Nuclear Factor-inducing Kinase Plays a Crucial Role in Osteopontin-induced MAPK/IκBα Kinase-dependent Nuclear Factor κB-mediated Promatrix Metalloproteinase-9 Activation

Hema Rangaswami,Anuradha Bulbule,Gopal C Kundu

doi:10.1074/jbc.m404674200

Hema Rangaswami, Anuradha Bulbule + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m404674200

Copy DOI

Abstract

We have recently demonstrated that osteopontin (OPN) induces nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 activation through IkappaBalpha/IkappaBalpha kinase (IKK) signaling pathways. However, the molecular mechanism(s) by which OPN regulates promatrix metalloproteinase-9 (pro-MMP-9) activation, MMP-9-dependent cell motility, and tumor growth and the involvement of upstream kinases in regulation of these processes in murine melanoma cells are not well defined. Here we report that OPN induced alpha(v)beta(3) integrin-mediated phosphorylation and activation of nuclear factor-inducing kinase (NIK) and enhanced the interaction between phosphorylated NIK and IKKalpha/beta in B16F10 cells. Moreover, NIK was involved in OPN-induced phosphorylations of MEK-1 and ERK1/2 in these cells. OPN induced NIK-dependent NFkappaB activation through ERK/IKKalpha/beta-mediated pathways. Furthermore OPN enhanced NIK-regulated urokinase-type plasminogen activator (uPA) secretion, uPA-dependent pro-MMP-9 activation, cell motility, and tumor growth. Wild type NIK, IKKalpha/beta, and ERK1/2 enhanced and kinase-negative NIK (mut NIK), dominant negative IKKalpha/beta (dn IKKalpha/beta), and dn ERK1/2 suppressed the OPN-induced NFkappaB activation, uPA secretion, pro-MMP-9 activation, cell motility, and chemoinvasion. Pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibody drastically inhibited the OPN-induced cell migration and chemoinvasion, whereas cells pretreated with anti-MMP-2 antibody had no effect on OPN-induced pro-MMP-9 activation suggesting that OPN induces pro-MMP-2 and pro-MMP-9 activations through two distinct pathways. The level of active MMP-9 in the OPN-induced tumor was higher compared with control. To our knowledge, this is the first report that NIK plays a crucial role in OPN-induced NFkappaB activation, uPA secretion, and pro-MMP-9 activation through MAPK/IKKalpha/beta-mediated pathways, and all of these ultimately control the cell motility, invasiveness, and tumor growth.

Highlights

OPN Induces ␣v␤3 Integrin-dependent nuclear factor-inducing kinase (NIK) Phosphorylation—Because we have reported earlier that OPN induces nuclear factor ␬B (NF␬B)-mediated pro-MMP-2 activation through IKK/I␬B␣-mediated pathway [20], we first examined whether any upstream kinase(s) such as NIK plays any role in OPN-induced NF␬B activation in B16F10 cells
We investigated whether OPN regulates proMMP-9 activation and MMP-9-dependent cell motility, invasiveness, and tumor growth
We examined whether any upstream kinase such as NIK is involved in OPN-induced NF␬B activation, NF␬B-mediated urokinase-type plasminogen activator (uPA) secretion, pro-MMP-9 activation, and cell motility through activation of MAPK/IKK in B16F10 cells

Summary

EXPERIMENTAL PROCEDURES

Materials—The rabbit polyclonal anti-phospho-NIK (Thr-559), antiNIK, anti-IKK␣/␤, anti-NF␬B p65 X TransCruz, anti-p-MEK-1, antiMEK-1, anti-ERK1/2, anti-I␬B␣, anti-uPA, anti-MMP-9, and anti-actin and the mouse monoclonal anti-phospho-ERK1/2, anti-phospho-I␬B␣ antibodies, and I␬B␣ recombinant protein were purchased from Santa Cruz Biotechnology. The cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM iodoacetamide, and 2 mM phenylmethylsulfonyl fluoride), and the lysates containing an equal amount of total proteins were analyzed by Western blot with rabbit anti-phospho-NIK antibody. Negative staining showed the zones of gelatinolytic activity In another experiment, cells were transfected with wild type NIK, kinase-negative NIK, or the super-repressor form of I␬B␣ in the presence of LipofectAMINE Plus and treated with OPN for 24 h. Cells were individually transfected with wild type and kinase-negative NIK, wild type and dn IKK␣ and IKK␤, or the super-repressor form of I␬B␣ as described above and used for invasion assays. The levels of pro- and active MMP-9 in tumor samples were analyzed by zymography as described above

RESULTS

DISCUSSION

TABLE I OPN induces tumor growth in nude mice

No nude mice