Filamin A in the ovary: a mediator of granulosa cell functions.

ABSTRACT

Filamin A (FLNA) regulates the mechanical properties and shape of cells by cross-linking actin filaments orthogonally. It also serves as a scaffold for numerous interacting proteins, thereby coordinating cell differentiation and morphogenesis. The role of FLNA in the human ovary is unknown. Immunohistochemistry indicated its expression in granulosa cells (GC) and oocytes in human and nonhuman primate ovaries. Studies in cultured human GC and KGN granulosa tumor cells showed that FLNA colocalizes with actin filaments, as expected. Forskolin decreased FLNA transcript and protein levels, and caused a loss of filamentous FLNA and actin staining. In GC, this was accompanied by a pronounced change in cell shape and a massive mRNA increase in steroidogenic enzymes. Further putative interaction partners of FLNA were identified by immunoprecipitation followed by mass spectrometry. Cytoskeleton-associated proteins (e.g. FLNB and plectin), but also unexpected proteins (e.g. major vault protein and mitochondrial stress-70 protein) were found in both cellular models, while e.g. cholesterol monooxygenase and gap junction α-1 were exclusively enriched in GC. Immunofluorescence revealed that plectin, one of the interaction partners identified in KGN cells, colocalized with FLNA. A siRNA-mediated knockdown of FLNA in KGN cells led to an increase in cell size, supporting a role in the regulation of the cytoskeleton. These studies demonstrate the widespread expression of FLNA in human ovarian cells in situ, provide insight into its regulation, and identify its potential interaction partners. Our data indicate that FLNA has specific roles in GC in regulating cytoskeletal activities including cell size and steroidogenic competence.

LAY SUMMARY

The ovary produces hormones and egg cells. Eggs are enclosed in cellular spheres, called follicles, and are nursed by the cells around them (granulosa cells). Follicles increase in size and then acquire the ability to produce hormones. How this is regulated in an orderly way in women is not fully known and involves the interaction of many players. We describe an as yet unknown player, filamin A. While many roles for filamin A have been described in other organs, such roles in the ovary were unknown. We therefore studied isolated human granulosa cells and granulosa tumor cells. We examined how filamin A is regulated and found that filamin A has specific roles in granulosa cell maturation and initiation of their hormone-producing function.