Regulation of somatotroph development & function by C-type natriuretic peptide (CNP) in humans, cats, mice and zebrafish

Lead Research Organisation: Royal Veterinary College
Department Name: Comparative Biomedical Sciences CBS

Abstract

The growth of an organism is one of the major components of homeostasis. As such, controlling growth appropriately is vital for the health of all animals. The endocrine system, through various hormones, plays a critical role in regulating when an organism needs to grow and develop. The major endocrine control centre that regulates growth is the pituitary gland, which is a pea-sized organ that lies at the base of the brain. Many genes have been identified that are known to be important in the development of the pituitary gland, and several of these are specific to the cells that make growth hormone (GH). Tumours of the pituitary gland represent the most common intracranial tumour in humans, with 1 in 6 people displaying evidence of these growths at the time of their death. Pituitary tumours, as well as other pituitary abnormalities, can cause disrupted release of many hormones, which consequently affects the quality of life of these patients. We have recently found that companion animals, such as dogs and cats, are known to be susceptible to these pituitary tumours. Growth hormone, one of the major pituitary hormones, is extremely important for the development and growth of an individual, and the release of GH is normally under tight control by the hypothalamus (part of the brain), as well as from other hormones released from tissues such as the liver. Inappropriately high or low GH release can cause a series of disorders, ranging from developmental abnormalities in infants, to dwarfism or metabolic complications in adults, and even an increased risk of certain types of colon cancer. We have recently discovered that another hormone found in the pituitary gland, C-type natriuretic peptide (CNP), is produced very early on in the development of human, mice and fish pituitaries. Our recent investigation of 30 human pituitary adenomas found that each one of these tumours expressed CNP, and the receptor that controls its effects, called GC-B. In addition, our preliminary studies have shown that treating pituitary cells with CNP can cause a dramatic increase in the amount of GH that is made. The work we propose to perform, detailed within this application, will extend our understanding of how CNP might influence the way in which the pituitary gland develops and functions normally. We shall use five different models to examine the effects of CNP; cultured pituitary cells, mice that specifically lack CNP in their pituitary glands, human and cat pituitary tumours and the highly versatile Zebrafish, in which we will silence the genes that encode CNP and establish the consequences for normal growth. In addition, our laboratory is equipped with an extremely efficient genetic analyser, that allows us to measure the amounts of up to 15 different genes in a single sample, greatly increasing our productivity from these very small amounts of tissue. These studies may reveal a role for CNP in the treatment of growth disorders, either as a way to increase GH release in individuals with impaired growth, or by developing drugs to block the effects of CNP and, therefore, reduce GH release. Such findings could lead to an improved quality of life, and a reduced susceptibility to subsequent endocrine disease.

Technical Summary

In this study, we propose to dissect the role of C-type natriuretic peptide (CNP) on pituitary somatotroph development and function, and establish its potential as a regulator of growth hormone (GH) expression and secretion. Our previous BBSRC project grant established the expression of a functional natriuretic peptide system in human pituitaries (both normal foetal tissue and adult adenomas) and somatotroph cell lines, but we have now established that Zebrafish express a complete natriuretic peptide system during development. We will use a range of in vitro and in vivo approaches, and five vertebrate model systems (human and feline pituitary tumours, mice, zebrafish, and rodent cell lines) to examine the direct effects of CNP on somatotroph development, gene expression and function. We have developed multiplex RT-PCR assays for each species, that allows us to analyse up to 25 individual genes in a single PCR reaction. In vitro studies with cell lines and primary cultures will determine the effects of CNP on somatotroph-specific gene expression and hormone secretion, as well as establish the effects of physiological regulators of somatotrophs (GHRH, TRH) on the local natriuretic peptide system. Having cloned four CNP transcripts in the Zebrafish, we will target two of these, nppcl and nppc4 (both highly homologous to mammalian CNP), with a morpholino silencing approach and determine the effect on pituitary development and growth of the larvae. We will attempt to reverse any phenotypes with a long-acting CNP analogue. Finally, we are breeding a pituitary-specific CNP-null mouse, in which we will investigate pituitary development and subsequent effects on growth, maturation and GH targets in these mice. This combination of studies, using both normal, genetically modified and diseased tissues, will provide a comprehensive understanding of the role of CNP in somatotroph development and function, and may yield novel approaches to deal with GH excess or insufficiency.

