Rho/ROCK signaling and α-catenin mediate β-catenin–driven hyperplasia in the adrenal cortex via adherens junctions
Mesut Berber, Betul Haykir, Nick A. Guagliardo, Vasileios Chortis, Kleiton Silva Borges, Paula Q. Barrett, Felix Beuschlein, Diana L. Carlone, David T. Breault
Mesut Berber, Betul Haykir, Nick A. Guagliardo, Vasileios Chortis, Kleiton Silva Borges, Paula Q. Barrett, Felix Beuschlein, Diana L. Carlone, David T. Breault
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Research Article Cardiology Development Endocrinology

Rho/ROCK signaling and α-catenin mediate β-catenin–driven hyperplasia in the adrenal cortex via adherens junctions

Abstract

How β-catenin (βCat) mediates tissue hyperplasia is poorly understood. To explore this, we employed the adrenal cortex as a model system given its stereotypical spatial organization and the important role βCat plays in homeostasis and disease. For example, excessive production of aldosterone by the adrenal cortex (primary aldosteronism [PA]) is a major cause of cardiovascular morbidity and is associated with βCat gain of function (βCat-GOF). Adherens junctions (AJs) connect the actin cytoskeletons of adjacent zona glomerulosa (zG) cells via a cadherin–βCat–α-catenin complex and mediate aldosterone production. Whether βCat-GOF drives zG hyperplasia, a key feature of PA, via AJs is unknown. Here, we showed that aldosterone secretagogues (K+ and AngII) and βCat-GOF mediated AJ formation via Rho/ROCK/actomyosin signaling. In addition, Rho/ROCK inhibition led to altered zG rosette morphology and decreased aldosterone production. Mice with zG-specific βCat-GOF demonstrated increased AJ formation and zG hyperplasia, which was blunted by Rho/ROCK inhibition and deletion of α-catenin (αCat). βCat also impacted AJ formation independently of its role as a transcription factor. Furthermore, analysis of human aldosterone-producing adenomas revealed high levels of βCat expression were associated with increased membranous expression of K-cadherin. Together, our findings identified Rho/ROCK signaling and αCat as key mediators of AJ formation and βCat-driven hyperplasia.

Authors

Mesut Berber, Betul Haykir, Nick A. Guagliardo, Vasileios Chortis, Kleiton Silva Borges, Paula Q. Barrett, Felix Beuschlein, Diana L. Carlone, David T. Breault

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Figure 1

Aldosterone secretagogues increase AJ formation via the Rho/ROCK/NMII pathway.

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Aldosterone secretagogues increase AJ formation via the Rho/ROCK/NMII pa...
(A) Schematic illustrating AJ regulation by the K+/AngII-mediated Ca2+/ROCK/NMII pathway. Red arrow denotes AJ formation and stabilization. Nif, nifedipine; Fas, fasudil; Bleb, blebbistatin. (B) αCat co-IP in NCI-H295R cells treated with vehicle (11 mM NaCl), K+ (11 mM KCl), or AngII (100 nM) for 48 hours. KCad and αCat immunoblots. Input: whole-cell lysates. β-Actin: loading control. (C) Densitometric quantification of KCad/αCat co-IP (n = 3 independent experiments). (D) Representative image of αCat (green) and KCad (magenta) immunofluorescence in NCI-H295R cells stimulated with vehicle (11 mM NaCl), K+ (11 mM KCl) ± nifedipine (10 μM), fasudil (10 μM), or blebbistatin (10 μM) for 48 hours (1 hour preincubation with inhibitors or DMSO). Low-power images are in Supplemental Figure 2A. (E) Schematic of qLPA methodology. (F) qLPA of αCat and KCad fluorescence intensities represented in D presented as mean ± SEM (n = 90 cell–cell interfaces pooled from 3 independent experiments). (G) Average peak fluorescence intensity per experiment for KCad and αCat from D and F (n = 3). (H) RhoA activity in NCI-H295R cells treated with vehicle, K+, or AngII for 3 hours was assessed by pull-down assay. Immunoblots show active RhoA (RhoA-GTP) in precipitates (top), total RhoA in lysates (middle), and β-actin in lysates (bottom). Statistical significance determined by unpaired 2-tailed Student’s t test in C and 1-way ANOVA with Tukey’s multiple-comparison post test in G. *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM. Scale bar: 20 μm.