title: Cathepsin D is the primary protease for the generation of adenohypophyseal vasoinhibins: cleavage occurs within the prolactin secretory granules.
abstract: Vasoinhibins are a family of N-terminal prolactin (PRL) fragments that inhibit blood vessel growth, dilation, permeability, and survival. The aspartyl endoprotease cathepsin D is active at acidic pH and can cleave rat PRL to generate vasoinhibins. We investigated whether and where vasoinhibins could be generated by cathepsin D in the adenohypophysis of rats and mice and whether their production could be gender dependent. Vasoinhibins were detected in primary cultures of rat adenohypophyseal cells by Western blot with antibodies directed against the N terminus of PRL but not the C terminus. Ovariectomized, estrogen-treated females show greater levels of adenohypophyseal vasoinhibins than males. Peptide sequencing analysis revealed that the cleaved form of PRL in rat adenohypophyseal extracts contains the PRL N terminus and a second N terminus starting at Ser(149), the reported cleavage site of cathepsin D in rat PRL. In addition, cathepsin D inhibition by pepstatin A reduced vasoinhibin levels in rat adenohypophyseal cell cultures. Confocal and electron microscopy showed the colocalization of cathepsin D and PRL within rat adenohypophyseal cells and secretory granules, and a subcellular fraction of rat adenohypophysis enriched in secretory granules contained cathepsin D activity able to generate vasoinhibins from PRL. Of note, vasoinhibins were absent in the adenohypophysis of mice lacking the cathepsin D gene but not in wild-type mice. These findings show that cathepsin D is the main protease responsible for the generation of adenohypophyseal vasoinhibins and that its action can take place within the secretory granules of lactotrophs.
authors.affiliation: Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus UNAM-Juriquilla, 76230 Querétaro, México.
authors.forename: Martha E
authors.initials: ME
authors.lastname: Cruz-Soto
authors.forename: Gabriela
authors.initials: G
authors.lastname: Cosío
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jeziorski
authors.forename: Víctor
authors.initials: V
authors.lastname: Vargas-Barroso
authors.forename: Manuel B
authors.initials: MB
authors.lastname: Aguilar
authors.forename: Alfonso
authors.initials: A
authors.lastname: Cárabez
authors.forename: Peter
authors.initials: P
authors.lastname: Berger
authors.forename: Paul
authors.initials: P
authors.lastname: Saftig
authors.forename: Edith
authors.initials: E
authors.lastname: Arnold
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Thebault
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Martínez de la Escalera
authors.forename: Carmen
authors.initials: C
authors.lastname: Clapp
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D005455:Fluorescent Antibody Technique; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D011388:Prolactin / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D010446:Peptide Fragments; D011388:Prolactin; D002402:Cathepsin D