Planned Impact

Whilst the immediate impact of this project will be scientific advancement, increased knowledge and skills, improved training and provision of local jobs at the RVC, there are many medium to long-term areas on which the proposed research will impact. As we propose to take a multifaceted approach to answer our research questions, we benefit from gaining information from several biological models. Our use of human & feline pituitary adenomas is an important translational aspect of our proposal, and our findings could impact upon the work of clinicians involved in treating neuroendocrine tumour patients (by providing novel therapies to regulate hormone secretion), but will be of importance in the understanding of normal pituitary control of growth. Such findings could be used in the treatment of growth disorders, and perhaps reduce some of the financial burden associated with current therapies. The economic consequence of improving both therapies for pituitary tumours, and for growth disorders, could impact upon the areas of the NHS that are involved in providing these services (normally tertiary care facilities).

The findings of our research will not only be of interest in generating new therapies for growth disorders in humans, but may also establish a potential role for CNP in the regulation and treatment of human pituitary adenomas that may lead to alternative therapies for these tumours.

The outcomes of this project will also directly impact upon the careers of the applicants, and of the employed post-doctoral scientist who will gain an impressive range of techniques as part of this project. It is likely that a graduate student would also be involved in some aspects of the work, and hence the attainment of their docotoral degree from these studies would be a significant impact on their own career. Beyond the local environment of the RVC, additional beneficiaries would be those involved in breeding fish, either for the aquaculture industry (fish farms) or those scientists engaged in captive breeding programmes for endangered species.

As the PI and Co-applicant (McGonnell) are actively engaged in outreach work with the RVC, and within their roles in the Society for Endocrinology (SfE), the Anatomical Society and the Society of Biology (all on a national scale). Therefore, findings of the proposed work will be disseminated rapidly within the public domain. Examples of this impact would be the involvement in annual public engagement science fairs that the SfE arrange, and involvement in Society of Biology linked events such as "The Big Bang Fair" as wells as the widening participation events that RVC staff (including both applicants) take part in at both primary and secondary school level.

Collaborative work resulting from these studies would initially involve other academic partners (at the University of Oxford and the PI's collaborator at University of Michigan), hence impacting upon increased international collaborations. Links to US pharmaceutical companies (Biomarin (CA), Gentropin (NY) and Novo Nordisk (NJ) being obvious candidates, with products in growth hormone therapies) would be more medium term impacts as the work develops. However, research firms such as Evotec, would be interested in the morpholino-treated Zebrafish data to inform them of possible homeostatic targets. Aside from its potential role in regulating growth, CNP is a major regulator of bone development, so the use of the new conditional mouse model may well establish a pituitary-dependent effect of CNP on bone disorders (such as osteoporosis,arthritis etc.).

Finally, the establishment of new intellectual property may result from determining the mechanism(s) by which CNP regulates growth. The novel use of CNP, GC-B receptor modulators,or cGMP mimetics to treat pituitary growth disorders would be attractive to the pharmaceutical industry, as all of these targets are 'druggable'.

Publications

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Fowkes R (2014) Natriuretic peptides in pituitary development and function in Endocrine Abstracts

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Scudder CJ (2019) Pituitary Pathology and Gene Expression in Acromegalic Cats. in Journal of the Endocrine Society

 
Description Our project investigates how the peptide hormone, CNP, may influence the production of growth hormone in the pituitary gland, and the overall growth and reproductive capacity of the organism. Our experiments have used a combination of model organisms; zebrafish, mice, and cell cultures. The initial Zebrafish experiments have involved manipulating the levels of one of the CNP genes that Zebrafish express, using a novel technique called CRISPR; fish that have reduced levels of CNP exhibit stunted growth and other developmental abnormalities.

Our mice studies have focussed on generating pituitary-specific CNP knock-out animals; we continue to struggle to gain sufficient homozygote mice (mostly males to date, although we have obtained n=4 homozygous conditional females in the past 12 months, too); the males that we have analysed have exhibited stunted growth, compared with heterozygous and wild-type litter mates, which suggests that loss of CNP in the pituitary is influencing growth hormone expression/secretion in vivo. This growth discrepancy is not as pronounced in the females.

