Integrin-linked kinase is involved in lactoferrin-induced anchorage-independent cell growth and survival in PC12 cells.

AIMS: Bovine lactoferrin (bLf) causes anchorage-independent cell growth in PC12 cells. The present study investigated the mechanisms involved in bLf-induced anchorage-independent cell growth and survival in PC12 cells. MAIN METHODS: The number of adherent cells and suspended cells was estimated separately by using a methyl thiazol tetrazolium (MTT) assay, and the sum of both optical density (O.D.) (570 nm) values was used as a measure of the total number of cells. KEY FINDINGS: Integrin-linked kinase (ILK) plays an important role in integrin and growth factor signaling pathways. Stable transfection of PC12 cells with a dominant negative kinase-deficient mutant of ILK (DN-ILK) inhibited bLf-induced anchorage-independent cell growth. The ILK activity in the parental cells was transiently activated after addition of bLf, whereas bLf-induced activation of ILK was blocked in DN-ILK-transfected cells. bLf also activated p38 mitogen-activated protein kinase (MAPK); however, the p38 MAPK activation was inhibited by stable DN-ILK transfection. Moreover, cell viability in the suspended cells by bLf strongly decreased after treatment with SB203580, an inhibitor of p38 MAPK. SIGNIFICANCE: These results suggest that ILK is involved in bLf-induced anchorage-independent cell growth and viability via activation of p38 MAPK.

Bovine lactoferrin stimulates anchorage-independent cell growth via membrane-associated chondroitin sulfate and heparan sulfate proteoglycans in PC12 cells.

Bovine lactoferrin (bLf) is an iron-binding secretory protein present in breast milk, mucosal secretions, and the secondary granules of neutrophils. Although bLf has multiple functions, including antimicrobial and immunomodulatory activities, its effect on neuronal cells is not fully understood. We report that bLf prevents cell adhesion of PC12 cells and allows them to be cultivated in suspension. PC12 cells normally adhere well to plastic culture plates and show anchorage-dependent cell growth, but we found that soon after adding bLf, they detach from culture plates and begin to grow in suspension. When bLf was removed from the medium, the cells began to re-adhere to the plates. Thus, bLf inhibits cell adhesion and stimulates anchorage-independent growth in PC12 cells. On the other hand, bLf-induced cell suspension growth was not observed when cells were grown on a laminin matrix, suggesting that bLf does not affect integrin-mediated cell adhesion on a laminin matrix. Treatment of cells with heparin or chondroitin sulfate A or C inhibited bLf-induced growth in cell suspension. Furthermore, pretreatment of cells with heparinase and/or chondroitinase prevented direct binding of bLf to the cell membrane. These results suggest that bLf binds to the membrane of PC12 cells via membrane-associated proteoglycans and leads to anchorage-independent growth.