Gnrh Induced Citrullination of the Cytoskeleton Is Important for LH Secretion in Gonadotrope Cells
Presentation Number: SUN 493
Date of Presentation: April 2nd, 2017
Shaihla Amin Khan*1, Stanley B DeVore1, Brian S Edwards1, Aaron Muth2, Paul R Thompson2, Brian D Cherrington1 and Amy Marie Navratil1
1University of Wyoming, Laramie, WY, 2University of Massachusetts Medical School, Worcester, MA
Our recent work shows that peptidylarginine deiminase 2 (PAD2) is highly expressed in mouse gonadotropes cells during estrus, precisely when high levels of LH secretion are critical for inducing ovulation. PADs are a family of Ca2+ dependent enzymes that catalyze the conversion of positively charged peptidyl arginines to neutrally charged citrulline, which can alter protein structure and function. Known cytoplasmic targets of citrullination include the cytoskeletal proteins, actin and tubulin. Our work and others, suggests that GnRH engagement of the cytoskeleton not only facilitates the exocytosis of LH but also organizes these cells into a favorable spatial orientation to achieve an increase in circulating LH in vivo. Thus, we hypothesized that post-translational modification of the cytoskeleton by PADs is important for modulating gonadotrope plasticity and function. To test this, we first examined if the GnRH agonist Buserelin (GnRHa) induces citrullination of the cytoskeleton in the gonadotrope derived LβT2 cell line. Using a biotin-phenylglyoxal (Biotin-PG) probe, we selectively enriched citrullinated proteins from our lysates. Western blot analysis reveals that GnRHa temporally induces citrullination of β-actin in LβT2 cells, with maximal levels occurring at 10 minutes. The rapid citrullination kinetics are consistent with actin remodeling events occurring within seconds following GnRH activation. Citrullination of actin induced by GnRHa was blunted when cells were pre-treated with a pan PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA). To identify function, imaging studies examined actin in LβT2 cells and primary mouse gonadotropes illustrate that BB-ClA attenuated GnRHa induced actin reorganization. At issue is how citrullination of actin mediates gonadotrope secretory events. To address this, pituitary primary cultures were pre-treated with BB-ClA, then received pulses of GnRHa at 30 and 60 minutes. Cell culture medium was harvested following subsequent 30 and 60 min pulses of GnRHa and LH levels were analyzed by RIA. Our results show that inhibition of PAD catalyzed citrullination results in a decrease in LH secretion following BB-ClA pre-treatment in the presence of GnRHa. Taken together, these observations suggest that GnRH induced actin citrullination is a novel post-translational mechanism that can regulate cellular architecture and LH release in gonadotrope cells.
Nothing to Disclose: SAK, SBD, BSE, AM, PRT, BDC, AMN