Elsevier

Growth Hormone & IGF Research

Volumes 42–43, October–December 2018, Pages 58-65
Growth Hormone & IGF Research

Review article
A useful model to compare human and mouse growth hormone gene chromosomal structure, expression and regulation, and immune tolerance of human growth hormone analogues

https://doi.org/10.1016/j.ghir.2018.09.001Get rights and content

Highlights

  • Partially humanized mice allow comparison of human and mouse GH synthesis.

  • Human and mouse GH expression is associated with multiple Pit-1 site interactions.

  • Human but not mouse GH gene expression is affected acutely by a high fat diet.

  • Acute clinical effects of overeating on GH is modeled in partially humanized mice.

  • Reduced GH with overeating in puberty is consistent with accelerated somatopause.

  • Immune tolerance of human GH analogues can be tested in partially humanized mice.

Abstract

Human (h) pituitary growth hormone (GH) is both physiologically and clinically important. GH reaches its highest circulatory levels in puberty, where it contributes to energy homeostasis and somatogenic growth. GH also helps to maintain tissues and organs and, thus, health and homeostasis. A reduction in the rate of hGH production begins in middle age but if GH insufficiency occurs this may result in tissue degenerative and metabolic diseases. As a consequence, hGH is prescribed under conditions of GH deficiency and, because of its lipolytic activity, stimulation of hGH release has also been used to treat obesity. However, studies of normal GH production and particularly synthesis versus secretion are not feasible in humans as they require sampling normal pituitaries from living subjects. Furthermore, human (or primate) GH structure and, as such, regulation and potential function, is distinct from non-primate rodent GH. As a result, most information about hGH regulation comes from measurements of secreted levels of GH in humans. Thus, partially humanized hGH transgenic mice, generated containing fragments of human chromosome 17 that include the intact hGH gene locus and many thousands of flanking base pairs as well as the endogenous mouse (m) GH gene provide a potentially useful model. Here we review this mouse model in terms of its ability to allow comparison of hGH versus mGH gene expression, and specifically: (i) GH locus structure as well as regulated and rhythmic expression; (ii) their ability to model a clinical assessment of hGH production in response to overeating and hyperinsulinemia as well as a possible effect of exercise, and (iii) their hGH-related immune tolerance and thus potential for testing hGH-related analogue immunogenicity.

Section snippets

Where and when is growth hormone produced?

Growth hormone (GH), also referred to as somatotropin, is the product of the somatotrophs of the anterior pituitary gland. It is the most abundant hormone produced by the anterior pituitary gland [66]. GH is a 22-k Dalton (kDa) polypeptide hormone composed of 191 amino acid residues forming a single chain with four helical regions and two disulfide bridges [17]. Pituitary GH is generally regarded to be an essential factor for regulating somatic growth in vertebrates from fishes to mammals [34,57

What limits studies of hGH synthesis?

Studies of GH production and particularly synthesis versus secretion are not feasible in humans as they require sampling pituitaries from living subjects. Human GH-secreting pituitary adenomas are available but are relatively rare compared to prolactinomas [13]. When assessed for a response in primary culture, it is not always clear if effects are on hGH secretion and/or cell survival as a result of cell death, lysis and release of intracellular contents, particularly if a marker such as

What sequences are required for pituitary and regulated expression of the hGH gene in vivo?

Duplications of the pituitary GH gene in higher primates have given rise to a family of GH-related genes. The human (h) GH-related gene family consists of five members including pituitary growth hormone (hGH-N or hGH1) as well as placental GH (hGH-V), and the chorionic somatomammotropin (CS) genes (hCS-A, hCS-B and pseudogene hCS-L), contained within a single 47 kb locus on chromosome 17 [12]. The family is flanked upstream by the lymphocyte-specific CD79b and skeletal muscle sodium channel

What is the phenotype and genotype of partially humanized hGH transgenic mice?

