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Anti-proliferative Activity of T-bet.

Oh YJ, Shin JH, Won HY, Hwang ES - Immune Netw (2015)

Bottom Line: Therefore, we examined the effect of T-bet on Th cell proliferation and found that T-bet deficiency significantly enhanced Th cell proliferation under non-skewing, Th1-skewing, and Th2-skewing conditions.By using IFN-γ- mice to eliminate the anti-proliferative effect of IFN-γ, T-bet deficiency still enhanced Th cell proliferation under both Th1- and Th2-skewing conditions.We demonstrated that T-bet expression induced by ecdysone treatment in human embryonic kidney (HEK) cells increased IFN-γ promoter activity in a dose dependent manner, and sustained T-bet expression considerably decreased cell proliferation in HEK cells.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Korea.

ABSTRACT
T-bet is a critical transcription factor that regulates differentiation of Th1 cells from CD4(+) precursor cells. Since T-bet directly binds to the promoter of the IFN-γ gene and activates its transcription, T-bet deficiency impairs IFN-γ production in Th1 cells. Interestingly, T-bet-deficient Th cells also display substantially augmented the production of IL-2, a T cell growth factor. Exogenous expression of T-bet in T-bet deficient Th cells rescued the IFN-γ production and suppressed IL-2 expression. IFN-γ and IL-2 reciprocally regulate Th cell proliferation following TCR stimulation. Therefore, we examined the effect of T-bet on Th cell proliferation and found that T-bet deficiency significantly enhanced Th cell proliferation under non-skewing, Th1-skewing, and Th2-skewing conditions. By using IFN-γ- mice to eliminate the anti-proliferative effect of IFN-γ, T-bet deficiency still enhanced Th cell proliferation under both Th1- and Th2-skewing conditions. Since the anti-proliferative activity of T-bet may be influenced by IL-2 suppression in Th cells, we examined whether T-bet modulates IL-2-independent cell proliferation in a non-T cell population. We demonstrated that T-bet expression induced by ecdysone treatment in human embryonic kidney (HEK) cells increased IFN-γ promoter activity in a dose dependent manner, and sustained T-bet expression considerably decreased cell proliferation in HEK cells. Although the molecular mechanisms underlying anti-proliferative activity of T-bet remain to be elucidated, T-bet may directly suppress cell proliferation in an IFN-γ- or an IL-2-independent manner.

No MeSH data available.


Related in: MedlinePlus

Ecdysone-inducible expression of functional T-bet in non-T cells. EcR-HEK cells were transiently transfected with T-bet expression vector and subsequently treated with PonA for 24 hours. (A) Whole cell protein extracts were resolved by SDS-PAGE and subjected to immunoblotting with Ab against Flag, T-bet (4B10), or actin. (B) EcR-HEK cells were transiently transfected with T-bet expression vector together with pIFN-γ-luciferase reporter gene and pCMVβ as an internal control. After treatment with different concentrations of PonA, cells were harvested for determination of luciferase activity. At least three independent reporter assays were performed and data are given as the average ±SD. ***p<0.0005.
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Figure 5: Ecdysone-inducible expression of functional T-bet in non-T cells. EcR-HEK cells were transiently transfected with T-bet expression vector and subsequently treated with PonA for 24 hours. (A) Whole cell protein extracts were resolved by SDS-PAGE and subjected to immunoblotting with Ab against Flag, T-bet (4B10), or actin. (B) EcR-HEK cells were transiently transfected with T-bet expression vector together with pIFN-γ-luciferase reporter gene and pCMVβ as an internal control. After treatment with different concentrations of PonA, cells were harvested for determination of luciferase activity. At least three independent reporter assays were performed and data are given as the average ±SD. ***p<0.0005.

Mentions: In contrast to IFN-γ, which has no regulatory effect on T-bet-mediated inhibition of Th cell proliferation, IL-2 is a critical factor for regulating Th cell proliferation (18). Additionally, we found that T-bet suppresses IL-2 levels. To rule out a T-bet-mediated effect on IL-2 suppression that influences Th cell proliferation, we established an inducible T-bet expression system in IL-2-independent EcR-HEK cells that were transfected with ecdysone receptor. Subsequent transfection of ecdysone receptor-binding elementlinked T-bet expression vector into EcR-HEK cells induced T-bet expression proportionate to the concentration of PonA (Fig. 5A). IFN-γ promoter activity was increased in a dose dependent manner, mirroring T-bet expression levels (Fig. 5B). Thus, we confirmed functionality of the ecdysone-inducible expression system of T-bet in a non-T cell population that proliferates in an IL-2-independent manner.


