Gonadotropin-Releasing Hormone (GnRH) Metabolite, GnRH-(1-5), Mediates Cell Migration and Invasion in the Ishikawa Cell Line

Presentation Number: SUN-0041
Date of Presentation: June 22nd, 2014

Madelaine J Cho-Clark*1, Darwin Omar Larco1, Maya Jane Sorini2, Shailaja K Mani3 and Tao-Yiao John Wu1
1Uniformed Services University, Bethesda, MD, 2Holton-Arms School, Bethesda, MD, 3Baylor College of Medicine, Houston, TX

Abstract

GnRH is a decapeptide known for its key role in regulating the hypothalamic-pituitary-gonadal (HPG) axis.  While its major role in HPG regulation has been well characterized, its secondary level of regulation in peripheral tissues remains elusive.  GnRH is processed by zinc metalloendopeptidase EC 3.4.24.15 (EP24.15) that cleaves the hormone at the Tyr5-Gly6 bond to form a pentapeptide, GnRH-(1-5).  This pentapeptide’s actions differ from those of its parent peptide, GnRH, in both neural and peripheral cells.  We have previously shown that GnRH-(1-5) stimulates epidermal growth factor (EGF) release, increases the phosphorylation of EGFR, and promotes cellular migration in the Ishikawa endometrial cancer cell line.  We also demonstrated that the actions of GnRH-(1–5) are mediated by the orphan G protein-coupled receptor 101 (GPR101).  The down-regulation of GPR101 expression by antisense oligonucleotide and by siRNA blocked the GnRH-(1–5)-mediated transactivation of EGFR and increases in cellular migration.  Furthermore, the global MMP inhibitor, Batimastat, blocked GnRH-(1-5) mediated EGFR phosphorylation.  This suggests that activation of GPR101 may mobilize MMPs at the membrane level to release membrane bound EGF.  In this study, we investigated the effect of GnRH-(1-5) on cellular migration and invasion by examining its regulation of collagenases MMP2 and MMP9.  Using the matrigel invasion assay, we examined the effects of GnRH-(1-5) and its parental analogs on migration and invasion in the Ishikawa cell line and compared them to the vehicle (VEH) control. Treatment with GnRH-(1-5) (100nM) increased both migration and invasion into the matrigel by ~20% and 30% (p<0.05), respectively at t= 48h whereas treatment with 100nM GnRH had no effect (p>0.10) on either parameters. Interestingly, treatment with (D-Ser6)-GnRH (an agonist of GnRH), resulted in an anti-migratory effect (p<0.05), while having no effect on invasion.  Furthermore, treatment with GnRH-(1-5) increased (p<0.05) the levels of MMP9 in the crude membrane fraction, whereas treatment with 100nM GnRH or 100nM (D-Ser6)-GnRH had no effect (p>0.10).  No change (p>0.10) in MMP2 levels was observed in the lysate and crude membrane fractions with all treatments.  Our results suggest that GnRH-(1-5) regulate the recruitment of MMP9 to the membrane level to stimulate migration and invasion. 

 

Nothing to Disclose: MJC, DOL, MJS, SKM, TYJW