Endocrine Abstracts (2017) 49 EP37 | DOI: 10.1530/endoabs.49.EP37

Visinin-Like Protein-1 in the regulation of aldosterone biosynthesis

Celso Gomez-Sanchez1, Maniselvan Kuppusamy1, Jeanne Ishimwe1, Tracy Williams2, Paolo Mulatero2 & Elise Gomez-Sanchez1

1University of Mississippi Med Center, Jackson, MS, USA; 2University of Turin, Turin, Italy.

Visinin-Like Protein-1 (Vsnl1) is a member of the EF-hand calcium sensor protein family that is expressed in the zona glomerulosa of the rat and human adrenal and upregulated in aldosterone-producing adenomas.

We studied the expression pattern of Vsnl1 and co-localization with the CYP11B2 enzyme and the zona glomerulosa (ZG) marker Dlk1 (ZOG) using double and triple immunofluorescence in the adrenal of rats on a normal, high and low sodium diets. We also studied the role Vsnl1 in aldosterone biosynthesis and calcium in the HAC15 cell.

In the rat adrenal, Vsnl1 was expressed only in cells of the ZG and in cells expressing Dlk1, including undifferentiated cells between the ZG and zona fasciculata. Nearly all of the ZG cells from adrenals of rats on a low sodium diet expressed both Vsnl1 and CYP11B2. However in adrenals of rats on a normal and high sodium diet the staining for Vsnl1 was in many more ZG cells than those expressing CYP11B2. Vsnl1 and Dlk1 immunoreactivity was found not only cells that are steroidogenically active and producing aldosterone, but also in ZG and undifferentiated cells in the rat adrenal subcapsular area.

Human adrenal cortical carcinoma cell line HAC15 was transduced with a lentivirus carrying Vsnl1. Three days after transduction cells were incubated with vehicle, angiotensin II (A-II) 10 nM, Forskolin 10 μM and potassium 16 mM and compared with control cells transduced with an empty lentivirus. Aldosterone and cortisol were measured after 24 h. The basal secretion of aldosterone was not increased, but that of cortisol was. Aldosterone and cortisol secretion were greater after A-II, forskolin and potassium stimulation in cells overexpressing Vsnl1. Proliferation of the HAC15 cells measured by the XXT and crystal violet methods and intracellular calcium, as measured by the Fluo-4 AM dye, were also increased after transduction with Vsnl1.

Transduction of HAC15 cells with shRNA for Vsnl1 that reduced Vsnl1 expression decreased their basal and A-II stimulated aldosterone synthesis. The Vsnl1 has a myristoylation consensus sequence at the N-terminal domain. Site directed mutagenesis of Vsnl1 at the 2 position (G2A) lentivirus transduced in HAC15 cells resulted in a significant decrease in the aldosterone response to A-II.

In summary, the Vsnl1 is a zona glomerulosa protein that participates in calcium mobilization and increases the various secretagogue-induced aldosterone synthesis.

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