Our cell cultures studies have examined the effects of CNP on the expression levels of genes in pituitary somatotroph cells, using our highly versatile multiplex RT-qPCR assays. Analyses of over 40 targets has established that CNP can alter the expression level of genes involved in growth hormone secretion, responsiveness as well as those responding to metabolic signals (e.g. insulin and leptin).

Finally, we have completed our initial analyses of pituitary adenomas collected from a cohort of cats with poorly controlled type II diabetes, secondary to growth hormone secreting tumours. These patients have undergone elective hypophysectomy (to remove the pituitary and tumour), and enabling our molecular analyses of the natriuretic peptide system. As predicted, feline pituitary samples (both normal and adenoma tissue) express natriuretic peptides and receptors. Of great interest, however, is that CNP expression negatively correlates with the size of the acromegalic tumour, suggesting that increased CNP might serve to reduce tumour size.
Exploitation Route Once we have completed our gene expression analyses, the data sets will be available for others to examine potential targets for pharmacological treatments of growth hormone disorders. We will publish the details of our bespoke multiplex RT-qPCR assays such that others may use them as well. Furthermore, if the pituitary-specific CNP mice are reproductively viable, these animals will be available to the research community for further studies.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The PI (Fowkes) has already visited our potential industrial collaborators (BioMarin, CA, USA) to present the initial findings of our studies. BioMarin have a long-acting CNP analogue in phase II clinical trials to treat achondroplasia, but have yet to investigate its' potential as a GH mimic. We will remain in close contact with them to establish the potential for further therapeutic targets of this compound.
First Year Of Impact 2014
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Title Conditional mouse model 
Description We have established a pituitary-specific Nppc-null mouse. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Our initial characterisation is being performed, and the phenotype is being determined. 
 
Title Multiplex RT-qPCR assays 
Description We have designed bespoke multiplex RT-qPCR assays to examine between 15-20 individual genes per PCR reaction. Our assays to date cover multiple species (mouse, rat, zebrafish, fruit fly, cow, human, cat, horse, dog), and have provided quantitative gene expression data for over 300 gene targets. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact We are providing a gene expression assay service to collaborators. New pilot data has been rapidly generated, and potential diagnostics applications are being developed. 
 
Description Role of CNP in protecting astrocytes during hepatic encephalopathy 
Organisation Royal Veterinary College (RVC)
Department Department of Veterinary Clinical Sciences (formerly Veterinary Clinical Sciences)
Country United Kingdom 
Sector Academic/University 
PI Contribution We have setup an in vitro model in which to examine CNP as a protective agent during the neurological condition of hepatic encephalopathy. The multiplex assays used for the main BBSRC grant have been adapted to investigate the effects of hyperammonaemia on astrocyte gene expression, and we have also designed new multiplexes to analyse hepatic samples from feline patients with congenital portosystemic shunts.
Collaborator Contribution Our partners are soft tissue surgeons, who are performing the corrective surgery (ligations) and collecting biopsies. They are also contributing intellectual input in to the in vitro modelling aspect of this project.
Impact Oral presentation at ECVS2016 in Lisbon this July: Altered hepatic gene expression in cats with congenital portosystemic shunts. A. Charlesworth, S.M. Mirczuk, M.S. Tivers, R.C. Fowkes, V.J. Lipscomb.
Start Year 2014
 
Description Schools science days 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I have run an annual science day at Grange Park Primary School (year intake of 120 children) over the past 7 years. Each year, we present activities to at least two year groups (240 children/year), which have covered the range of natural sciences. Children have the opportunity to handle samples, participate in experiments, and critically interpret the observations - the age range has been from 4yrs to 11 yrs. Every year the children have written about the day, and I have been shown examples of their 'scientific writing'. All sessions are interactive - although not all children can participate with the hands-on aspects, every activity has at least two children acting as demonstrators, and reporter their findings to their peers. I have also run science master-classes at East Barnet School (Secondary), to highlight the benefits of the multiplex RT-qPCR machine that is currently supported by this BBSRC grant.

Feedback from the school is always excellent. Individual parents always comment on the engagement of their children, and this has led to request for visits to the RVC and to my lab.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012