and those regulated by the distal part of the GHR, particularly by STAT5.

scholarly journals In Vivo Analysis of Growth Hormone Receptor Signaling Domains and Their Associated Transcripts

2005 ◽  
Vol 25 (1) ◽  
pp. 66-77 ◽  
Author(s):  
Jennifer E. Rowland ◽  
Agnieszka M. Lichanska ◽  
Linda M. Kerr ◽  
Mary White ◽  
Elisabetta M. d’Aniello ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Mouse Models ◽  
Microarray Analysis ◽  
Hormone Receptor ◽  
Growth Hormone Receptor ◽  
Target Genes ◽  
Postnatal Growth ◽  
In Vivo Analysis ◽  
Signaling Domains

ABSTRACT The growth hormone receptor (GHR) is a critical regulator of postnatal growth and metabolism. However, the GHR signaling domains and pathways that regulate these processes in vivo are not defined. We report the first knock-in mouse models with deletions of specific domains of the receptor that are required for its in vivo actions. Mice expressing truncations at residue m569 (plus Y539/545-F) and at residue m391 displayed a progressive impairment of postnatal growth with receptor truncation. Moreover, after 4 months of age, marked male obesity was observed in both mutant 569 and mutant 391 and was associated with hyperglycemia. Both mutants activated hepatic JAK2 and ERK2, whereas STAT5 phosphorylation was substantially decreased for mutant 569 and absent from mutant 391, correlating with loss of IGF-1 expression and reduction in growth. Microarray analysis of these and GHR−/− mice demonstrated that particular signaling domains are responsible for the regulation of different target genes and revealed novel actions of growth hormone. These mice represent the first step in delineating the domains of the GHR regulating body growth and composition and the transcripts associated with these domains.

scholarly journals The microRNA miR-8 is a conserved negative regulator of Wnt signaling

2008 ◽  
Vol 105 (40) ◽  
pp. 15417-15422 ◽  
Author(s):  
Jennifer A. Kennell ◽  
Isabelle Gerin ◽  
Ormond A. MacDougald ◽  
Ken M. Cadigan
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathway ◽  
Target Genes ◽  
Wnt Signaling Pathway ◽  
Negative Regulator ◽  
Animal Development ◽  
Protein Levels ◽  
Evolutionarily Conserved ◽  
Multiple Levels

Wnt signaling plays many important roles in animal development. This evolutionarily conserved signaling pathway is highly regulated at all levels. To identify regulators of the Wnt/Wingless (Wg) pathway, we performed a genetic screen in Drosophila. We identified the microRNA miR-8 as an inhibitor of Wg signaling. Expression of miR-8 potently antagonizes Wg signaling in vivo, in part by directly targeting wntless, a gene required for Wg secretion. In addition, miR-8 inhibits the pathway downstream of the Wg signal by repressing TCF protein levels. Another positive regulator of the pathway, CG32767, is also targeted by miR-8. Our data suggest that miR-8 potently antagonizes the Wg pathway at multiple levels, from secretion of the ligand to transcription of target genes. In addition, mammalian homologues of miR-8 promote adipogenesis of marrow stromal cells by inhibiting Wnt signaling. These findings indicate that miR-8 family members play an evolutionarily conserved role in regulating the Wnt signaling pathway.

scholarly journals Id1 is a common downstream target of oncogenic tyrosine kinases in leukemic cells

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 1981-1992 ◽  
Author(s):  
Winnie F. Tam ◽  
Ting-Lei Gu ◽  
Jing Chen ◽  
Benjamin H. Lee ◽  
Lars Bullinger ◽  
...  
Keyword(s):  
Cell Lines ◽  
Tyrosine Kinases ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Hematopoietic Malignancy ◽  
Downstream Target ◽  
Models Of Disease ◽  
Induced Apoptosis

