Endocrine Abstracts (2012) 28 OC5.6

SUMOylation genes in pituitary gonadotrophs: wrestling with GnRH signalling

Samantha Mirczuk1, Samantha Wilkinson1, Alyssa Savage1, Andrew Sunters1, Bigboy Simbi1, Craig McArdle2, Martin Lee3 & Robert Fowkes1

1Veterinary Basic Sciences, Royal Veterinary College, London, United Kingdom; 2Laboratories for Integrative Neuroscience & Endocrinology, University of Bristol, Bristol, United Kingdom; 3Division of Nephrology, National University Hospital, Singapore, Singapore.

The orphan nuclear receptor, SF-1, is sumoylated in adrenal and gonadal tissues, but the role of sumoylation in pituitary gonadotrophs is currently unknown. We performed expression studies for key components of sumoylation machinery in αT3-1 and LβT2 gonadotroph cell lines, using RT-PCR. Expression of the genes encoding the SUMO proteins (Sumo1, Sumo2, Sumo3) as well as the E3 ligase enzymes responsible for their covalent linkage (Pias1, Pias2/xα, Pias4/γ) was detected in both αT3-1 and LβT2 cells. To determine whether GnRH could alter the expression of sumoylation genes, αT3-1 cells were treated with 0 or 100 nM GnRH chronically, or using a pulsatile stimulation (1×5 min pulse/h), for 4 h before analyzing gene expression by qPCR. Chronic exposure to GnRH failed to alter the expression of any Sumo gene, or Pias1 or Pias4/γ genes, but did cause a 3.5±0.9-fold increase in Pias2/xα. However, pulsatile GnRH resulted in altered expression of Sumo2, Sumo3, Pias1, Pias2/xα, and Pias4/γ (by 0.45±0.1, 0.46±0.16, 6.1±3.1, 7.4±3.5 and 0.45±0.2-fold, respectively). Protein studies in LβT2 cells showed enhanced expression of SF-1 in response to both chronic and pulsatile GnRH stimulation. Interestingly, additional protein species, consistent with monosumoylated SF-1 were observed in all samples, and the intensity of this band increased following exposure to GnRH for 4 h. By comparison, the levels of phospho-ERK1/2 were only elevated in pulsatile, but not chronically-stimulated LβT2 cells, after 4 h exposure to GnRH. Finally, the functional consequence of SUMO proteins on SF-1-target gene expression was examined, by co-transfecting the human αGSU promoter with either empty vector or a SUMO-1 expression vector. Remarkably, the presence of elevated SUMO-1 completely blocked GnRH-stimulated αGSU promoter activity in αT3-1 cells. These data reveal that sumoylation genes are sensitive to physiologically relevant GnRH stimulation in gonadotroph cell lines, and indicate a potential mechanism to regulate SF-1 function.

Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.

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