JunB/AP-1 and NF-κB-mediated induction of nitric oxide synthase by bovine type I collagen in serum-stimulated murine macrophages

Type I collagen comprises the majority of the total body collagens. In particular, bovine type I collagen is utilized for medical purposes and used widely in a variety of cell culture models as a fibrous component of extracellular matrix. This study was designed to explore the effects of type I collagen on the expression of inducible nitric oxide synthase (iNOS) in serum-stimulated Raw264.7 cells and to study the molecular mechanistic basis. Bovine, but not rat or murine, type I collagen increased NO production in serum-stimulated cells, which resulted from the induction of iNOS, as monitored by Northern and Western blot analyses. Bovine type I collagen in combination with serum activated JunB and JunB/AP-1 transcription complex, as evidenced by supershift and immunodepletion of the retarded AP-1 band with anti-JunB antibody. AP-1 complex was immunodepleted in part by anti-c-Jun or anti-JunD antibody. Extracellular signal-regulated kinase1/2 (ERK1/2), p38 kinase, and c-Jun N-terminal kinase (JNK) were all activated by bovine type I collagen in serum-stimulated cells. PD98059, but not SB203580 or JNK1(-) transfection, inhibited both ERK1/2 phosphorylation and JunB/AP-1 activation. Either PD98059 or MKK1(-) transfection suppressed the iNOS induction. The induction of iNOS accompanied activation of NF-kappaB with degradation of I-kappaBalpha. AP-1 and/or NF-kappaB decoy oligonucleotides and pyrrolidine dithiocarbamate suppressed the iNOS induction, which confirmed involvement of AP-1 and NF-kappaB as transcription factors. These results demonstrated that bovine type I collagen induces iNOS in serum-stimulated murine macrophages through JunB/AP-1 and NF-kappaB activation and that activation of ERK1/2 plays an essential role in JunB/AP-1 activation. (C) 2002 Elsevier Science (USA).