Endocrine Abstracts

Annexin 1 (ANXA1) plays an essential role in the 'early-delayed' feedback effects of glucocorticoids (GCs) on ACTH release. Evidence that ANXA1 is externalised from pituitary cells in response to GCs, together with the finding that the protein is localised mainly to the folliculo-stellate (FS) cells, has led us to propose that ANXA1 is a paracrine mediator of GC action in the anterior pituitary. To address this hypothesis we have examined ANXA1-dependent GC actions in co-cultures of murine corticotroph (AtT20 D1) and FS (TtT/GF) cell lines. AtT20 cells responded to CRH (1micromolar, 2h) with a marked increase in ACTH release that was inhibited by pre-incubation with an N-terminal fragment of ANXA1 (ANXA1_1-188, 1ng/ml; 2.5h) but not by dexamethasone (100nM, 2.5h). By contrast, dexamethasone inhibited CRH-induced ACTH from co-cultures of TtT/GF and AtT20; its effects were increased with the ratio of AtT20:TtT/GF cells reaching a maximum of 70% inhibition at a cell ratio of 4:1. The inhibitory actions of dexamethasone on the co-cultures were reduced by the GC receptor antagonist mifepristone (1micromolar) and by an antisense oligodeoxynucleotide (ODN) to ANXA1; the corresponding sense or scramble sequences were without effect. The antisense ODN also prevented the dexamethasone-induced externalisation from TtT/GF cells, as shown by western blot analysis. ANXA1 protein was not detectable in AtT20 cells, however immunofluorescence labelling of co-cultures revealed punctate staining of ANXA1 on the boundaries between the TtT/GF and AtT20 cells suggesting that ANXA1 is indeed a paracrine mediator of GCs in this model. These results provide functional and histological evidence that the inhibitory actions of dexamethasone on ACTH release in the co-cultures are mediated via the GC receptor and depend on the exportation of ANXA1 from the TtT/GF cells. They thus support the premise that ANXA1 serves as a paracrine mediator of GC action in the anterior pituitary gland.

We are grateful to The Wellcome Trust for generous financial support.