Abstract Oncogenic tyrosine kinases, such as BCR-ABL, TEL-ABL, TEL-PDGFβR, and FLT3-ITD, play a major role in the development of hematopoietic malignancy. They activate many of the same signal transduction pathways. To identify the critical target genes required for transformation in hematopoietic cells, we used a comparative gene expression strategy in which selective small molecules were applied to 32Dcl3 cells that had been transformed to factor-independent growth by these respective oncogenic alleles. We identified inhibitor of DNA binding 1 (Id1), a gene involved in development, cell cycle, and tumorigenesis, as a common target of these oncogenic kinases. These findings were prospectively confirmed in cell lines and primary bone marrow cells engineered to express the respective tyrosine kinase alleles and were also confirmed in vivo in murine models of disease. Moreover, human AML cell lines Molm-14 and K562, which express the FLT3-ITD and BCR-ABL tyrosine kinases, respectively, showed high levels of Id1 expression. Antisense and siRNA based knockdown of Id1-inhibited growth of these cells associated with increased p27Kip1 expression and increased sensitivity to Trail-induced apoptosis. These findings indicate that Id1 is an important target of constitutively activated tyrosine kinases and may be a therapeutic target for leukemias associated with oncogenic tyrosine kinases.

Overexpression of Foxf2 in adipose tissue is associated with lower levels of IRS1 and decreased glucose uptake in vivo

2010 ◽  
Vol 298 (3) ◽  
pp. E548-E554 ◽  
Author(s):  
Rickard Westergren ◽  
Daniel Nilsson ◽  
Mikael Heglind ◽  
Zahra Arani ◽  
Mats Grände ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Glucose Tolerance ◽  
Glucose Uptake ◽  
Target Genes ◽  
Whole Body ◽  
Wild Type ◽  
Intravenous Glucose ◽  
Downstream Target ◽  
Protein Levels

Many members of the forkhead genes family of transcription factors have been implicated as important regulators of metabolism, in particular, glucose homeostasis, e.g., Foxo1, Foxa3, and Foxc2. The purpose of this study was to exploit the possibility that yet unknown members of this gene family play a role in regulating glucose tolerance in adipocytes. We identified Foxf2 in a screen for adipose-expressed forkhead genes. In vivo overexpression of Foxf2 in an adipose tissue-restricted fashion demonstrated that such mice display a significantly induced insulin secretion in response to an intravenous glucose load compared with wild-type littermates. In response to increased Foxf2 expression, insulin receptor substrate 1 (IRS1) mRNA and protein levels are significantly downregulated in adipocytes; however, the ratio of serine vs. tyrosine phosphorylation of IRS1 seems to remain unaffected. Furthermore, adipocytes overexpressing Foxf2 have a significantly lower insulin-mediated glucose uptake compared with wild-type adipocytes. These findings argue that Foxf2 is a previously unrecognized regulator of cellular and systemic whole body glucose tolerance, at least in part, due to lower levels of IRS1. Foxf2 and its downstream target genes can provide new insights with regard to identification of novel therapeutic targets.

scholarly journals C-terminal truncation of Pik3r1 in mice models human lipodystrophic insulin resistance uncoupled from dyslipidemia

2017 ◽  
Author(s):  
Albert Kwok ◽  
Ilona Zvetkova ◽  
Sam Virtue ◽  
Isabel Huang-Doran ◽  
Patsy Tomlinson ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Tyrosine Kinases ◽  
Target Genes ◽  
Ex Vivo ◽  
Liver Weight ◽  
Postnatal Growth ◽  
Regulatory Subunit ◽  
Severe Insulin Resistance ◽  
Triglyceride Content ◽  
Short Syndrome

