Osteopontin Stimulates Cell Motility and Nuclear Factor κB-mediated Secretion of Urokinase Type Plasminogen Activator through Phosphatidylinositol 3-Kinase/Akt Signaling Pathways in Breast Cancer Cells

Riku Das,Ganapati H Mahabeleshwar,Gopal C Kundu

doi:10.1074/jbc.m303445200

Riku Das, Ganapati H Mahabeleshwar + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m303445200

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Abstract

We have recently reported that osteopontin (OPN) induces nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 activation through IkappaBalpha/IKK signaling pathways and that curcumin (diferulolylmethane) down-regulates these pathways (Philip, S., and Kundu, G. C. (2003) J. Biol. Chem. 278, 14487-14497). However, the molecular mechanism by which upstream kinases regulate the OPN-induced NFkappaB activation and urokinase type plasminogen activator (uPA) secretion in human breast cancer cells is not well defined. Here we report that OPN induces the phosphatidylinositol 3'-kinase (PI 3'-kinase) activity and phosphorylation of Akt in highly invasive MDA-MB-231 and low invasive MCF-7 cells. The OPN-induced Akt phosphorylation was inhibited when cells were transfected with a dominant negative mutant of the p85 domain of PI 3-kinase (Deltap85) and enhanced when cells were transfected with an activated form of PI 3-kinase (p110CAAX), indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IkappaBalpha kinase (IKK) and phosphorylated Akt. OPN also induces NFkappaB activation through phosphorylation and degradation of IkappaBalpha by inducing the IKK activity. However, both pharmacological (wortmannin and LY294002) and genetic (Deltap85) inhibitors of PI 3'-kinase inhibited OPN-induced Akt phosphorylation, IKK activity, and NFkappaB activation through phosphorylation and degradation of IkappaBalpha. OPN also enhances uPA secretion, cell motility, and extracellular matrix invasion. Furthermore, cells transfected with Deltap85 or the super-repressor form of IkappaBalpha suppressed the OPN-induced uPA secretion and cell motility, whereas cells transfected with p110CAAX enhanced these effects. Pretreatment of cells with PI 3-kinase inhibitors or NFkappaB inhibitory peptide (SN-50) reduced the OPN-induced uPA secretion, cell motility, and invasion. To our knowledge, this is first report that OPN induces NFkappaB activity and uPA secretion by activating PI 3'-kinase/Akt/IKK-mediated signaling pathways and further demonstrates a functional molecular link between OPN-induced PI 3'-kinase-dependent Akt phosphorylation and NFkappaB-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.

Highlights

Cell migration and extracellular matrix invasion are two of the major steps in embryonic development [1, 2] and wound healing and cancer cell metastasis [3, 4]
The results indicated that OPN induces the cell migration in a dose-dependent manner in both these cells (Fig. 8, A and B). ␣v␤3 integrin antibody, GRGDSP, LY294002, wortmannin, SN-50, and anti-urokinase type plasminogen activator (uPA) antibody suppressed the OPN-induced cell migration in MCF-7 cells (Figs. 8C and 9A) and MDA-MB-231 cells (Figs. 8D and 9B)
The results showed that OPN induced extracellular matrix (ECM) invasion and that ␣v␤3 integrin antibody, LY294002, wortmannin, and SN-50 suppressed the OPN-induced ECM invasion in MCF-7 cells (Fig. 10A) and MDA-MB231 cells (Fig. 10B)

Summary

Introduction

Cell migration and extracellular matrix invasion are two of the major steps in embryonic development [1, 2] and wound healing and cancer cell metastasis [3, 4]. To ascertain the role of OPN on Akt phosphorylation, both these cells were treated with 5 ␮M OPN for 0 –90 min at 37 °C, the cell lysates containing equal amounts of total proteins were resolved by SDS-PAGE, and the level of phosphorylated Akt was detected by Western blot analysis using anti-phospho-Akt antibody (Ser-473).

Results

Conclusion