Tomosyn Negatively Regulates Arginine Vasopressin Secretion in Embryonic Stem Cell-Derived Neurons

• Published in: PloS one Vol. 11; no. 10; p. e0164544
• Main Authors: Takeuchi, Seiji, Iwama, Shintaro, Takagi, Hiroshi, Kiyota, Atsushi, Nakashima, Kohtaro, Izumida, Hisakazu, Fujisawa, Haruki, Iwata, Naoko, Suga, Hidetaka, Watanabe, Takashi, Kaibuchi, Kozo, Oiso, Yutaka, Arima, Hiroshi, Sugimura, Yoshihisa
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 12-10-2016; Public Library of Science (PLoS)
• Subjects: Animals; Arginine; Arginine Vasopressin - metabolism; Argipressin; Bands; Binding; Biology and Life Sciences; Cell culture; Cell Line; Chlorides; Diabetes; Embryonic stem cells; Endocrinology; Exocytosis; Glutathione; Glutathione transferase; Hormones; Immunohistochemistry; Male; Mass spectrometry; Medicine; Medicine and Health Sciences; Mice; Molecular weight; Mouse Embryonic Stem Cells - cytology; Mouse Embryonic Stem Cells - metabolism; N-Ethylmaleimide-sensitive protein; Nerve Tissue Proteins - analysis; Nerve Tissue Proteins - metabolism; Neurogenesis; Neurons; Neurons - cytology; Neurons - metabolism; Neuropeptides; Pituitary (posterior); Pituitary Gland, Posterior - metabolism; Pituitary Gland, Posterior - ultrastructure; Potassium chloride; Protein Binding; Proteins; Qa-SNARE Proteins - metabolism; R-SNARE Proteins - analysis; R-SNARE Proteins - metabolism; Rats, Sprague-Dawley; Research and Analysis Methods; Rodents; Scientific imaging; siRNA; SNAP receptors; SNAP-25 protein; Stem cells; Synaptosomal-Associated Protein 25 - metabolism; Syntaxin; Systems analysis; Tomosyn; University graduates; Vasopressin; Vasopressins
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0164544

Arginine vasopressin (AVP) is secreted via exocytosis; however, the precise molecular mechanism underlying the exocytosis of AVP remains to be elucidated. To better understand the mechanisms of AVP secretion, in our study we have identified proteins that bind with a 25 kDa synaptosomal-associated protein (SNAP25). SNAP25 plays a crucial role in exocytosis, in the posterior pituitary. Embryonic stem (ES) cell-derived AVP neurons were established to investigate the functions of the identified proteins. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, we identified tomosyn-1 (syntaxin-binding protein 5) as a SNAP25-binding protein in the posterior pituitary. Coimmunoprecipitation assays indicated that tomosyn formed N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with SNAP25 and syntaxin1. Immunohistochemistry showed that tomosyn localized to the posterior pituitary. Mouse ES cells self-differentiated into AVP neurons (mES-AVP) that expressed tomosyn and two transmembrane SNARE proteins, including SNAP25 and syntaxin1. KCl increased AVP secretion in mES-AVP, and overexpression of tomosyn-1 reduced KCl-stimulated AVP secretion. Downregulation of tomosyn-1 with siRNA increased KCl-stimulated AVP secretion. These results suggested that tomosyn-1 negatively regulated AVP secretion in mES-AVP and further suggest the possibility of using mES-AVP culture systems to evaluate the role of synaptic proteins from AVP neurons.