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G Norstedt

Publications and source records attributed to G Norstedt.

At least 19 recordsLinked to original sources
PubMed2001

Microarray analysis of the in vivo effects of hypophysectomy and growth hormone treatment on gene expression in the rat.

Complementary DNA microarrays containing 3000 different rat genes were used to study the consequences of severe hormonal deficiency (hypophysectomy) on the gene expression patterns in heart, liver, and kidney. Hybridization signals were seen from a majority of the arrayed complementary DNAs; nonetheless, tissue-specific expression patterns could be delineated. Hypophysectomy affected the expression of genes involved in a variety of cellular functions. Between 16-29% of the detected transcripts from each tissue changed expression level as a reaction to this condition. Chronic treatment of hypophysectomized animals with human GH also caused significant changes in gene expression patterns. The study confirms previous knowledge concerning certain gene expression changes in the above-mentioned situations and provides new information regarding hypophysectomy and chronic human GH effects in the rat. Furthermore, we have identified several new genes that respond to GH treatment. Our results represent a first step toward a more global understanding of gene expression changes in states of hormonal deficiency.

Animals↗
PubMed2001

Gene expression profile of the aging process in rat liver: normalizing effects of growth hormone replacement.

The mechanisms that control life span and age-related phenotypes are not well understood. It has been suggested that aging or at least some of its symptoms are related to a physiological decline in GH levels with age. To test this hypothesis, and to improve our understanding of the cellular and molecular mechanisms behind the aging process, we have analyzed age-induced changes in gene expression patterns through the application of DNA chip technology. In the present study, the aging process was analyzed in rat liver in the presence or absence of GH replacement. Out of 3,000 genes printed on the microarrays, approximately 1,000 were detected in the rat liver. Among these, 47 unique transcripts were affected by the aging process in male rat livers. The largest groups of age-regulated transcripts encoded proteins involved in intermediary metabolism, mitochondrial respiration, and drug metabolism. Approximately 40% of the differentially expressed gene products were normalized after GH treatment. The majority of those transcripts have previously not been shown to be under GH control. The list of gene products that showed normalized expression levels in GH-treated old rats may shed further insight on the action and mechanism behind the positive effects of GH on, for example, fuel metabolism and body composition observed in different animal and human studies.

Aging↗
PubMed2001

Endoplasmic reticulum stress prolongs GH-induced Janus kinase (JAK2)/signal transducer and activator of transcription (STAT5) signaling pathway.

The desensitization of the GH-induced Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) signaling pathway plays a crucial role in GH regulation of hepatic genes. Previous studies have demonstrated that the inactivation of the GH-induced JAK2/STAT5 pathway is regulated by protein translation and suppressors of cytokine signaling (SOCS). In this study we sought to explore the relationships between endoplasmic reticulum stress, GH-induced JAK2/STAT5 activity and SOCS expression. 1,2-bis(o-Aminophenoxy)ethane-N,N,N,N-tetraacetic acid (acetoxymethyl)ester (BAPTA-AM), used to provoke endoplasmic reticulum stress, caused a drastic inhibition of protein translation that correlated with the phosphorylation of the eukaryotic translation initiation factor 2alpha. Both GH and BAPTA-AM caused a rapid induction of the transcription factor C/EBP homology protein (CHOP) and an additive effect was observed with combined treatment, which suggests a regulatory role of GH on endoplasmic reticulum stress. Endoplasmic reticulum stress did not interfere with the rapid GH activation of STAT5 DNA binding activity. However, BAPTA-AM prolonged the DNA binding activity of STAT5 without affecting STAT5 or JAK2 protein levels. GH-induced phosphorylation of JAK2 and STAT5 DNA binding activity were prolonged in the presence of BAPTA-AM, suggesting that endoplasmic reticulum stress prevents the inactivation of STAT5 DNA binding activity by modulating the rate of JAK2/STAT5 dephosphorylation. Like BAPTA-AM, the endoplasmic reticulum stressors dithiothreitol and A23187 also prolonged the GH-induced STAT5 DNA binding activity. We were not able to correlate BAPTA-AM effects to the GH-dependent expression of SOCS proteins or SOCS mRNA, suggesting that endoplasmic reticulum stress modulates the rate of JAK2/STAT5 dephosphorylation through mechanisms other than inhibition of SOCS expression. This study indicates that cellular stress may modulate transcription through the JAK/STAT pathway.

