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Transcriptional and functional regulation of the pituitary guanylyl cyclase-B/cyclic GMP (GC-B/cGMP) system

Reference	BB/D001560/1
Principal Investigator / Supervisor	Professor Robert Fowkes
Co-Investigators / Co-Supervisors
Institution	Royal Veterinary College
Department	Comparative Biomedical Sciences CBS
Funding type	Research
Value (£)	230,105
Status	Completed
Type	Research Grant
Start date	10/10/2005
End date	09/10/2008
Duration	36 months

Abstract

The main objectives and employed methodologies of the proposed work are to: 1. Determine the transcriptional regulation of GC-B expression in anterior pituitary cell lines, identifying the transcription factors involved in mediating basal and hormone-dependent expression of GC-B. Human GC-B promoter constructs (ranging from -2129 to -34bp) will be transiently transfected, using lipofection, into aT3-1, LbT2 (gonadotrophs) and GH3 (somatotroph) cells, along with a Beta-Galactosidase expression vector as a transfection efficiency control. Transfected cells will be stimulated with media alone or a range of peptide hormones or pharmacological agents. Luciferase and Beta-galactosidase assays will be performed to establish promoter activity. Once identified, responsive regions of the GC-B promoter will be further analysed by electrophoretic mobility shift assays to determine which transcription factors are involved in regulating GC-B expression under basal and hormone-stimulated conditions. These experiments will be supported by kinetic analysis of endogenous transcription factor binding by performing chromatin immunoprecipitation. Site-directed mutagenesis will be performed to establish the effect of mutating relevant response elements on GC-B transcription. 2. Determine the mechanism of CNP action on pituitary hormone expression. Transient transfection of pituitary hormone reporter genes will be performed by lipofection. Transfected cells will be pre-treated with 0 or 100nM CNP 30min prior to stimulation with peptide hormones or pharmacological agents. Reporter gene assays will be performed as above. Secondary experiments will be conducted using specific pharmacological inhibitors of the PKA, PKC, PKG, MAPK and calcium pathways, to identify the signalling mechanisms that mediate GC-B effects on pituitary gene transcription. 3. Examine the mechanisms behind homologous and heterologous desensitisation of endogenous GC-B receptors in aT3-1, LbT2 and GH3 cells, and elucidate the mechanism of receptor down-regulation. Cells will be pre-treated with a range of peptide and steroid hormones prior to subsequent stimulation with 0, 100nM CNP or 1uM SNP (soluble guanylyl cyclase activator) in the presence of 1mM isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor). Total cGMP concentrations will be assayed by cyclic GMP enzymeimmunoassay. To establish the molecular mechanism regulating GC-B receptor desensitisation, we will transiently transfect NIH3T3 fibroblasts (no endogenous GC-B) with wild-type and phosphorylation site mutant forms of the GC-B receptor. Pre-treatment with CNP (homologous) or PKC/PKA/PKG activators (heterologous) will be performed prior to subsequent stimulation with 0, 100nM CNP or 1uM SNP. To determine whether receptor down-regulation and internalisation occurs, wild-type GC-B will be cloned into an enhanced green fluorescent protein expression vector to create a GC-B-eGFP fusion protein. aT3-1,LbT2, GH3 and NIH3T3 cells will be transiently transfected with this construct by lipofection. GC-B-eGFP function will be confirmed by cGMP-EIA following 100nM CNP stimulation. To determine GC-B receptor internalisation, transfected cells will be pre-treated with 0, 100nM CNP or 1mM SNP (for 0, 15min, 30min, 1h, 4h, 8h, 24h). Confocal microscopy will be performed to analyse GC-B-eGFP down-regulation and internalisation. 4. Characterise novel signalling pathways activated by CNP in aT3-1, LbT2 and GH3 cells. Cells will be loaded with the calcium chelating dye, FURA2-A/M for 30 min prior to exposure with 0, 10pM,1nM or 100nM CNP and subsequent stimulation with either 56mM KCl, 100nM PACAP or 100nM TRH. Intracellular calcium concentrations will be calculated by the Magical/TARDIS software. The signalling mechanisms involved in mediating GC-B effects on calcium entry will be determined by pre-treating FURA2-A/M loaded cells with pharmacological inhibitors, prior to CNP exposure and KCL/PACAP/TRH stimulation.

Summary

The behaviour of many mammalian cells is influenced by a family of peptide hormones known as natriuretic peptides. These peptides bind to specific cell surface natriuretic peptide receptors, known as guanylyl cyclase-A and -B (GC-A, GC-B). Activation of these receptors increases the intracellular concentration of the signalling molecule, cyclic GMP (cGMP). In the pituitary gland, C-type natriuretic peptide (CNP) activates the GC-B receptor to produce cGMP in many endocrine cell types, including gonadotroph and somatotroph cells. These cells, respectively, regulate reproductive function, growth and metabolism under normal conditions. cGMP alters other intracellular signalling pathways, but its role in regulating gene expression is poorly understood. This proposal seeks to: 1)investigate how expression of the GC-B receptor is regulated in gonadotroph and somatotroph cell lines, under normal conditions and in the presence of peptide hormones. 2)establish whether GC-B/cGMP directly controls the expression of pituitary genes in gonadotroph and somatotroph cell lines. 3)determine the regulatory mechanisms that affect the activity of the GC-B receptor and cGMP generation, particularly the role of natriuretic peptides and other peptide and steroid hormones. 4)characterise novel signalling pathways that GC-B/cGMP can influence in gonadotrophs and somatotrophs, to determine their role in mediating cell function. Understanding the regulation of GC-B receptor expression and action under normal conditions will help to identify mechanisms to enhance or decrease the activity of these receptors in clinical disorders, such as tumour growth, cardiovascular and endocrine dysfunction (in particular dwarfism and infertility).

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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