Insulin-like Growth Factor I Prevents Mannitol-induced Degradation of Focal Adhesion Kinase and Akt

Bhumsoo Kim,Eva L Feldman

doi:10.1074/jbc.m201963200

Bhumsoo Kim, Eva L Feldman

Open Access

https://doi.org/10.1074/jbc.m201963200

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Abstract

In our laboratory, we are interested in hyperosmolarity-induced apoptosis in neuronal cells. We have shown that high concentrations of glucose or mannitol induce apoptotic cell death in dorsal root ganglia in culture and in SH-SY5Y and SH-EP human neuroblastoma cells. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that has a critical role for transmitting integrin-mediated-signals. In this study, we report that hyperosmolar treatment mediates FAK dephosphorylation and cleavage, which is prevented by insulin-like growth factor I (IGF-I) treatment. Mannitol treatment of SH-EP cells transfected with vector (SH-EP/pSFFV) results in concentration- and time-dependent dephosphorylation and degradation of FAK. Dephosphorylation and degradation of FAK are tightly correlated with apoptotic morphological changes, including the disruption of actin stress fibers, the loss of focal adhesion sites, membrane blebbing, and cell detachment. Treatment of SH-EP/pSFFV cells with IGF-I or transfection of IGF-I receptor prevents these changes. Treatment of cells with pharmacologic inhibitors of the mitogen-activated protein kinase or phosphatidylinositol 3-kinase pathways does not affect mannitol-induced FAK dephosphorylation and degradation. However, phosphatidylinositol 3-kinase is necessary for IGF-I-mediated protection against FAK alteration. Mannitol treatment also results in the degradation of Akt. Mannitol induces the activation of caspases-3 and -9 in a time course similar to the dephosphorylation and degradation of FAK. Treatment of the cells with ZVAD, a general caspase inhibitor, blocks the mannitol-induced FAK and Akt degradation as well as cell detachment and apoptosis. These results suggest that one of the pathways of mannitol-mediated apoptosis is through the degradation of FAK and Akt and that IGF-I protects the cells from apoptosis by blocking the activation of caspases, which may be responsible for the loss of FAK and Akt.

Highlights

Less than 15% of IGF-I receptor (IGF-IR)-transfected SH-EP (SH-EP/IGF-IR) cells detach from the dishes under serum-free conditions (Fig. 1A). When both SH-EP/pSFFV and SH-EP/IGF-IR cells are stained with Texas Red-conjugated phalloidin, well-organized actin stress fibers are seen in attached cells
The cells start to detach, significant numbers of SH-EP/IGF-IR cells still adhere to the dishes after 4 h of mannitol treatment (Ͻ40% detached cells) (Fig. 1B)
The current study investigates the effects of hyperosmotic stress induced by mannitol on cytoskeletal changes and Focal adhesion kinase (FAK) and Akt preceding apoptosis

Summary

Introduction

Dephosphorylation and degradation of FAK are tightly correlated with apoptotic morphological changes, including the disruption of actin stress fibers, the loss of focal adhesion sites, membrane blebbing, and cell detachment. Treatment of the cells with ZVAD, a general caspase inhibitor, blocks the mannitol-induced FAK and Akt degradation as well as cell detachment and apoptosis. Because our results suggest an anoikis-like cell death induced by mannitol treatment, we investigated changes in FAK in SH-EP/pSFFV and SH-EP/ IGF-IR cells.

Results

Conclusion