ERK1/2 and JNKs, but not p38 kinase, are involved in reactive oxygen species‐mediated induction of osteopontin gene expression by angiotensin II and interleukin‐1β in adult rat cardiac fibroblasts

Abstract

Osteopontin (OPN), also called cytokine Eta-1, expressed in the myocardium co-incident with heart failure plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Angiotensin II (Ang II) and inflammatory cytokines are increased in the heart following MI. We studied the involvement of mitogen-activated protein kinases (ERK1/2, JNKs, p38 kinase) and reactive oxygen species (ROS) in Ang II- and cytokine-induced OPN gene expression in adult rat cardiac fibroblasts. Ang II alone increased OPN mRNA (3.3 +/- 0.3-folds; P < 0.05; n = 7), while interleukin-1beta (IL-1beta), tumor necrosis factor (TNF-alpha), and interferon-gamma (IFN-gamma) had no effect. A combination of Ang II with IL-1beta or TNF-alpha, not IFN-gamma, increased OPN mRNA more than Ang II alone. Nitric oxide donor, S-nitrosoacetylpenicillamine (SNAP), alone or in combination with Ang II had no effect. Diphenylene iodonium (DPI), inhibitor of NAD(P)H oxidase, and tiron, superoxide scavenger, inhibited Ang II- and Ang II+ IL-1beta-stimulated increases in OPN mRNA. Ang II activated ERK1/2 within 5 min of treatment, not JNKs. IL-1beta activated ERK1/2 and JNKs within 15 min of treatment. A combination of Ang II and IL-1beta activated ERK1/2 within 5 min of treatment. None of these stimuli activated p38 kinase. DPI almost completely inhibited Ang II + IL-1beta-stimulated activation of ERK1/2, while partially inhibiting JNKs. PD98059, ERK1/2 pathway inhibitor, and SP600125, JNKs inhibitor, partially inhibited Ang II + IL-1beta-stimulated increases in OPN mRNA. A combination of PD98059 and SP600125 almost completely inhibited Ang II + IL-1beta-stimulated increases in OPN mRNA. Thus, Ang II alone increases OPN expression, while IL-1beta and TNF-alpha act synergistically with Ang II to increase OPN mRNA possibly via NO independent mechanisms. The synergistic increase in OPN mRNA involves ROS-mediated activation of ERK1/2 and JNKs, not P38 kinase, pathways in cardiac fibroblasts.

Differential Regulation of Matrix Metalloproteinase-2 and -9 Expression and Activity in Adult Rat Cardiac Fibroblasts in Response to Interleukin-1β

“…SP600125 from 2-20 M concentration had no effect on IL-1␤-stimulated activation of ERK1/2 (18). To explore the possibility of whether a combination of PD98059 and SP600125 could be effective in the inhibition of MMP-2 activity and protein expression, cells were pretreated with PD98059 ϩ SP600125 (PD ϩ SP) followed by treatment with IL-1␤ for 48 h. In-gel zymography and Western blot analyses indicated that inhibition of both ERK1/2 and JNKs together significantly inhibits IL-1␤-stimulated MMP-9 activity and protein levels but has no significant effect on MMP-2 (Fig.…”

“…IL-1␤ activates ERK1/2 and JNKs, not p38 kinase, in adult rat cardiac fibroblasts (18). Recently, we found that IL-1␤ activates PKC and that activation of PKC plays a critical role in the regulation of MMP-2 and -9 expression and activity (12).…”

“…Western Blot Analyses-Activation of MAPKs (ERK1/2 and JNKs), IKK␣/␤, and I B-␣ was studied using Western blot analyses as described (18). Cells lysates were prepared using lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, and 0.5% Nonidet P-40), and protein contents were measured using the Bradford assay (Bio-Rad).…”

“…The membranes were then probed with phospho-specific ERK1/2, JNKs, c-Jun, IKK␣/␤, or I B-␣ antibodies. The membranes were then incubated with appropriate secondary antibody and analyzed as described (18). The protein loading of each sample was verified by staining the membrane with 0.1% Ponceau S solution.…”

“…Recently, we have shown that IL-1␤ activates ERK1/2 and JNKs, but not p38, in cardiac fibroblasts (18). To determine whether activation of ERK1/2 and JNKs plays a role in the regulation of MMP expression and activity, cells were pretreated with PD98059 (PD, a selective inhibitor of ERK1/2 pathway, 10 M) or SP600125 (SP, a selective inhibitor of JNKs, 10 M) for 30 min followed by treatment with IL-1␤ for 48 h. Analysis of conditioned media demonstrated that PD98059 significantly inhibits MMP-9 activity (-fold increase versus control; IL-1␤, 3.7 Ϯ 0.04; IL-1␤ ϩ PD, 1.9 Ϯ 0.2, p Ͻ 0.001; n ϭ 4; Fig.…”