TNF-α enhances the effect of TGF-β on Gli2 expression in the KG-1 leukemic cell line

Abstract

The Hedgehog (Hh) signaling pathway regulates a variety of tumor-related diseases, including leukemia. The present study aimed to determine whether there was an interaction between the Hh signaling pathway and transforming growth factor (TGF)-β in the KG-1 cell line. KG-1 cells were treated with TGF-β, tumor necrosis factor (TNF)-α and specific inhibitor of smad3 (SIS3). The expression level of Gli family zinc finger 2 (Gli2) was detected by quantitative polymerase chain reaction (qPCR) and western blot analyses. The results revealed that TGF-β significantly decreased the expression level of Gli2 in KG-1 cells, and that TNF-α and TGF-β together further reduced Gli2 expression in KG-1 cells. SIS3 inhibited the effect of TGF-β. These results suggest that Gli2 expression in KG-1 cells is suppressed by TGF-β in a Smad3-dependent manner, TNF-α can enhance the effect of TGF-β on Gli2 expression and that this occurs independently of Hh receptor signaling.

Keywords: Gli family zinc finger 2; specific inhibitor of smad3; transforming growth factor-β; tumor necrosis factor α.

Figure 1

Effect of transforming growth factor β (TGF-β) or tumor necrosis factor α (TNF-α) alone on the expression level of Gli family zinc finger 2 (Gli2) mRNA in the KG-1 cell line. (A) KG-1 cells were divided into two treatment groups: control group (no treatment) and the TGF-β group which received 5 ng/ml transforming growth factor β1 (TGF-β1) for 6, 12 and 24 h. (B) KG-1 cells were divided into two treatment groups: control group (no treatment) and the TNF-α group which received 5 ng/ml TNF-α for 12, 24 and 48 h. The total mRNA in the KG-1 cells was extracted and quantitative PCR was performed. ^*P<0.05, TGF-β1 group vs. control group.

Figure 2

Tumor necrosis factor (TNF)-α increases transforming growth factor β type I receptor (TGF-βRI) and transforming growth factor β type II receptor (TGF-βRII) protein expression levels in KG-1 cells. KG-1 cells were treated with 5 ng/ml TNF-α (+ group) or not treated as the control (− group) for 24 h. The total proteins in the KG-1 cells were extracted and a western blot analysis was performed.

Figure 3

Effect of treatment with transforming growth factor β (TGF-β) with or without tumor necrosis factor (TNF)-α on the expression level of Gli family zinc finger 2 (Gli2) in KG-1 cells. KG-1 cells were treated with: no treatment as a control (−), 5 ng/ml transforming growth factor β1 (TGF-β1; +) or 5 ng/ml TGF-β1+5 ng/ml TNF-α (+), respectively for 24 h. (A) The total mRNA in the KG-1 cells was extracted and quantitative PCR was carried out. (B) The total proteins in the KG-1 cells were extracted and western blot analyses were performed. ^*P<0.05, treatment group (+) vs. control (−) group; ^#P<0.05, TGF-β1 + TNF-α group vs. TGF-β1 group.

Figure 4

Effect of treatment with transforming growth factor (TGF)-β and tumor necrosis factor α (TNF-α) with or without smad3 inhibitor (SIS3) on the expression level of Gli family zinc finger 2 (Gli2) in KG-1 cells. KG-1 cells were treated with: no treatment as a control (−), 5 ng/ml transforming growth factor β1 (TGF-β1) + 5 ng/ml TNF-α or 5 ng/ml TGF-β1 + 5 ng/ml TNF-α + 5 uM SIS3, respectively for 24 h. (A) The total mRNA in the KG-1 cells was extracted and quantitative PCR was performed. (B) The total proteins in the KG-1 cells were extracted and western blot analyses were carried out. ^*P<0.05, treatment group (+) vs. control (−) group.

Figure 5

Specific inhibitor of Smad 3 (SIS3) is able to increase the expression of Gli family zinc finger 2 (Gli2) in KG-1 cells at the protein and mRNA levels. The KG-1 cells were treated with 5 μM SIS3 or the same amount of control solution (dimethyl sulfoxide, DMSO) for 6, 12 and 24 h. (A) The total mRNA in the KG-1 cells was extracted and quantitative PCR was performed. (B) The total proteins in the KG-1 cells were extracted and western blot analyses were carried out. ^*P<0.05, SIS3 group vs. DMSO group.