Activation and mitogen-activated protein kinase regulation of transcription factors Ets and NF-kappaB in Mycobacterium-infected macrophages and role of these factors in tumor necrosis factor alpha and nitric oxide synthase 2 promoter function

Abstract

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

FIG. 1.

RAW 264.7 cells infected with M. avium 724 show decreased production of TNF-α and NOS2 and a corresponding decrease in promoter activities compared to macrophages infected with M. smegmatis. (A) RAW 264.7 cells were infected with mycobacteria as described in Materials and Methods. Supernatants were collected from cells infected for 1, 4, 9, or 24 h and used for TNF-α ELISA. (B and D) RAW 264.7 cells, transfected with either Basic-pGL3-luc, −1200 TNF-α-pGL3-luc, or −1700 NOS2-pGL3-luc, in combination with phRL-SV40, were infected with mycobacteria. Twenty-four hours postinfection, cell extracts were assayed for luciferase activity. Results represent firefly luciferase activities relative to Renilla luciferase activities and were normalized to those of cells transfected with Basic-pGL3-luc. (C) NOS2 expression from cell lysates was detected by Western blotting. Results are representative of three separate experiments. a, P < 0.01 compared to RC; b, P < 0.05 compared to RC; c, P < 0.05 compared to M. avium 724; d, P < 0.01 compared to M. avium 724. Smeg, M. smegmatis; luc., luciferase; RC, noninfected BMMφ.

FIG. 2.

Topography of murine TNF-α (A) and NOS2 (B) promoter regions. The promoter sequences used in this study were derived from a Mus musculus Tnf 5′-regulatory region (GenBank accession no. AB062426) and from the NOS2 promoter region sequence (GenBank accession no. L09126). (A) The transcription factors NF-κB, Ets/Elk, SP1, and CREB/ATF-2/c-jun are recruited to the TNF-α promoter following M. tuberculosis infection (2, 14). Egr, early growth response. (B) The NOS2 promoter contains numerous binding sites for nuclear transcription factors in two clusters: region I (+10 to −300) contains NF-IL-6, a TNF response element, NF-κB, and octamer binding sites, and region II (−100 to −800) contains interferon regulatory factor-1 (IRF-1), STAT1a, and NF-κB binding sites (8, 11, 12, 17). The numbers indicate the distance from the TNF-α or NOS2 transcription start site.

FIG. 3.

RAW 264.7 cells infected with M. smegmatis show higher Ets/Elk and NF-κB promoter activities relative to cells infected with M. avium 724. (A) The Ets/Elk and NF-κB reporter vectors contain five copies of the Ets-1 binding site, ACCGGAAGTT, and six copies of the NF-κB binding site, GGGAATTTC, respectively, in front of the TATA promoter and luciferase gene (luc⁺). (B) RAW 264.7 cells, transfected with Ets-pGL3-luc or NF-κB-pGL3-luc in combination with phRL-SV40, were infected with mycobacteria as described in Materials and Methods. At indicated times, cell extracts were assayed for luciferase activity. Results represent firefly luciferase activities relative to Renilla luciferase activities and were normalized to those of cells transfected with Basic-pGL3-luc. a, P < 0.05 compared to RC; b, P < 0.01 compared to RC; c, P < 0.01 compared to M. avium 724; d, P < 0.05 compared to M. avium 724. luc., luciferase; Smeg, M. smegmatis; RC, noninfected BMMφ.

FIG. 4.