Animals↗
PubMed2000

A cDNA RDA protocol using solid-phase technology suited for analysis in small tissue samples.

cDNA representational difference analysis (cDNA RDA) is a PCR-based subtractive enrichment procedure for the cloning of differentially expressed genes. In this study, we have further developed the procedure to take advantage of solid-phase technology, and to facilitate the use of RDA when starting material is limited. Several parameters of the PCR-based generation of cDNA representations were investigated, and a solid-phase based purification step was introduced to simplify removal of digested adapter-ends and uncleaved fragments. The use of magnetic particles increased the speed of the method, and also eliminated the risk of carry-over contamination between iterative steps of subtraction and PCR amplification. The modified protocol was evaluated in monitoring differences in gene expression in (i) a rat system consisting of livers with and without growth hormone treatment, and in (ii) a human system consisting of normal colon and colon cancer.

Acrylic Resins↗
PubMed2000

Comparison of STAT5 mRNA levels in GH-treated male and female rats analysed by a solution hybridization assay.

In this study we describe the development of a RNA:RNA solution hybridization-RNase protection assay to quantify STAT5 mRNA in total RNA extracts from rat tissues. The assay is sensitive and reproducible. We quantified STAT5 mRNA levels in liver and thymus lymphocytes from male and female control rats and from rats treated with a single dose of recombinant human growth hormone (rhGH). No significant sex differences in the expression pattern were observed in both studied tissues, but STAT5 mRNA levels were significantly (P< 0.05) higher in liver than in thymus lymphocytes. STAT5 mRNA levels were significantly (P< 0.05) increased by a pulse of GH given to either male or female normal rats, suggesting a regulation of STAT5 gene expression in the studied tissues. In conclusion, quantitative solution hybridization-RNase protection assay of STAT5 mRNA provides a tool to further advance the study of the regulatory mechanisms involved in STAT5 gene expression.

Animals↗
PubMed2000

Differential cloning of growth hormone-regulated hepatic transcripts in the aged rat.

It has been suggested that aging or at least some of its symptoms are related to a physiological decline in GH levels with age. This study was performed to elucidate age-related changes in GH-dependent effects at the level of gene expression. Through the application of complementary DNA representational difference analysis (RDA) we have identified gene products that are reduced during aging in rat liver. The expression of these genes was restored upon GH treatment. Results from reverse Northern and ribonuclease protection analysis confirmed that the RDA products were truly differentially expressed. In addition to well characterized GH-regulated genes, including CYP2C12, CYP2C13, and alpha2u-globulin, we demonstrate the differential expression of at least 11 genes previously not known to be under GH control. Several hepatic transcripts encoding enzymes and receptors involved in the metabolism of protein, carbohydrates, and lipids were identified. Other RDA products consisted of transcripts encoding proteins involved in ATP synthesis, detoxification of reactive oxygen species, or immune responses. This list of GH-regulated genes in the old rat may shed further light on the action and mechanism behind the positive effects of GH on, for example, body composition and the immune system that have been observed in different animal and human studies.

Aging↗
PubMed2000

Characterisation of novel missense mutations in the GH receptor gene causing severe growth retardation.

Two Swedish brothers, 2.5 and 4 years of age, were found to fulfil all the clinical and laboratory characteristics of Laron's syndrome. They were shown to have unique missense mutations in the GH receptor gene. Both of their parents were of normal height, but they both separately carried one of the identified gene alterations. A molecular model of the first receptor alteration suggests that a collapse in three-dimensional receptor structure most likely contributed to the GH insensitivity in these patients.

Amino Acid Sequence↗
PubMed1999

Growth hormone regulation of SOCS-2, SOCS-3, and CIS messenger ribonucleic acid expression in the rat.

