FOXO1 regulates early cytoskeletal dynamics in human gingival fibroblasts

Objectives: Gingival connective tissue healing involves changes in cell adhesion, migration and the differentiation of a transient population of collagen-producing cells known as myofibroblasts. FOXO1 is a transcription factor that actively participates in the wound healing process. However, its precise role in the regulation of gingival fibroblasts is still poorly understood. The aim of this study was to identify the role of FOXO1 on the adhesion, size and myofibroblastic differentiation in gingival fibroblasts.

Methods: Primary cultures of human gingival fibroblasts were obtained from healthy young donors.
Myofibroblastic differentiation and cell size were analyzed in the presence or absence of a pharmacological FOXO1 inhibitor and/or Transforming Growth Factor-beta 1. Changes in cell size and actin distribution were studied at different times by staining cells with phalloidin and DAPI. FOXO1 and Alpha Smooth Muscle Actin protein levels and distribution were analyzed through immunofluorescence and Western-blot. Cell adhesion over a collagen substrate was assessed through anti-vinculin and anti-integrin beta 1 staining. Images were analyzed using ImageJ.

Results: Myofibroblastic differentiation was characterized by an increase in cell size and in the protein levels of Alpha Smooth Muscle Actin. Interestingly, the FOXO1 inhibitor significantly reduced these two responses. Moreover, FOXO1 inhibitor diminished the adhesion and spreading of fibroblasts at 24h and 48h.

Conclusions: The present study reveals an important role for the transcription factor FOXO1 through the modulation of cytoskeletal changes in gingival fibroblasts. These responses may have an impact in connective tissue wound healing in oral tissues.
Funding: FONDECYT 1170555 (PS).