The −117 and −84 ETS binding sites on the TNF-α promoter are required for TNF-α promoter activity in mycobacterium-infected RAW 264.7 cells. (A) The −117 Ets/Elk mt (−117 to −114; CTTC to ACCG), the −84 Ets/Elk mt (−84 to −81; AAGG to TGCT), and the −76 Ets/Elk mt (−76 to −73; TTTC to CACT) were created by site-directed mutagenesis as described in Materials and Methods. The numbers indicate the distance from the TNF-α transcription start site. (B) RAW 264.7 cells, transfected with wild-type (WT) or mutated TNF-α-pGL3-luc in combination with phRL-SV40, were infected with mycobacteria. Twenty-four hours postinfection, cell extracts were assayed for luciferase activity, and the results represent firefly luciferase activities relative to Renilla luciferase activities and were normalized to those of cells transfected with Basic-pGL3-luc. a, P < 0.05 compared to RC; b, P < 0.05 compared to M. avium 724; c, P < 0.01 compared to RC; d, P < 0.01 compared to −76 Ets/Elk mt plus M. avium 724; e, P < 0.01 compared to WT plus M. smegmatis (Smeg). luc., luciferase; RC, noninfected BMMφ.

FIG. 5.

The −971 NF-κB site on the NOS2 promoter is required for promoter activity in RAW 264.7 cells following a mycobacterial infection. (A) 390 NOS2-pGL3-luc containing the mouse NOS2 promoter (−230 to +160) and −971 NF-κB mt (−971 to −969; GGG to CTC) were generated as described in Materials and Methods. The numbers indicate the distance from the NOS2 transcription start site. (B) RAW 264.7 cells, transfected with wild-type (WT) promoter, 390 NOS2-pGL3-luc, or −971 NF-κB mt NOS2-pGL3-luc in combination with phRL-SV40, were infected with mycobacteria. Twenty-four hours after infection, cell extracts were assayed for luciferase activity. Results represent firefly luciferase activities relative to Renilla luciferase activities and were normalized to those of cells transfected with Basic-pGL3-luc. a, P < 0.05 compared to RC; b, P < 0.01 compared to M. avium 724. Smeg, M. smegmatis; luc., luciferase; RC, noninfected BMMφ.

FIG. 6.

Elevated p65 NF-κB and ERK1/2 phosphorylation in M. smegmatis-infected RAW 264.7 cells. Western blot analyses of p65 NF-κB and ERK1/2 phosphorylation following 1-, 4-, 9-, or 24-h infections with either M. avium 724 or M. smegmatis. Levels of total p38 are shown to indicate equal protein loading in each lane. Smeg, M. smegmatis.

FIG. 7.

ERK1/2 is essential for TNF-α, but not NOS2, production following a mycobacterium infection. (A) The cells were pretreated with 20 μM PD98059 (PD) or DMSO as a solvent control for 1 h and were then infected with mycobacteria for 4 h. The percentages of mycobacterium-associated macrophages were determined using microscopy as described in Materials and Methods. (B) TNF-α in the respective cell culture supernatants was analyzed by ELISA. (C) Western blot analysis of NOS2 production in the respective cell lysates. a, P < 0.01 compared to RC; b, P < 0.05 compared to M. avium 724 plus DMSO; c, P < 0.01 compared to M. avium 724 plus DMSO; d, P < 0.01 compared to M. smegmatis plus DMSO. Smeg, M. smegmatis.

FIG. 8.

ERK1/2 promotes TNF-α and Ets but not NOS2 or NF-κB promoter activity. (A to D) RAW 264.7 cells were transfected with either Basic-pGL3-luc, −1200 TNF-α-pGL3-luc, −1700 NOS2-pGL3-luc, Ets-pGL3-luc, or NF-κB-pGL3-luc, in combination with phRL-SV40. Twenty-four hours after transfection, the cells were pretreated with 20 μM PD98059 (PD) or DMSO for 1 h and then infected with mycobacteria for 4 h. Cell extracts were assayed for luciferase activity as described in Materials and Methods. Results represent firefly luciferase activities relative to Renilla luciferase activities and were normalized to those of cells transfected with Basic-pGL3-luc. a, P < 0.05 compared to RC plus DMSO; b, P < 0.01 compared to RC plus DMSO; c, P < 0.05 compared to M. avium 724 plus DMSO; d, P < 0.01 compared to M. smegmatis plus DMSO. Smeg, M. smegmatis; luc., luciferase.