[Frontiers in Bioscience E3, 1249-1258, June 1, 2011]

Novel molecular mechanisms by inorganic phosphate in osteosarcoma U2OS cells

Silvio Naviglio, Davide Di Gesto, Vittoria Borrelli, Mafalda Forni, Fausto Illiano, Raffaella D'Auria, Annunziata Sorrentino, Emilio Chiosi, Gennaro Illiano, Annamaria Spina

Department of Biochemistry and Biophysics, Second University of Naples, Medical School, Via L. De Crecchio 7, 80138 Naples, Italy

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Materials
3.2. Cell culture and treatments
3.3. Cell adhesion assay
3.4. Cell proliferation assay
3.5. Rap1 activation assay and phosphorylation of ERK and CREB
3.6. Preparation of cell lysates
3.7. Immunodetection of proteins
3.8. Statistical analysis
4. Results
4.1. Inorganic phosphate inhibits the trypsin-induced detachment of U2OS cells
4.2. Inorganic phosphate affects integrin expression and enhances cell adhesion
4.3. Inorganic phosphate affects multiple signalling pathways and mostly up-regulates the cAMP/Epac/Rap1 axis.
4.4. cAMP/Epac/Rap1 up-regulation is relevantly involved in the Pi-induced enhancement of cell adhesion
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

Osteosarcoma is the most common malignant primary bone tumor in children and adolescents and is characterized by a high metastatic potential. Its clinical outcome remains discouraging despite aggressive treatments. Thus, novel therapeutic approaches are needed. Recent results indicate that inorganic phosphate (Pi) is capable of affecting specific signal transduction pathways and of acting as an active regulator of cell behaviour. Previously, we found that Pi inhibits proliferation of human osteosarcoma U2OS cells via an adenylate cyclase/cAMP mediated mechanism. Here, we report that upon Pi treatment, U2OS cells become extremely hard to dislodge with trypsin. The lack of sensitivity to the trypsin action was paralleled by relevant changes in integrin subunits expression and accompanied by an increase of cell adhesion in cell-matrix adhesion assays. Interestingly, exposure of U2OS cells to Pi results also in a strong activation and protein level up-regulation of Rap1 small GTPase and in an early increase followed by a sustained inhibition of Erk1/2 phosphorylation. Importantly, the Pi-induced increase of cell adhesion was enforced by a cAMP analogue which specifically activated Epac/Rap1 and insensitive to PKA and MEK1/2 inhibitors. Our results enforce the evidences of inorganic phosphate as a signalling molecule, identify beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected by Pi in osteosarcoma U2OS cells The clinical significance and potential therapeutic applications by our data will be discussed.