Annexin A1 (ANXA1), a Ca2+ and phospholipid binding protein, serves as a paracrine/justacrine mediator of glucocorticoid action in the neuroendocrine system. Within the anterior pituitary gland ANXA1 is produced by folliculo-stellate (FS) cells and, following serine-27 phosphorylation (Ser27-P) and membrane translocation, acts on adjoining corticotroph cells to suppress ACTH release via a mechanism involving formyl peptide receptor (FPR) activation. To explore this signalling mechanism further, we have used the AtT20 corticotroph-like cell line (subclone D16:16) as a model. RT-PCR and western blot analysis confirmed that, like primary corticotrophs, AtT20 cells do not express ANXA1 but readily release ACTH in response to CRH. Clones of cells stably transfected with a wild-type ANXA1 DNA construct showed localisation of ANXA1 to the plasma membrane, a diminished response to CRH and significant actin polymerisation vs. controls (empty pRCMV plasmid vector or untransfected). Cells expressing mutant ANXA1 constructs in which Ser5 or Ser34 were replaced with alanine also showed actin polymerisation and a diminished ACTH response to CRH but those transfected with mutants in which Ser27 or Ser45 were substituted did not. The inhibitory effects of the wild-type construct on ACTH release were countered by inclusion of the FPR antagonists Boc1 and Boc2 in the medium. Furthermore, human recombinant ANXA1 and the FPR ligands fMLF and lipoxins A4 induced actin polymerisiation in untransfected cells.
Our data show for the first time that Ser27 and Ser45 play an essential role in effecting the inhibitory effects of ANXA1 on ACTH release. They also consistent with premise that the actions of ANXA1 in the pituitary gland are effected by an FPR family member and provide novel insight to the potential role of actin polymerisation, and hence rho/rac small G proteins, in the signal transduction mechanism used by ANXA1 to suppress exocytosis.
Generously supported by the Wellcome Trust.