Activation and Mitogen-Activated Protein Kinase Regulation of Transcription Factors Ets and NF- B in Mycobacterium-Infected Macrophages and Role of These Factors in Tumor Necrosis Factor Alpha and Nitric Oxide Synthase 2 Promoter Function

Previous studies have shown that primary murine macrophages infected with Mycobacterium avium produced lower levels of tumor necrosis factor alpha (TNF) and inducible nitric oxide synthase 2 (NOS2) compared to cells infected with nonpathogenic Mycobacterium smegmatis. TNFand NOS2 levels correlated with and were dependent on the activation of mitogen-activated protein kinases (MAPKs) p38 and extracellular signalregulated kinase 1/2 (ERK1/2). To define the macrophage transcriptional responses dependent on ERK1/2 activation following a mycobacterial infection, we used RAW 264.7 cells transfected with a TNFor NOS2 promoter vector. We determined that macrophages infected with M. avium compared to M. smegmatis showed diminished TNFand NOS2 promoter activity. A more pronounced difference in promoter activity was observed when only the consensus ETS and NFB binding sites were used as promoters. Mutational analysis of the ETS and NFB binding sites present on the TNFand NOS2 promoters, respectively, showed that these sites were essential for a functional promoter. Moreover, the Ets/Elk but not the NFB transcriptional response was dependent on ERK1/2. This correlated with the requirement for ERK1/2 in TNFbut not NOS2 promoter activity. Our data indicate that the increased Ets/Elk and NFB promoter activities associated with M. smegmatis-infected macrophages are responsible, at least in part, for the increased TNFand NOS2 production observed in these infected cells and that ERK1/2 is required for Ets/Elk activity and full TNFproduction.