Abstract
Purpose: :
Maspin, a non–classical serpin (serine protease inhibitor), regulates cell migration, adhesion and invasion. It is synthesized by normal epithelial cells and down regulated in invasive tumor epithelial cells. Normal human corneal stromal cells were the first non–epithelial cells shown to synthesize maspin. Like carcinoma cells, human corneal stromal fibroblasts down regulate the synthesis of maspin. However, these cells respond to maspin resulting in an increase in adhesion to extracellular matrix molecules and inhibition of migration. The mechanism of these maspin induced effects may involve alteration of the actin cytoskeleton. The current study explores whether maspin alters the actin cytoskeleton of corneal stromal cells, whether maspin is localized to the cytoplasm and whether maspin can interact with actin.
Methods: :
Human donor corneal stromal cells were converted to fibroblasts by treatment with FGF–2 or to myofibroblasts with TGF–ß. Corneal stromal cells, treated with and without maspin were stained with monoclonal antibodies to maspin and FITC–phalloidin, for maspin and F–actin, respectively. Maspin treated corneal stromal fibroblasts were lysed and the proteins separated by gradient centrifugation. The samples were analyzed for maspin by western blotting. Maspin binding molecules were identified on a ligand blot of cellular proteins followed by LC–ESI–MS–MS. Immuno–pull down assays were used to confirm complex formation.
Results: :
Actin stress fibers present in corneal stromal fibroblasts were decreased by maspin treatment but were not affected in stromal myofibroblasts. Maspin was taken up by the stromal fibroblasts and observed as punctate staining surrounding the nucleus but not associated with the stress–fibers. In addition, cell fractionation studies showed maspin is also associated with the membrane, cytoplasmic and the nuclear fractions. Maspin but not the homologous molecule, ovalbumin, bound to a 42 kDa band on ligand blots of the cytoplasmic fraction. This binding protein was identified as ß–actin by LC–ESI–MS–MS. The presence of maspin–ß–actin complexes was confirmed using an immuno–pull–down assay.
Conclusions: :
Alterations in the mobility of corneal stromal fibroblasts by maspin may involve changes in the actin cytoskeleton through direct interaction of maspin with actin within the cytoplasm inhibiting the formation of actin stress fibers.
Keywords: cornea: basic science • cornea: stroma and keratocytes • protein structure/function