Murine Growth Hormone Research Articles

IGF-1/GH axis enhances losartan treatment in Lama2-related muscular dystrophy.

As the complexities of dystrophic pathology have been elucidated over the last few years, it has become increasingly clear that primary monogenetic defects result in multiple secondary pathologies capable of autonomously driving disease progression. Consequently, single-mode therapies fail to comprehensively ameliorate all aspects of pathology. Lama2-related muscular dystrophy (MDC1A) is a devastating congenital muscular dystrophy caused by mutations in the LAMA2 gene that results in multi-faceted secondary pathologies that include inflammation, fibrosis, apoptosis, and necrosis leading to severe muscle weakness and minimal postnatal growth. This study sought to implement a novel combinatorial treatment utilizing losartan, previously shown to ameliorate fibrosis and inflammation in conjunction with transgenic IGF-1 overexpression to improve postnatal growth. We found that dual-therapy rescued inflammation and fibrosis, improved weight gain, and led to remarkable restoration of muscle architecture and locomotory function in DyW mice (mouse model of MDC1A). We further showed using murine growth hormone that postnatal intervention with both therapies also yielded impressive amelioration of dystrophic pathology. Our results suggest for the first time that a combinatorial anti-fibrotic and pro-myogenic therapy could be the foundation of future therapies to a population of afflicted children in serious need.

Silicone Oil Microdroplets Can Induce Antibody Responses Against Recombinant Murine Growth Hormone in Mice

Therapeutic protein products can cause adverse immune responses in patients. The presence of subvisible particles is a potential contributing factor to the immunogenicity of parenterally administered therapeutic protein formulations. Silicone oil microdroplets, which derive from silicone oil used as a lubricating coating on barrels of prefilled glass syringes, are often found in formulations. In this study, we investigated the potential of silicone oil microdroplets to act as adjuvants to induce an immune response in mice against a recombinant murine protein. Antibody responses in mice to subcutaneous injections of formulations of recombinant murine growth hormone (rmGH) that contained silicone oil microdroplets were measured and compared to responses to oil-free rmGH formulations. When rmGH formulations containing silicone oil microdroplets were administered once every other week, anti-rmGH antibodies were not detected. In contrast, mice exhibited a small IgG1 response against rmGH when silicone oil–containing rmGH formulations were administered daily, and an anti-rmGH IgM response was observed at later time points. Our findings showed that silicone oil microdroplets can act as an adjuvant to promote a break in immunological tolerance and induce antibody responses against a recombinant self-protein.

Energy homeostasis targets chromosomal reconfiguration of the human GH1 locus.

Levels of pituitary growth hormone (GH), a metabolic homeostatic factor with strong lipolytic activity, are decreased in obese individuals. GH declines prior to the onset of weight gain in response to excess caloric intake and hyperinsulinemia; however, the mechanism by which GH is reduced is not clear. We used transgenic mice expressing the human GH (hGH) gene, GH1, to assess the effect of high caloric intake on expression as well as the local chromosome structure of the intact GH1 locus. Animals exposed to 3 days of high caloric intake exhibited hyperinsulinemia without hyperglycemia and a decrease in both hGH synthesis and secretion, but no difference in endogenous production of murine GH. Efficient GH1 expression requires a long-range intrachromosomal interaction between remote enhancer sequences and the proximal promoter region through "looping" of intervening chromatin. High caloric intake disrupted this interaction and decreased both histone H3/H4 hyperacetylation and RNA polymerase II occupancy at the GH1 promoter. Incorporation of physical activity muted the effects of excess caloric intake on insulin levels, GH1 promoter hyperacetylation, chromosomal architecture, and expression. These results indicate that energy homeostasis alters postnatal hGH synthesis through dynamic changes in the 3-dimensional chromatin structure of the GH1 locus, including structures required for cell type specificity during development.

