The role of Hippo pathway signaling and A-kinase anchoring protein 13 in primordial follicle activation and inhibition

Objective: To determine whether the mechanotransduction and pharmacomanipulation of A-kinase anchoring protein 13 (AKAP13) altered Hippo signaling pathway transcription and growth factors in granulosa cells. Primary ovarian insufficiency is the depletion or dysfunction of primordial ovarian follicles. In vitro activation of ovarian tissue in patients with primary ovarian insufficiency alters the Hippo and phosphatase and tensin homolog/phosphatidylinositol 3-kinase/protein kinase B/forkhead box O3 pathways. A-kinase anchoring protein 13 is found in granulosa cells and may regulate the Hippo pathway via F-actin polymerization resulting in altered nuclear yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif coactivators and Tea domain family (TEAD) transcription factors.

Design: Laboratory studies.

Setting: Translational science laboratory.

Patient(s): None.

Intervention(s): COV434 cells, derived from a primary human granulosa tumor cell line, were studied under different cell density and well stiffness conditions. Cells were transfected with a TEAD-luciferase (TEAD-luc) reporter as well as expression constructs for AKAP13 or AKAP13 mutants and then treated with AKAP13 activators, inhibitors, and follicle-stimulating hormone.

Main outcome measure(s): TEAD gene activation or inhibition was measured by TEAD-luciferase assays. The messenger ribonucleic acid levels of Hippo pathway signaling molecules, including connective tissue growth factor (CTGF), baculoviral inhibitors of apoptosis repeat-containing 5, Ankyrin repeat domain-containing protein 1, YAP1, and TEAD1, were measured by quantitative real-time polymerase chain reaction. Protein expressions for AKAP13, CTGF, YAP1, and TEAD1 were measured using Western blot.

Result(s): Increased TEAD-luciferase activity and expression of markers for cellular growth were associated with decreased cell density, increased well stiffness, and AKAP13 activator (A02) treatment. Additionally, decreased TEAD-luc activity and expression of markers for cellular growth were associated with AKAP13 inhibitor (A13) treatment, including a reduced expression of the BIRC5 and ANKRD1 (YAP-responsive genes) transcript levels and CTGF protein levels. There were no changes in TEAD-luc with follicle-stimulating hormone treatment, supporting Hippo pathway involvement in the gonadotropin-independent portion of folliculogenesis.

Conclusion(s): These findings suggest that AKAP13 mediates Hippo-regulated changes in granulosa cell growth via mechanotransduction and pharmacomanipulation. The AKAP13 regulation of the Hippo pathway may represent a potential target for regulation of follicle activation.