The SOCS (suppressors of cytokine signaling) proteins have been suggested to function as inhibitors of cytokine receptor signaling. We have analyzed SOCS-2, SOCS-3, and CIS expression in relation to GH actions in the rat. SOCS-2, SOCS-3, and CIS transcripts were detected in various GH responsive tissues, including liver, muscle, and fat. In addition to the finding that different tissues express different levels of SOCS-2, SOCS-3, and CIS messenger RNA (mRNA), the steady-state levels of these SOCS transcripts were dependent on the endocrine status of the animal. SOCS-3 expression was 5-fold higher in fat from old compared with younger rats. Hypophysectomy reduced the levels of SOCS-2 and CIS mRNA in liver, muscle, and fat, whereas SOCS-3 expression was unchanged. Using primary cultures of rat hepatocytes, GH was shown to increase SOCS-2, SOCS-3, and CIS mRNA levels with different kinetics. SOCS-3 was rapidly and transiently induced, whereas SOCS-2 and CIS were increased in a slower fashion. Glucocorticoids blocked GH-induced SOCS-3 expression in cultured hepatocytes, whereas SOCS-2 and CIS expression was potentiated. Our data fit well with a concept of SOCS proteins acting as modulators of GH signal transduction.

Animals↗
PubMed1998

In vitro interaction between STAT 5 and JAK 2; dependence upon phosphorylation status of STAT 5 and JAK 2.

A working model for haematopoietic cytokine signal transduction has been hypothesised as follows. Binding of cytokines to specific receptor molecules leads to phosphorylation and activation of receptor associated members of the Janus kinase family. This is followed by tyrosine phosphorylation of the associated receptor and members of the STAT (signal transducer and activator of transcription) family of DNA-binding transcription factors. Phosphorylation is accompanied by STAT dimerisation, nuclear transport and activation of gene transcription. Activation of gene transcription is mediated by the binding of STAT dimers to palindromic STAT response elements. A number of areas of confusion remain; not least the mechanism by which multiple cytokines signal via a limited number of STATs. A role has been suggested for phosphorylated receptor tyrosine residues as STAT docking sites on activated receptor-JAK complexes. According to this model the amino acid sequence context of key tyrosine residues confers receptor specificity upon STAT activation. There is some controversy as to whether this model applies to STAT 5. The heterologous expression of STAT 5 in Sf 9 insect cells using the baculovirus expression system is described here. Protein of the correct molecular weight was expressed and found to be phosphorylated on tyrosine residues and to bind to a STAT response DNA element. This binding was dependent upon the phosphorylation status of the STAT protein. DNA binding could be abolished in vitro by treatment with a phosphotyrosine phosphatase and restored in vitro by treatment with activated recombinant JAK 2. The protein was purified to near homogeneity using a simple ion exchange/gel filtration chromatography procedure. The interaction between purified recombinant STAT 5 and JAK 2, either expressed by baculovirus or endogenously expressed in Buffalo rat liver cells, was studied. In both cases STAT 5 in its non-phosphorylated form was found to form a stable complex with activated JAK 2. Non-activated JAK 2 and phosphorylated STAT 5 were unable to participate in complex formation. The results presented provide a mechanistic basis for the activation of STAT 5 by a wide range of cytokines capable of activating JAK 2.

Animals↗
PubMed1998

Desensitization of the growth hormone-induced Janus kinase 2 (Jak 2)/signal transducer and activator of transcription 5 (Stat5)-signaling pathway requires protein synthesis and phospholipase C.