UV photodegradation of murine growth hormone: Chemical analysis and immunogenicity consequences

During manufacturing, therapeutic proteins may be exposed to ultraviolet (UV) radiation. Such exposure is of concern because UV radiation may cause photooxidative damage to proteins, which in turn could lead to physical changes such as aggregation and enhanced immunogenicity. We exposed murine growth hormone (mGH) to controlled doses of UV radiation, and examined the resulting chemical, physical and immunogenic changes in the protein. mGH chemical structure was analyzed by mass spectrometry after UV irradiation. Photooxidation products detected by mass spectrometry included methionine sulfoxide formed at Met[127] and Met[149] residues, and, tentatively assigned by MS/MS analysis, ether cross-links between original Ser[78] and Cys[188], and Cys[206] and Ser[213], and a thioether cross-link between Cys[17] and Cys[78] residues, transformation of Cys[189] into Ala, and various hydrolytic fragments. Physical damage to UV-irradiated mGH was monitored by infrared spectrometry, chromatographic analyses, and particle counting by micro-flow imaging. UV radiation caused mGH to aggregate, forming insoluble microparticles containing mGH with non-native secondary structure. When administered subcutaneously to Balb/c or Nude Balb/c mice, UV-irradiated mGH provoked antibodies that cross-reacted with unmodified mGH in a fashion consistent with a T-cell dependent immune response. In wildtype Balb/c mice, titers for anti-mGH IgG1 antibodies increased with increasing UV radiation doses.

Recombinant Murine Growth Hormone Particles are More Immunogenic with Intravenous than Subcutaneous Administration

Evaluation and mitigation of the risk of immunogenicity to protein aggregates and particles in therapeutic protein products remains a primary concern for drug developers and regulatory agencies. To investigate how the presence of protein particles and the route of administration influence the immunogenicity of a model therapeutic protein, we measured the immune response in mice to injections of formulations of recombinant murine growth hormone (rmGH) that contained controlled levels of protein particles. Mice were injected twice over 6 weeks with rmGH formulations via the subcutaneous, intraperitoneal, or intravenous (i.v.) routes. In addition to soluble, monomeric rmGH, the samples prepared contained either nanoparticles of rmGH or both nano- and microparticles of rmGH. The appearance of anti‐rmGH IgG1, IgG2a, IgG2b, IgG2c, and IgG3 titers following the second injection of both preparations implies that multiple mechanisms contributed to the immune response. No dependence of the immune response on particle size and distribution was observed. The immune response measured after the second injection was most pronounced when i.v. administration was used. Despite producing high anti‐rmGH titers mice appeared to retain the ability to properly regulate and use endogenous growth hormone. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:128–139, 2014

Effect of ghrelin and anamorelin (ONO-7643), a selective ghrelin receptor agonist, on tumor growth in a lung cancer mouse xenograft model

PurposeAnamorelin (ONO-7643) is an orally active ghrelin receptor agonist in development for non-small cell lung cancer (NSCLC)-related anorexia/cachexia. It displays both orexigenic and anabolic properties via ghrelin mimetic activity and transient increases in growth hormone (GH). However, increasing GH and insulin-like growth factor-1 in cancer patients raises concerns of potentially stimulating tumor growth. Therefore, we investigated the effect of ghrelin and anamorelin on tumor growth in a murine NSCLC xenograft model.MethodsFemale nude mice (15–21/group) with established A549 tumors were administered ghrelin (2 mg/kg i.p.), anamorelin (3, 10, or 30 mg/kg p.o.), or vehicle controls daily for 28 days. Tumor growth, food consumption, and body weight were monitored. Murine growth hormone (mGH) and murine insulin-like growth factor-1 (mIGF-1) were measured in plasma.ResultsTumor growth progressed throughout the study, with no significant differences between treatment groups. Daily food consumption was also relatively unchanged, while the percentage of mean body weight gain at the end of treatment was significantly increased in animals administered 10 and 30 mg/kg compared with controls (p < 0.01). Peak mGH levels were significantly higher in ghrelin- and anamorelin-treated animals than in controls, while peak mIGF-1 levels were slightly elevated but not statistically significant. All regimens were well tolerated.ConclusionsThese findings demonstrate that neither anamorelin nor ghrelin promoted tumor growth in this model, despite increased levels of mGH and a trend of increased mIGF-1. Together with anamorelin’s ability to increase body weight, these results support the clinical development of ghrelin receptor agonist treatments for managing NSCLC-related anorexia/cachexia.