Anti-proliferative Activity of T-bet.

Oh YJ, Shin JH, Won HY, Hwang ES - Immune Netw (2015)

Ecdysone-inducible expression of functional T-bet in non-T cells. EcR-HEK cells were transiently transfected with T-bet expression vector and subsequently treated with PonA for 24 hours. (A) Whole cell protein extracts were resolved by SDS-PAGE and subjected to immunoblotting with Ab against Flag, T-bet (4B10), or actin. (B) EcR-HEK cells were transiently transfected with T-bet expression vector together with pIFN-γ-luciferase reporter gene and pCMVβ as an internal control. After treatment with different concentrations of PonA, cells were harvested for determination of luciferase activity. At least three independent reporter assays were performed and data are given as the average ±SD. ***p<0.0005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4553258&req=5

Figure 5: Ecdysone-inducible expression of functional T-bet in non-T cells. EcR-HEK cells were transiently transfected with T-bet expression vector and subsequently treated with PonA for 24 hours. (A) Whole cell protein extracts were resolved by SDS-PAGE and subjected to immunoblotting with Ab against Flag, T-bet (4B10), or actin. (B) EcR-HEK cells were transiently transfected with T-bet expression vector together with pIFN-γ-luciferase reporter gene and pCMVβ as an internal control. After treatment with different concentrations of PonA, cells were harvested for determination of luciferase activity. At least three independent reporter assays were performed and data are given as the average ±SD. ***p<0.0005.
Mentions: In contrast to IFN-γ, which has no regulatory effect on T-bet-mediated inhibition of Th cell proliferation, IL-2 is a critical factor for regulating Th cell proliferation (18). Additionally, we found that T-bet suppresses IL-2 levels. To rule out a T-bet-mediated effect on IL-2 suppression that influences Th cell proliferation, we established an inducible T-bet expression system in IL-2-independent EcR-HEK cells that were transfected with ecdysone receptor. Subsequent transfection of ecdysone receptor-binding elementlinked T-bet expression vector into EcR-HEK cells induced T-bet expression proportionate to the concentration of PonA (Fig. 5A). IFN-γ promoter activity was increased in a dose dependent manner, mirroring T-bet expression levels (Fig. 5B). Thus, we confirmed functionality of the ecdysone-inducible expression system of T-bet in a non-T cell population that proliferates in an IL-2-independent manner.

Bottom Line: Therefore, we examined the effect of T-bet on Th cell proliferation and found that T-bet deficiency significantly enhanced Th cell proliferation under non-skewing, Th1-skewing, and Th2-skewing conditions.By using IFN-γ- mice to eliminate the anti-proliferative effect of IFN-γ, T-bet deficiency still enhanced Th cell proliferation under both Th1- and Th2-skewing conditions.We demonstrated that T-bet expression induced by ecdysone treatment in human embryonic kidney (HEK) cells increased IFN-γ promoter activity in a dose dependent manner, and sustained T-bet expression considerably decreased cell proliferation in HEK cells.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Korea.

ABSTRACT
T-bet is a critical transcription factor that regulates differentiation of Th1 cells from CD4(+) precursor cells. Since T-bet directly binds to the promoter of the IFN-γ gene and activates its transcription, T-bet deficiency impairs IFN-γ production in Th1 cells. Interestingly, T-bet-deficient Th cells also display substantially augmented the production of IL-2, a T cell growth factor. Exogenous expression of T-bet in T-bet deficient Th cells rescued the IFN-γ production and suppressed IL-2 expression. IFN-γ and IL-2 reciprocally regulate Th cell proliferation following TCR stimulation. Therefore, we examined the effect of T-bet on Th cell proliferation and found that T-bet deficiency significantly enhanced Th cell proliferation under non-skewing, Th1-skewing, and Th2-skewing conditions. By using IFN-γ- mice to eliminate the anti-proliferative effect of IFN-γ, T-bet deficiency still enhanced Th cell proliferation under both Th1- and Th2-skewing conditions. Since the anti-proliferative activity of T-bet may be influenced by IL-2 suppression in Th cells, we examined whether T-bet modulates IL-2-independent cell proliferation in a non-T cell population. We demonstrated that T-bet expression induced by ecdysone treatment in human embryonic kidney (HEK) cells increased IFN-γ promoter activity in a dose dependent manner, and sustained T-bet expression considerably decreased cell proliferation in HEK cells. Although the molecular mechanisms underlying anti-proliferative activity of T-bet remain to be elucidated, T-bet may directly suppress cell proliferation in an IFN-γ- or an IL-2-independent manner.

No MeSH data available.


Related in: MedlinePlus