SummaryHeterodimeric class IA phosphatidylinositol-3-kinases (PI3K) transduce signals from many receptor tyrosine kinases including the insulin receptor. PI3K recruitment to phosphotyrosines is mediated by Pik3r1 gene products including the most intensely studied PI3K regulatory subunit, p85α, which also binds and regulates the PIP3 phosphatase Pten, and the lipogenic transcription factor Xbp1. Mutations in human PIK3R1 cause SHORT syndrome, featuring lipodystrophy and severe insulin resistance which, uniquely, are uncoupled from fatty liver and dyslipidemia. We describe a novel mouse model of SHORT syndrome made by knock in of the Pik3r1 Y657X mutation. Homozygous embryos die at E11.5, while heterozygous mice exhibit pre-and postnatal growth impairment with diminished placental vascularity. Adipose tissue accretion on high fat feeding was reduced, however adipocyte size was unchanged and preadipocyte differentiation ex vivo unimpaired. Despite severe insulin resistance, heterozygous mice were hypolipidemic, and plasma adiponectin, liver weight, cholesterol, glycogen and triglyceride content were unchanged. Mild downregulation of lipogenic Srebp1, Srebp2 and Chrebp transcriptional activity but no suppression of Xbp1 target genes was seen after fasting. These findings give new insights into the developmental role of Pik3r1, and establish a model of lipodystrophic insulin resistance dissociated from dyslipidemia as seen in SHORT syndrome.

scholarly journals DOT1L-Mediated H3K79 Methylation in Chromatin Is Dispensable for Wnt Pathway-Specific and Other Intestinal Epithelial Functions

2013 ◽  
Vol 33 (9) ◽  
pp. 1735-1745 ◽  
Author(s):  
Li-Lun Ho ◽  
Amit Sinha ◽  
Michael Verzi ◽  
Kathrin M. Bernt ◽  
Scott A. Armstrong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Intestinal Epithelium ◽  
Target Genes ◽  
Intestinal Stem Cells ◽  
Intestinal Epithelial ◽  
Aberrant Expression ◽  
Genome Wide ◽  
Transcript Profiles ◽  
H3k79 Methylation

Methylation of H3K79 is associated with chromatin at expressed genes, though it is unclear if this histone modification is required for transcription of all genes. Recent studies suggest that Wnt-responsive genes depend particularly on H3K79 methylation, which is catalyzed by the methyltransferase DOT1L. Human leukemias carrying MLL gene rearrangements show DOT1L-mediated H3K79 methylation and aberrant expression of leukemogenic genes. DOT1L inhibitors reverse these effects, but their clinical use is potentially limited by toxicity in Wnt-dependent tissues such as intestinal epithelium. Genome-wide positioning of the H3K79me2 mark in Lgr5 + mouse intestinal stem cells and mature intestinal villus epithelium correlated with expression levels of all transcripts and not with Wnt-responsive genes per se . Selective Dot1l disruption in Lgr5 + stem cells or in whole intestinal epithelium eliminated H3K79me2 from the respective compartments, allowing genetic evaluation of DOT1L requirements. The absence of methylated H3K79 did not impair health, intestinal homeostasis, or expression of Wnt target genes in crypt epithelium for up to 4 months, despite increased crypt cell apoptosis. Global transcript profiles in Dot1l -null cells were barely altered. Thus, H3K79 methylation is not essential for transcription of Wnt-responsive or other intestinal genes, and intestinal toxicity is not imperative when DOT1L is rendered inactive in vivo .

scholarly journals Direct Interaction of Jak1 and v-Abl Is Required for v-Abl-Induced Activation of STATs and Proliferation

1998 ◽  
Vol 18 (11) ◽  
pp. 6795-6804 ◽  
Author(s):  
Nika N. Danial ◽  
Julie A. Losman ◽  
Tianhong Lu ◽  
Natalie Yip ◽  
Kartik Krishnan ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Leukemia Virus ◽  
Direct Interaction ◽  
Janus Kinase ◽  
Signaling Proteins ◽  
Interaction Domain ◽  
Signal Transducers ◽  
Stat Proteins ◽  
Immature B Cells