Based on the presence of a distant LCR as characterized by five nuclease hypersensitive sites, multiple independent partially humanized hGH-TG mouse lines containing hGH-N and the upstream LCR in single continuous ~100–200 kb fragments of human chromosome 17 were generated [29,32]. These fragments include hGH-N as well as the lymphocyte CD79B gene and part of the skeletal muscle sodium channel SCN4A gene upstream and placental chorionic somatomammotropin (CS) genes (including hCS-L and hCS-A)

Does formation of a chromosomal ‘expression’ loop play a role in hGH-N activation?

While cells of an organism contain the same genomic DNA [15], cell-specific gene activation results from appropriate physical chromatin-chromatin contacts and thus spatial arrangements of chromosomes being made within the interphase nucleus of differentiated cells [18,19]. Activation of hGH-N in developing somatotrophs requires global acetylation and opening of a 32-kb chromatin domain centered and greatest at HS I/II that spreads outwards in an ‘umbrella-like’ manner to include HS V upstream

Is hGH-N expression rhythmic in partially humanized hGH transgenic mice?

Human GH RNA and secreted protein levels were shown to oscillate over a 24-h  cycle using 141hGH/CS-TG and 171hGH/CS-TG mice [74]. Mutation analysis revealed an E-box element (5´-CACGTG-3′) at nucleotides −264/−259, relative to the hGH-N transcription initiation site [72], which binds the circadian transcription factors Clock/Bmal1, and regulates the hGH-N promoter [74]. There was also a difference between the levels of Bmal1 associated with the hGH-N promoter in the light versus dark phase [74

Can a clinical study showing decreased hGH secretion in response to acute overeating and excess insulin be modeled by partially humanized hGH transgenic mice?

Hyperinsulinemia was suggested to be the signal leading to reduced serum hGH levels in young adult males overfed for 3 days, in the absence of hyperglycemia and obesity [14]. This clinical human study was subsequently modeled in hGH/CS-TG mice that were fed a high fat diet of fat - 60 kcal%, carbohydrate - 20 kcal% and protein - 20 kcal%. Here it was demonstrated for the first time that overeating, resulting in excess insulin, has a negative effect on hGH synthesis as well as secretion [73]. A

Do the hGH and mGH gene-related chromatin structure and expression respond similarly to common stimuli in partially humanized hGH transgenic mice?

Multiple studies indicate that mGH gene expression, as least in mice with a C57BL/6 genetic background, is suppressed by diet-induced obesity over 8–16 weeks [41,70]. However, unlike hGH-N, mGH expression was not similarly rhythmic or affected by an acute high fat diet for three days with resulting hyperinsulinemia in CD-1-based hGH/CS-TG mice [73,74]. It appears unlikely that this is due to the absence of any long-range physical interaction between the mGH promoter and more remote upstream

Do hGH transgenic mice show immune tolerance for hGH-related antigens?

Humoral responses, resulting in anti-drug antibodies (ADAs), often occur in patients treated with therapeutic proteins. Protein aggregates are one of the product attributes that have been discussed in the recent literature as a potential risk factor related to immunogenicity. Animal models can be beneficial for assessing the immunogenic potential of therapeutic protein preparations. While in silico and in vitro methods assess, for example, the conditions for T-cell activation, transgenic in vivo

Final observations

Partially humanized hGH transgenic mice provide an additional model to study hGH synthesis and secretion and underlying regulatory mechanisms in vivo and in vitro with primary cultures. Specifically, pituitary somatotroph-specific and rhythmic production of hGH can be modeled in mice using a fragment of human chromosome 17 as the transgene, which includes the hGH-N promoter and coding sequences as well as ~32 kb upstream flanking DNA containing the HS I-III/V (pituitary hGH LCR). Young adult

Conflicts of interest

The authors declare no conflict of interest. However, James Zanghi was an employee of Versartis, Inc. when Genotropin was assessed in hGH/CS-TG mice at the University of Manitoba under a service contract.

Acknowledgements

Funding from the Canadian Institutes of Health Research (MT-10853), Research Manitoba, University of Manitoba and Versartis Inc. (Menlo Park, CA, USA) helped to support this work. The manuscript does not contain clinical studies or patient data. All procedures involving animals conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011), and were approved by the Animal Protocol Management and Review

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