Effect of anamorelin/ONO-7643, a selective ghrelin receptor agonist, on tumor growth in a lung cancer mouse xenograft model.

e19577 Background: Anamorelin/ONO-7643 is an orally-active ghrelin receptor agonist in development for non-small cell lung cancer (NSCLC)-related cachexia/anorexia. It displays both anabolic and orexigenic properties via its ghrelin and growth hormone (GH) secretagogue activity. However, increasing GH and insulin-like growth factor-1 (IGF-1) in cancer patients raises potential concerns of stimulating tumor growth. In this study, we investigated the effect of ghrelin and Anamorelin/ONO-7643 on tumor growth in a NSCLC xenograft model. Methods: On Day 1 (D1), 21 days after implanting A549 tumors, female nude mice were sorted into six groups (n=15/group) and administered ghrelin (2 mg/kg i.p.), Anamorelin/ONO-7643 (3, 10, or 30 mg/kg p.o.) or vehicles (saline i.p. or de-ionized water p.o.) for 28 days, starting on D3. Tumor growth, body weight, and food consumption were monitored. Mice used to assess plasma levels of murine GH (mGH) and IGF-1 (mIGF-1) were sorted into three groups (n=21/group) and treated for 28 days with ghrelin, the high dose of Anamorelin/ONO-7643 or vehicle (de-ionized water p.o.). Results: After 28 days of treatment, there was no difference in median tumor volumes (D30 values: 1008, 936, 1080, 666 and 847 mm3 for vehicle, ghrelin and Anamorelin/ONO-7643 at 3, 10 and 30 mg/kg, respectively). Ghrelin significantly increased mGH compared to controls, while Anamorelin/ONO-7643 modestly increased mGH. Peak mIGF-1 levels were slightly higher in animals given ghrelin or Anamorelin/ONO-7643 compared to vehicle, although not significantly. Anamorelin/ONO-7643 at 10 and 30 mg/kg/day showed a statistically significant (p&lt;0.01) increase in body weight from D1 to D30 compared to control animals, with no change in food consumption. Ghrelin treatment had no effect on body weight or food consumption. Conclusions: Anamorelin/ONO-7643 or ghrelin treatment for 28 days had no effect on tumor growth in A549 tumor-bearing nude mice, despite increased mGH and a trend of increased mIGF-1. Anamorelin/ONO-7643 also significantly increased body weight at 10 and 30 mg/kg/day. These results support using ghrelin receptor agonist-based treatments in managing NSCLC-related cachexia/anorexia.

Glass particles as an adjuvant: A model for adverse immunogenicity of therapeutic proteins

Unwanted immune responses to parenterally administered therapeutic proteins pose serious safety and economic risks, but the mechanism(s) by which these responses are generated are unknown. We measured immune responses to aggregates of recombinant murine growth hormone (mGH) formed by agitation or freeze-thawing, two pharmaceutically relevant stresses, as well as to mGH adsorbed on microscopic glass or alum particles. Insoluble aggregates, even at levels below the detection limits of size-exclusion high-performance liquid chromatography analysis (<1%), induce immune responses when administered subcutaneously. Furthermore, we show that application of high hydrostatic pressures (200 MPa) reduces aggregate levels to 0.02 ng/dose and eliminates immunogenicity.

