The transcription factor C/EBPbeta is essential for inducible expression of the cox-2 gene in macrophages but not in fibroblasts.

Barbara Gorgoni,Matilde Caivano,Carmen Arizmendi,Valeria Poli

doi:10.1074/jbc.m106865200

Abstract

Cyclooxygenase-2 (COX-2) is the rate-limiting enzyme for the inducible synthesis of prostaglandins, and its up-regulated activity is thought to play a pathological role in diseases such as inflammatory bowel disease, rheumatoid arthritis, and cancer. Regulation of COX-2 expression is complex and appears to involve diversified mechanisms in different cell types and conditions. Here we make use of immortalized macrophages and fibroblasts that we have generated from C/EBPbeta-deficient mice to directly test and compare the specific role played by this factor in inducible COX-2 expression in these two cell types. We could demonstrate that COX-2 mRNA induction and promoter activity were profoundly impaired in C/EBPbeta(-/-) macrophages and could be rescued by expression of C/EBPbeta. The obligatory role of C/EBPbeta in COX-2 expression appeared to be mediated exclusively by the C/EBP element located at positions -138/-130 of the murine cox-2 promoter, and did not involve altered activity at the level of the other promoter elements described previously (the -402/-392 NF-kappaB site, the -59/-48 CRE/E box element, and a potential second C/EBP site located at positions -93/-85). In contrast, COX-2 induction was completely normal in C/EBPbeta-deficient fibroblasts, thus highlighting the diversity of cell-specific molecular mechanisms in determining inducible COX-2 expression and prostaglandins production.

Highlights

Role in diseases such as inflammatory bowel disease, rheumatoid arthritis, and cancer
We make use of immortalized macrophages and fibroblasts that we have generated from C/EBP␤-deficient mice to directly test and compare the specific role played by this factor in inducible COX-2 expression in these two cell types
COX-2 induction was completely normal in C/EBP␤-deficient fibroblasts, highlighting the diversity of cell-specific molecular mechanisms in determining inducible COX-2 expression and prostaglandins production

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Treatments—The generation of C/EBP␤Ϫ/Ϫ- and C/EBP␤ϩ/ϩ-immortalized macrophages is described elsewhere. The cells were maintained at 37 °C in a 5% CO2 atmosphere in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and containing 2 mM L-glutamine, 100 units/ml penicillin, and 100 ␮g/ml streptomycin (standard medium). Bone marrow macrophages were treated with 100 units/ml IFN␥ for 16 h, followed by 1 ␮g/ml of LPS for 4 h prior to RNA extraction. 2 ␮l of polyclonal purified antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was incubated with nuclear extracts and poly(dI-dC) for 30 min on ice, prior to probe addition. For transient transfection of immortalized macrophages, 107 cells were resuspended in 250 ␮l of RPMI 1640 supplemented with 20% fetal calf serum, together with 5 ␮g of the indicated firefly luciferase reporter plasmid and 1 ␮g of the internal control pRL-TK, encoding Renilla luciferase (Promega). And Renilla luciferase values were obtained by analyzing 20 ␮l of cell extract using the Dual Luciferase kit (Promega), according to manufacturer’s instructions, in a Lumat LB 9507 luminometer. Since C/EBP␤ activated the control pRL-TK plasmid, luciferase activity was normalized to protein content as measured by Bradford assay when C/EBP␤ was co-transfected

RESULTS

Findings

DISCUSSION