Signal transducers and activators of transcription (Stat) proteins are latent cytoplasmic transcription factors that are tyrosine phosphorylated by Janus kinases (Jak) in response to GH and other cytokines. GH activates Stat5 by a mechanism that involves tyrosine phosphorylation and nuclear translocation. However, the mechanisms that turn off the GH-activated Jak2/Stat5 pathway are unknown. Continuous exposure to GH of BRL-4 cells, a rat hepatoma cell line stably transfected with rat GH receptor, induces a rapid but transient activation of Jak2 and Stat5. GH-induced Stat5 DNA-binding activity was detected after 2 min and reached a maximum at 10 min. Continued exposure to GH resulted in a desensitization characterized by 1) a rapid decrease in Stat5 DNA-binding activity. The rate of decrease of activity was rapid up to 1 h of GH treatment, and the remaining activity declined slowly thereafter. The activity of Stat5 present after 5 h is still higher than the control levels and almost 10-20% with respect to maximal activity at 10 min; and 2) the inability of further GH treatment to reinduce activation of Stat5. In contrast, with transient exposures of BRL-4 cells to GH, Stat5 DNA-binding activity could repeatedly be induced. GH-induced Jak2 and Stat5 activities were independent of ongoing protein synthesis. However, Jak2 tyrosine phosphorylation and Stat5 DNA-binding activity were prolonged for at least 4 h in the presence of cycloheximide, which suggests that the maintenance of desensitization requires ongoing protein synthesis. Furthermore, inhibition of protein synthesis potentiated GH-induced transcriptional activity in BRL-4 cells transiently transfected with SPIGLE1CAT, a reporter plasmid activated by Stat5. GH-induced Jak2 and Stat5 activation were not affected by D609 or mepacrine, both inhibitors of phospholipase C. However, in the presence of D609 and mepacrine, GH maintained prolonged Jak2 and Stat5 activation. Transactivation of SPIGLE1 by GH was potentiated by mepacrine and D609 but not by the phospholipase A2 inhibitor AACOCF3. Thus, a regulatory circuit of GH-induced transcription through the Jak2/Stat5-signaling pathway includes a prompt GH-induced activation of Jak2/Stat5 followed by a negative regulatory response; ongoing protein synthesis and intracellular signaling pathways, where phospholipase C activity is involved, play a critical role to desensitize the GH-activated Jak2/Stat5-signaling pathway.

Animals↗
PubMed1997

Mode of the autocrine/paracrine mechanism of growth hormone action.

GH is synthesized at multiple extrapituitary sites suggestive of an autocrine/paracrine mechanism of action. We have investigated a possible autocrine/paracrine mechanism of GH action, compared the cellular response to exogenous versus endogenously produced GH, and determined the nature of the interaction between external stimuli and endogenously produced GH. BRL cells expressing the GH receptor were transiently transfected with expression plasmids containing either the hGH or the bGH gene and the response of the cell was measured by CAT reporter plasmids requiring either STATs 1 and 3 or STAT5 for their response. Transient transfection of the hGH gene resulted in hGH accumulation in the cell and secretion into the media. The functional response through STATs 1 and 3 and STAT5 obtained with endogenously produced hGH was comparable or greater in magnitude to that obtained with the maximal stimulatory dose of exogenous hGH. Similar results were obtained with an expression plasmid containing the bGH gene. Endogenously produced hGH interacted in an additive manner when combined with submaximal doses of both exogenous hGH and serum. Such results were also observed in a more physiologically relevant mammary carcinoma cell line (MCF-7). The nonreceptor-dimerizing hGH antagonist, hGH-G120R, used in cells expressing the homologous receptor extracellular domain was able to only partially inhibit the response of the cell to endogenously produced hGH, in contrast to full inhibition of exogenous hGH. We therefore conclude that GH can function in an autocrine/paracrine manner, additive in effect to external stimuli.

Animals↗
PubMed1997

Mitogen-activated protein kinase kinase inhibition decreases growth hormone stimulated transcription mediated by STAT5.