Recombinant murine growth hormone from E. coli inclusion bodies: Expression, high-pressure solubilization and refolding, and characterization of activity and structure

We expressed recombinant murine growth hormone (rmGH) in E. coli as a cost-effective way to produce large quantities (gram scale) of the protein for use in murine studies of immunogenicity to therapeutic proteins. High hydrostatic pressure was used to achieve high solubility and high refolding yields of rmGH protein produced in E. coli inclusion bodies. A two-step column purification protocol was used to produce 99% pure monomeric rmGH. Secondary and tertiary structures of purified rmGH were investigated using circular dichroism and 2D-UV spectroscopy. The purified rmGH produced was found to be biologically active in hypophysectomized rats.

Developmentally Regulated Activation of a SINE B2 Repeat as a Domain Boundary in Organogenesis

The temporal and spatial regulation of gene expression in mammalian development is linked to the establishment of functional chromatin domains. Here, we report that tissue-specific transcription of a retrotransposon repeat in the murine growth hormone locus is required for gene activation. This repeat serves as a boundary to block the influence of repressive chromatin modifications. The repeat element is able to generate short, overlapping Pol II-and Pol III-driven transcripts, both of which are necessary and sufficient to enable a restructuring of the regulated locus into nuclear compartments. These data suggest that transcription of interspersed repetitive sequences may represent a developmental strategy for the establishment of functionally distinct domains within the mammalian genome to control gene activation.

Growth Hormone and Development

During development, genes are often transcribed in a temporally and spatially regulated manner. The murine growth hormone gene is differentially expressed in the developing pituitary gland. Lunyak et al . now examine the region surrounding the growth hormone gene and show that a repeated DNA sequence (short interspersed nuclear element B2) in the growth hormone locus functions as an insulator to produce a boundary for chromatin domains and limit the action of regulatory factors such as enhancers and silencers. V. V. Lunyak, G. G. Prefontaine, E. Núñez, T. Cramer, B.-G. Ju, K. A. Ohgi, K. Hutt, R. Roy, A. García-Díaz, X. Zhu, Y. Yung, L. Montoliu, C. K. Glass, M. G. Rosenfeld, Developmentally regulated activation of a SINE B2 repeat as a domain boundary in organogenesis. Science 317 , 248-251 (2007). [Abstract] [Full Text]

Identification and characterization of a novel transcript of the murine growth hormone receptor gene exhibiting development- and tissue-specific expression

The growth hormone (GH) receptor gene is characterized by heterogeneity in the 5′-untranslated region (UTR). The technique of 5′-rapid amplification of cDNA ends (RACE) was employed to identify potentially novel 5′-UTRs for the GH receptor gene. One of the RACE clones displayed sequence homology to the human V5-UTR; hence this transcript was designated as L5. Sequence analysis of genomic DNA established that L5 was immediately upstream of exon 2. Northern blot analysis indicated that two bands of sizes ≅4.8 kb, corresponding to GH receptor mRNA, and ≅1.5 kb corresponding to GH binding protein mRNA, were detectable in liver, skeletal muscle, kidney and heart but not in brain, spleen, lung or testis. Fluorescent 5′-nuclease real-time RT-PCR based analysis indicated that in the placenta and fetal liver, the L5 transcript represented 10–15% of the GH receptor transcripts. In the adult liver, heart and kidney, the L5 transcript is less abundant accounting for 1–5% of the total GH receptor transcripts. Primer extension and ribonuclease protection assays were performed to identify the major transcription start site at 778 bp from the ATG codon. Transient transfection experiments revealed that the 5′-flanking sequence had promoter activity in rat placental trophoblast (HRP.1), Chinese hamster ovary (CHO) and mouse liver (BNL CL.2) cells. Analysis of expression of the L5 transcript in the non-obese diabetic (NOD) mouse, a model of spontaneous autoimmune diabetes, indicated that the expression of the L5 transcript was decreased in liver and kidney by 80–90 and 40–50%, respectively, but expression remained unchanged in the heart.

