Different effects of progesterone and estradiol on chimeric and wild type aldosterone synthase in vitro.
BACKGROUND
Familial hyperaldosteronism type I (FH-I) is caused by the unequal recombination between the 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, resulting in the generation of a CYP11B1/B2 chimeric gene and abnormal adrenal aldosterone production. Affected patients usually show severe hypertension and an elevated frequency of stroke at a young age. Aldosterone levels rise during pregnancy, yet in pregnant women with FH-1, their hypertensive condition either remains unchanged or may even improve. The purpose of this study was to investigate in vitro whether female sex steroids modulate the activity of chimeric (ASCE) or wild type (ASWT) aldosterone synthase enzymes.


METHODS
We designed an in vitro assay using HEK-293 cell line transiently transfected with vectors containing the full ASCE or ASWT cDNAs. Progesterone or estradiol effects on AS enzyme activities were evaluated in transfected cells incubated with deoxycorticosterone (DOC) alone or DOC plus increasing doses of these steroids.


RESULTS
In our in vitro model, both enzymes showed similar apparent kinetic parameters (Km = 1.191 microM and Vmax = 27.08 microM/24 h for ASCE and Km = 1.163 microM and Vmax = 36.98 microM/24 h for ASWT; p = ns, Mann-Whitney test). Progesterone inhibited aldosterone production by ASCE- and ASWT-transfected cells, while estradiol demonstrated no effect. Progesterone acted as a competitive inhibitor for both enzymes. Molecular modelling studies and binding affinity estimations indicate that progesterone might bind to the substrate site in both ASCE and ASWT, supporting the idea that this steroid could regulate these enzymatic activities and contribute to the decay of aldosterone synthase activity in chimeric gene-positive patients.


CONCLUSIONS
Our results show an inhibitory action of progesterone in the aldosterone synthesis by chimeric or wild type aldosterone synthase enzymes. This is a novel regulatory mechanism of progesterone action, which could be involved in protecting pregnant women with FH-1 against hypertension. In vitro, both enzymes showed comparable kinetic parameters, but ASWT was more strongly inhibited than ASCE. This study implicates a new role for progesterone in the regulation of aldosterone levels that could contribute, along with other factors, to the maintenance of an adequate aldosterone-progesterone balance in pregnancy.
Molecular Endocrinology Laboratory, Department of Endocrinology, School of Medicine, Pontificia Universidad Catolica de Chile, Lira 85, 5th Floor, Santiago, Chile. cfardella@med.puc.cl.
Andrea
A
Vecchiola
Carlos F
CF
Lagos
Cristóbal A
CA
Fuentes
Fidel
F
Allende
Carmen
C
Campino
Carolina
C
Valdivia
Alejandra
A
Tapia-Castillo
Tadashi
T
Ogishima
Kuniaki
K
Mukai
Gareth
G
Owen
Sandra
S
Solari
Cristian A
CA
Carvajal
Carlos E
CE
Fardella
D000450:Aldosterone / Q000378:metabolism; D019405:Cytochrome P-450 CYP11B2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004958:Estradiol / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D006929:Hyperaldosteronism / Q000235:genetics; D007700:Kinetics; D050506:Mutant Chimeric Proteins / Q000378:metabolism; D011374:Progesterone / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D050506:Mutant Chimeric Proteins; D000450:Aldosterone; D011374:Progesterone; D004958:Estradiol; D019405:Cytochrome P-450 CYP11B2