We have investigated the possible involvement of the MAPK pathway in the growth hormone(GH)-induced activation of one of the members of signal transducers and activators of transcription, STAT5, by using the MAPK kinase (MEK) inhibitor PD98059. PD98059 treatment of Chinese hamster ovarian cells, stably transfected with the GH receptor (CHOA cells), abolished the GH-induced MAPK activity. PD98059 decreased the amount of GH-induced STAT5 in nuclear extract with DNA-binding capacity. Furthermore, GH dependent transcription of a STAT5 regulated reporter gene was inhibited by PD98059. The MEK inhibitor did not reduce GH-stimulated nuclear translocation of STAT5. We also investigated if PD98059 differentially influences the activation of the two STAT5 homologs, STAT5a and STAT5b, which differ mainly at the C-terminal end, one of the differences being the presence of a possible MAPK phosphorylation site in STAT5a. Expression plasmids for these transcription factors were transfected into CHOA cells together with a reporter gene. GH-stimulated fold induction of transcription was reduced by PD98059 in STAT5a but not in STAT5b overexpressing cells. A MAPK phosphorylation site-mutated version of STAT5a was also transfected into CHOA cells. GH-stimulated fold induction of cotransfected reporter gene was not reduced by PD98059 in cells overexpressing mutant STAT5a. The above data show that the MAPK pathway is required for the full activation of one of the STAT5 isoforms (STAT5a).

Amino Acid Sequence↗
PubMed1997

Specificity of transcription enhancement via the STAT responsive element in the serine protease inhibitor 2.1 promoter.

The growth hormone regulated serine protease inhibitor (SPI) 2.1 and 2.2 gene promoters have been shown to contain a response element similar to the gamma-interferon activated sequence (GAS) family of signal transducer and activator of transcription (STAT) response elements. We have investigated the STAT and cytokine specificity of the SPI 2.1 STAT responsive element using a luciferase (LUC) reporter construct and a cDNA complementation strategy in the COS 7 cell line. Growth hormone was found to stimulate SPI-LUC reporter gene expression via activation of STAT 5, but not STATs 1 or 3, which indicates that the SPI 2.1 STAT responsive element is STAT 5 specific. In addition to the growth hormone receptor, the receptors for prolactin and erythropoietin enhanced gene transcription via the SPI 2.1 STAT responsive element, which indicates that this element is, on the other hand, not cytokine specific. Activation of STAT 5 was also observed after growth hormone treatment of cells transfected with cDNA expression plasmids for several different truncated growth hormone receptor mutants, although this activation was less efficient than with the wild type receptor. Point mutation of individual tyrosines in the growth hormone receptor intracellular domain to phenylalanines had no significant effect on signal transduction via STAT 5. These data, taken together with results from experiments using the phosphatase inhibitor sodium orthovanadate, suggest that STAT 5 may not have an absolute requirement for specific phosphorylated receptor tyrosine docking sites. That receptor tyrosine residues in a variety of amino acid contexts, or phosphorylated Janus kinase (JAK) 2 alone, can facilitate STAT 5 activation could explain the observed lack of cytokine specificity in STAT 5 activation.

Animals↗
PubMed1997

Growth hormone-induced reorganization of the actin cytoskeleton is not required for STAT5 (signal transducer and activator of transcription-5)-mediated transcription.

We have investigated the effect of GH on the organization of the actin cytoskeleton within the cell. Human GH (hGH) treatment (50 nM) of Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR(1-638)) resulted in a reorganization of actin filaments in the cell that was not observed upon GH treatment of the untransfected parental CHO cell line. hGH initially induced depolymerization of actin stress fibers similar in magnitude to that induced by treatment of the cells with 100 nM human insulin-like growth factor I. This loss of stress fibers was observed as early as 30 sec after addition of hGH to the medium, and maximal depolymerization of stress fibers was observed between 1-4 min after addition of hGH. This was followed by a slow, but submaximal, repolymerization of the stress fibers and the formation of localized focal filamentous actin containing complexes. Similar cytoskeletal changes were observed after hGH treatment in Swiss 3T3 fibroblasts and BRL cells stably transfected with rat GH receptor complementary DNA (BRL-GHR(1-6381)). Pretreatment of CHO-GHR(1-638) cells with wortmannin (a phosphatidylinositol 3-kinase inhibitor) and verapamil (a calcium channel antagonist) both inhibited the hGH-induced actin reorganization. The integrity of the actin cytoskeleton was not required for GH-induced STAT5 (signal transducer and activator of transcription-5)-mediated transcription, as treatment of cells with cytochalasins B and D did not alter the fold stimulation of the STAT5-mediated transcriptional response to GH. We conclude that GH induces a rapid reorganization of the actin cytoskeleton by a process requiring phosphatidylinositol 3-kinase activation and calcium influx, but this cytoskeletal reorganization is not required for the STAT5-mediated transcriptional response to GH.