Tumor necrosis factor a down regulates the murine growth hormone receptor promoter and spfamily transactivators

Tumor necrosis factor a down regulates the murine growth hormone receptor promoter and spfamily transactivators

Role of the Sp Family of Transcription Factors in the Ontogeny of Growth Hormone Receptor Gene Expression

The growth hormone (GH) receptor is essential for the actions of growth hormone on postnatal growth and metabolism. GH receptor transcripts are characterized by the presence of disparate 5'-untranslated exons. Factors regulating the expression of the GC rich L2 transcript of the murine GH receptor gene have hitherto remained unidentified. To characterize the mechanisms regulating expression of the L2 transcript, primer extension and ribonuclease protection assays were used to identify transcription start sites in RNA from liver of adult mice. Transient transfection experiments revealed that 2.0 kilobase pairs of the L2 5'-flanking sequence exhibited promoter activity in BNL CL.2 (mouse liver) cells, CV-1 (monkey kidney) cells, and HRP.1 trophoblasts. Deletional analysis localized a major regulatory region to within 75 base pairs of the 5' transcription start site. Sequence analysis revealed that the region contained consensus binding sites for the Sp family of transcription factors. Standard gel shift and supershift analysis using liver nuclear extracts established that Sp1 and Sp3 bound this regulatory element. Transfection of wild type but not mutant decoy oligonucleotides into BNL CL.2 cells decreased the activity of the L2 promoter. Overexpression of Sp1 and Sp3 protein in Drosophila Schneider cells established that Sp3 is more potent than Sp1 in transactivating the L2 promoter. Co-transfection experiments further established that Sp1 antagonizes the activity of Sp3 to transactivate the L2 promoter. Western blot analysis of liver nuclear extracts revealed that the levels of Sp3 increase significantly after birth, suggesting a role for the Sp family of transcription factors in controlling the fetal to postnatal increase in GH receptor gene expression.

Construction and in Vivo Efficacy of a Replication-Deficient Recombinant Adenovirus Encoding Murine Growth Hormone

Construction and in Vivo Efficacy of a Replication-Deficient Recombinant Adenovirus Encoding Murine Growth Hormone

Transcription Factor MSY-1 Regulates Expression of the Murine Growth Hormone Receptor Gene

Previous studies identified and partially characterized a 42-base pair regulatory element in the 5'-flanking region of the L1 transcript of the murine growth hormone (GH) receptor gene that interacted with both double- and single-stranded DNA-binding proteins. We present evidence that the double-stranded DNA-binding protein is NF-Y, a CCAAT box-binding protein. Experiments with a dominant negative form of NF-Y indicate that NF-Y does not play a direct role in regulating the activity of the FP42 element. A cDNA clone that specifically interacts with the upper (coding) strand of the regulatory element was isolated by screening a cDNA expression library using the Southwestern technique. DNA sequencing, electrophoretic mobility shift assay, Southwestern blot analysis, and supershift EMSA confirm the identity of the single-stranded binding protein to be MSY-1, a DNA-binding protein that is evolutionary conserved from prokaryotes to eukaryotes. Mapping of single-stranded DNA configurations reveals that MSY-1 can facilitate the formation of single-stranded DNA regions in the GH receptor 5'-flanking region. Transient transfection experiments support the role of MSY-1 as a repressor of GH receptor gene activation. Southwestern blot analysis indicates that the levels of nuclear MSY-1 are decreased in the livers of pregnant mice, suggesting a role for MSY-1 in the increased expression of the GH receptor during pregnancy.

A member of the CTF/NF-1 transcription factor family regulates murine growth hormone receptor gene promoter activity

Deletional analysis by transient transfection of COS-7 cells with the murine growth hormone (GH) receptor gene promoter for the L1 transcript identified a 16-bp enhancer element located approximately 3.0 kb upstream of the major transcription start sites. The sequence of this enhancer element suggested a binding motif for the CTF/NF-1 family of transcription factors. In electromobility shift assays (EMSA) this DNA-element formed a sequence-specific complex with nuclear proteins from COS-7 cells. Competition experiments with oligonucleotide containing a consensus binding motif for CTF/NF-1 protein(s) and supershift EMSA with an anti-CTF/NF-1 antibody established that a CTF/NF-1 like protein binds to this enhancer element.

