Immune Netw.  2012 Oct;12(5):207-212. 10.4110/in.2012.12.5.207.

T Cell Immunoglobulin Mucin Domain (TIM)-3 Promoter Activity in a Human Mast Cell Line

Affiliations
  • 1Department of Microbiology, Ajou University School of Medicine, Suwon 442-749, Korea. sinsun@ajou.ac.kr
  • 2Graduate Program of Molecular Medicine, Ajou University School of Medicine, Suwon 442-749, Korea.

Abstract

T cell immunoglobulin mucin domain (TIM)-3 is an immunomodulatory molecule and upregulated in T cells by several cytokines. TIM-3 also influences mast cell function but its transcriptional regulation in mast cells has not been clarified. Therefore, we examined the transcript level and the promoter activity of TIM-3 in mast cells. The TIM-3 transcript level was assessed by real-time RT-PCR and promoter activity by luciferase reporter assay. TIM-3 mRNA levels were increased in HMC-1, a human mast cell line by TGF-beta1 stimulation but not by stimulation with interferon (IFN)-alpha, IFN-lambda, TNF-alpha, or IL-10. TIM-3 promoter -349~+144 bp region relative to the transcription start site was crucial for the basal and TGF-beta1-induced TIM-3 promoter activities in HMC-1 cells. TIM-3 promoter activity was increased by overexpression of Smad2 and Smad4, downstream molecules of TGF-beta1 signaling. Our results localize TIM-3 promoter activity to the region spanning -349 to +144 bp in resting and TGF-beta1 stimulated mast cells.

Keyword

T cell immunoglobulin mucin domain-3; TGF-beta1; Transcription; Mast cells; Smad

MeSH Terms

Cytokines
Humans
Immunoglobulins
Interferons
Interleukin-10
Luciferases
Mast Cells
Mucins
RNA, Messenger
T-Lymphocytes
Transcription Initiation Site
Transforming Growth Factor beta1
Tumor Necrosis Factor-alpha
Cytokines
Immunoglobulins
Interferons
Interleukin-10
Luciferases
Mucins
RNA, Messenger
Transforming Growth Factor beta1
Tumor Necrosis Factor-alpha

Figure

  • Figure 1 TIM-3 mRNA expression was increased in HMC-1 cells by stimulation with TGF-β1 but not by stimulation with IFNs, IL-10, and TNF-α. HMC-1 cells were treated for the indicated time with IFN-α (100 U/ml), IFN-λ (100 ng/ml), TGF-β1 (2 ng/ml), IL-10 (10 ng/ml), and TNF-α (10 ng/ml), respectively and then TIM-3 transcript levels were analyzed by real-time RT-PCR. TIM-3 transcript levels were normalized to GAPDH transcript levels. Relative Tim-3 mRNA levels to control (Con, without cytokine treatment) were presented. *p<0.005.

  • Figure 2 Basal TIM-3 promoter activity in HEK293 and HMC-1 cells. (A) Luciferase reporter vectors containing various length of TIM-3 promoter region. (B) HEK293 (5×105) or (C) HMC-1 (1×106) cells were transfected with the indicated luciferase reporter vector together with pEGFP-N1 vector (1/10 of total plasmid) and 48 h later luciferase activity was measured and normalized to the frequencies of GFP-expressing cells. Relative luciferase activity to control (pGL-Basic) was presented. The data are the mean±standard deviation of 5 experiments. *p<0.01.

  • Figure 3 TIM-3 promoter activity in HMC-1 cells stimulated with TGF-β1. HMC-1 (1×106) cells were transfected with the indicated luciferase reporter vector together with pEGFP-N1 vector (1/10 of total plasmid) and 42 h later treated with TGF-β1 (2 ng/ml) for 6 h. Then luciferase activity was measured and normalized to the frequencies of GFP-expressing cells. Fold induction of luciferase activity by TGF-β1 treatment was presented for each luciferase reporter vector. The data are the mean±standard deviation of 5 experiments. *p<0.001.

  • Figure 4 TIM-3 promoter activity was increased by Smad overexpression in HEK293 cells. HEK293 cells (1×106) were transfected with T3U(1.8)-luc (1.8µg) together with the indicated Smad expression vector (+: 0.9µg, ++: 1.8µg) or control empty vector pcDNA (1.8µg). For normalization of transfection efficiency, pEGFP-N1 vector (1/10 of total plasmid) was also co-transfected. After 48 h, the luciferase activity was measured and normalized to the frequencies of GFP-expressing cells. Fold induction of luciferase activity compared to that in the absence of Smad overexpression was presented. The data are the mean±standard deviation of 5 experiments. *p<0.01.


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