3T3 Cells↗
PubMed1997

Insulin-like growth factor I increases bone formation in old or corticosteroid treated rats.

We studied bone induction in subcutaneous implants of demineralized bone matrix with or without insulin-like growth factor I (IGF-I) in aged or corticosteroid-treated rats. Each rat carried one pair of implants, one control and one experiment implant, containing IGF-I dissolved in a hyaluronan solution for slow release. The rats were killed after 3 weeks and the results were evaluated by measuring the calcium content of implants. Young (6-7 weeks) and old (19-27 months) rats were used. A group of young rats was treated for 1 week with subcutaneous injections of 140 micrograms/kg dexamethasone daily. Old rats produced only approximately 1% as much bone as young rats. Local delivery of IGF-I did not increase bone formation in young rats. In old rats, bone formation was increased by IGF-I, 3000 ng/implant. Corticosteroids reduced bone formation in young rats. This effect was partially reversed by local administration of IGF-I.

Age Factors↗
PubMed1996

Crystal structure of an antagonist mutant of human growth hormone, G120R, in complex with its receptor at 2.9 A resolution.

Human growth hormone binds two receptor molecules and thereby induces signal transduction through receptor dimerization. At high concentrations, growth hormone acts as an antagonist because of a large difference in affinities at the respective binding sites. This antagonist action can be enhanced further by reducing binding in the low affinity binding site. A growth hormone antagonist mutant Gly-120 --> Arg, has been crystallized with its receptor as a 1:1 complex and the crystal structure determined at 2.9 A resolution. The 1:1 complex is remarkably similar to the native growth hormone-receptor 1:2 complex. A comparison between the two structures reveals only minimal differences in the conformations of the hormone or its receptor in the two complexes, including the angle between the two immunoglobulin-like domains of the receptor. Further, two symmetry-related 1:1 complexes in the crystal form a 2:2 complex with a large solvent inaccessible area between two receptor molecules. In addition, we present here a native human growth hormone-human growth hormone-binding protein 1:2 complex structure at 2.5 A resolution. One important difference between our structure and the previously published crystal structure at 2.8 A is revealed. Trp-104 in the receptor, a key residue in the hormone-receptor interaction, has an altered conformation in the low affinity site enabling a favorable hydrogen bond to be formed with Asp-116 of the hormone.

Crystallography, X-Ray↗
PubMed1996

Growth hormone, interferon-gamma, and leukemia inhibitory factor utilize insulin receptor substrate-2 in intracellular signaling.

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated following stimulation of 3T3-F442A fibroblasts with growth hormone (GH), leukemia inhibitory factor and interferon-gamma. In response to GH and leukemia inhibitory factor, IRS-2 is immediately phosphorylated, with maximal phosphorylation detected at 15 min; the signal is substantially diminished by 60 min. In response to interferon-gamma, tyrosine phosphorylation of IRS-2 was prolonged, with substantial signal still detected at 60 min. Characterization of the mechanism of signaling utilized by GH indicated that tyrosine residues in GH receptor are not necessary for tyrosyl phosphorylation of IRS-2; however, the regions of GH receptor necessary for IRS-2 tyrosyl phosphorylation are the same as those required for JAK2 association and tyrosyl phosphorylation. The role of IRS-2 as a signaling molecule for GH is further demonstrated by the finding that GH stimulates association of IRS-2 with the 85-kDa regulatory subunit of phosphatidylinositol 3'-kinase and with the protein-tyrosine phosphatase SHP2. These results are consistent with the possibility that IRS-2 is a downstream signaling partner of multiple members of the cytokine family of receptors that activate JAK kinases.

3T3 Cells↗