A member of the CTF/NF-1 transcription factor family regulates murine growth hormone receptor gene promoter activity.

Deletional analysis by transient transfection of COS-7 cells with the murine growth hormone (GH) receptor gene promoter for the L1 transcript identified a 16-bp enhancer element located approximately 3.0 kb upstream of the major transcription start sites. The sequence of this enhancer element suggested a binding motif for the CTF/NF-1 family of transcription factors. In electromobility shift assays (EMSA) this DNA-element formed a sequence-specific complex with nuclear proteins from COS-7 cells. Competition experiments with oligonucleotide containing a consensus binding motif for CTF/NF-1 protein(s) and supershift EMSA with an anti-CTF/NF-1 antibody established that a CTF/NF-1 like protein binds to this enhancer element.

Cloning of the Promoter-Regulatory Region of the Murine Growth Hormone Receptor Gene: IDENTIFICATION OF A DEVELOPMENTALLY REGULATED ENHANCER ELEMENT

The growth hormone (GH) receptor is essential for the actions of GH on postnatal growth and metabolism. To identify DNA sequences involved in the regulation of transcription of the murine GH receptor gene, a 17-kilobase genomic clone containing the 5'-flanking region, exon 1, and part of intron 1 of the murine GH receptor gene was isolated. Utilizing primer extension and ribonuclease protection assays, two major transcription start sites were identified in RNA from liver of male, female, and pregnant mice. Transient transfection studies using a reporter gene demonstrated promoter activity in a variety of eukaryotic cells. Deletional analysis and DNA-protein binding assays led to the identification of a 30-base pair enhancer element located about 3.4 kilobases upstream of the transcription start sites. Computer analysis of the nucleotide sequence of the enhancer element did not reveal any potential DNA binding motifs for known transcription factors, and this DNA element failed to exhibit binding activity for some common transcription factors. Analysis of both functional activity and DNA-protein binding activity of this enhancer element in adult and fetal hepatocytes suggests that this DNA element may play a role in the developmental expression of the GH receptor gene.

Effect of recombinant porcine somatotropin and monoclonal antibody directed to ovine somatotrophic hormone on nitrogen retention and immune parameters in pigs.

Single and combined effects of administration and withdrawal of recombinant porcine somatotropin (rpST) and an enhancing murine antiovine growth hormone monoclonal antibody (OA15) on nitrogen retention, and serological and immunological measurements in pigs were examined in a placebo-controlled experiment. Thirty-six barrows were allotted to one of four treatments: control, rpST, OA15, and OA15+rpST. The trial phase was four balance periods: a preperiod, two periods of treatment, and a postperiod. Weight- and nitrogen gain were higher for the rpST group by 13% (P < .01) and 15% (P < .001), for the OA15 group by 8% (P < .05) and 9% (P < .05), and for the OA15+rpST group by 25% (P < .001) and 20% (P < .001), respectively compared with the control group. During the postperiod, weight gain of the OA15- and the OA15+rpST group was 23% (P < .001) and 22% (P < .001) lower than that of the control group. Nitrogen gain during the postperiod was decreased by 19% (P < .01) for the OA15 group compared with the control group. Single or combined administration of rpST or OA15 did not affect (P > .10) cellular constituents in the blood of all groups during the periods of observation. Animals treated solely with rpST mounted a humoral immune response directed to rpST. This anti-rpST antibody response was, however, decreased (P < .01) in barrows treated with rpST and OA15 simultaneously. Also, a slight anti-rpST antibody response was noticed in barrows solely treated with OA15.(ABSTRACT TRUNCATED AT 250 WORDS)