ABSTRACT In Abelson murine leukemia virus (A-MuLV)-transformed cells, members of the Janus kinase (Jak) family of non-receptor tyrosine kinases and the signal transducers and activators of transcription (STAT) family of signaling proteins are constitutively activated. In these cells, the v-Abl oncoprotein and the Jak proteins physically associate. To define the molecular mechanism of constitutive Jak-STAT signaling in these cells, the functional significance of the v-Abl–Jak association was examined. Mapping the Jak1 interaction domain in v-Abl demonstrates that amino acids 858 to 1080 within the carboxyl-terminal region of v-Abl bind Jak1 through a direct interaction. A mutant of v-Abl lacking this region exhibits a significant defect in Jak1 binding in vivo, fails to activate Jak1 and STAT proteins, and does not support either the proliferation or the survival of BAF/3 cells in the absence of cytokine. Cells expressing this v-Abl mutant show extended latency and decreased frequency in generating tumors in nude mice. In addition, inducible expression of a kinase-inactive mutant of Jak1 protein inhibits the ability of v-Abl to activate STATs and to induce cytokine-independent proliferation, indicating that an active Jak1 is required for these v-Abl-induced signaling pathways in vivo. We propose that Jak1 is a mediator of v-Abl-induced STAT activation and v-Abl induced proliferation in BAF/3 cells, and may be important for efficient transformation of immature B cells by the v-abloncogene.

scholarly journals Pregnenolone sulfate regulates prolactin production in the rat pituitary

2016 ◽  
Vol 230 (3) ◽  
pp. 339-346 ◽  
Author(s):  
Eun-Jin Kang ◽  
So-Hye Hong ◽  
Jae-Eon Lee ◽  
Seung Chul Kim ◽  
Hoe-Saeng Yang ◽  
...  
Keyword(s):  
Target Genes ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Pregnenolone Sulfate ◽  
Protein Levels ◽  
Rat Pituitary ◽  
Pituitary Prolactin ◽  
Gh3 Cell

Pregnenolone sulfate (PS) is a neuroactive steroid hormone produced in the brain. In this study, the effects of PS on synthesis and secretion of rat pituitary prolactin (PRL) were examined. To accomplish this, GH3 rat pituitary adenoma cells were treated with PS, which showed significantly increased mRNA and protein levels of PRL compared with the control. The mechanism of action responsible for the effects of PS on PRL synthesis and secretion was investigated by pretreating cells with inhibitors of traditional PRL- or the PS-related signaling pathway. PS-stimulated PRL transcription was significantly reduced by inhibitors of PKA, PKC and MAPK, but unchanged by GABAAR and NMDAR inhibitors. Western blotting analysis revealed that the total ERK1/2 level was upregulated in a time-dependent manner following PS treatment. An approximate 10% increase in GH3 cell proliferation was also observed in response to PS relative to the control. In the animal study, levels of PRL in the pituitary and in serum were elevated by PS. PS-stimulated PRL synthesis was also found to be associated with decreased expression of PRL target genes such as GNRH1, FSHB and LHB. These findings show that PS upregulates PRL synthesis and secretion in vivo and in vitro via MAPK signaling, suggesting that it has the potential for use as a therapeutic hormone to treat PRL-related disorders such as hypoprolactinemia and low milk supply.

scholarly journals The Somatomedin Hypothesis: 2001

2001 ◽  
Vol 22 (1) ◽  
pp. 53-74 ◽  
Author(s):  
Derek Le Roith ◽  
Carolyn Bondy ◽  
Shoshana Yakar ◽  
Jun-Li Liu ◽  
Andrew Butler
Keyword(s):  
Growth And Development ◽  
Gene Deletion ◽  
Somatic Growth ◽  
Postnatal Growth ◽  
Protein Levels ◽  
Factor I ◽  
Igf I ◽  
Igf Binding ◽  
Igf Binding Protein

Abstract Since the original somatomedin hypothesis was conceived, a number of important discoveries have allowed investigators to modify the concept. Originally somatic growth was thought to be controlled by pituitary GH and mediated by circulating insulin-like growth factor-I (IGF-I, somatomedin C) expressed exclusively by the liver. With the discovery that IGF-I is produced by most, if not all, tissues, the role of autocrine/paracrine IGF-I vs. the circulating form has been hotly debated. Recent experiments using transgenic and gene-deletion technologies have attempted to answer these questions. In the liver-specific igf-1 gene-deleted mouse model, postnatal growth and development are normal despite the marked reduction in circulating IGF-I and IGF-binding protein levels; free IGF-I levels are normal. Thus, the normal postnatal growth and development in these animals may be due to normal free IGF-I levels (from as yet unidentified sources), although the role of autocrine/paracrine IGF-I has yet to be determined.

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