Effect of HNE on signaling pathways. (a, b) Osteoblasts were treated with 20 μM HNE for the indicated times in the presence or absence of 1 ng/ml TNF-α. Total cell lysates or nuclear extracts (approximately 50 μg) were prepared and subjected to Western analysis with anti-phosphospecific antibodies anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-ERK1/2, anti-phospho-ATF-2 and anti-phospho-CREB-1, and anti-NF-κB/p65. (c, d) Osteoblasts were incubated in absence (control) or presence of 1 ng/ml TNF-α, 20 μM HNE, or 20 μM HNE combined with 1 ng/ml TNF-α in serum-free medium for 1 hour. Nuclear extracts were prepared and subjected to electrophoretic mobility shift assay using ATF/CRE (c) and NF-κB (d) oligonucleotide probes. Specificity of the binding was assayed by competition (comp) of the oligonucleotide with 50-fold of excess unlabeled ATF/CRE or NF-κB oligonucleotide or by the adding specific antibodies anti-ATF-2, anti-p50, or anti-p65. Arrows refer to specific DNA–protein complex. Data are representative of three to five independent expriments. ATF-2, activating transcription factor-2; CREB-1, CRE-binding factor-1; ERK, extracellular signal-regulated kinase; HNE, 4-hydroxynonenal; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; TNF-α, tumour necrosis factor-α.