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Abstract
Background
Mac-2 binding protein (Mac-2BP) is a secreted glycoprotein from the culture fluid of several human cancer cells, especially breast, lung, and gastric cells. Mac-2BP plays a role in immune response and cell adhesion activity in patients with various cancer and infectious diseases. In this study, we attempted to identify the regulators of Mac-2BP expression at the transcriptional level.
Methods
To determine the effect of epidermal growth factor (EGF) to Mac-2BP expression in gastric cancers, we constructed the different lengths of Mac-2BP promoter plasmids and measured the promoter activity and Mac-2BP expression. In addition to investigating the role of signal transducer and activator of transcription3 (STAT3) or human telomerase reverse transcriptase (hTERT) as a regulator of Mac-2BP, we transfected the small interfering RNA (siRNA) specific for STAT3 or hTERT, and Mac-2BP level was observed by a quantitative ELISA.
Results
EGF treatment could suppress the Mac-2BP transcription in HEK293 or gastric cancer cell lines (SNU-638 or AGS). In 5′-deleted promoter experiment, pGL3-Mac Pro-2377 transfected cells showed a decreased luciferase activity compared to pGL3-Mac Pro-2277. We also identified that (−2,366/-2,356) on Mac-2BP promoter is a putative STAT3 binding site and suppression of STAT3 with STAT3 specific siRNA increased the Mac-2BP level, suggesting the role of STAT3 as a negative regulator, in contrast to hTERT, which is known as a positive regulator.
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Fig. 1.
The effect of EGF treatment in Mac-2BP expression. HEK293 cells were serum-starved for 18 hr and treated with the indicated concentrations of EGF. And the cells were lysed and cell lysates were used to measure Mac-2BP by an ELISA. EGF treatment decreased the Mac-2BP expression; however, it didn't show the dosage dependency.
Abbreviations: EGF, epidermal growth factor; Mac-2BP, Mac-2 binding protein.
Fig. 2.
EGF treatment suppressed the Mac-2BP transcription. (A) HEK293 cells were transiently co-transfected with a pcDNA-hTERT wild type and a pGL3-Mac Pro-2377. After 24 hr, 10 ng/mL of EGF was treated for additional 24 hr. And the cells were lysed and the relative luciferase activity was measured. hTERT expression up-regulated the transcriptional activity of Mac-2BP, and EGF treatment adversely suppressed the activity. (B) SNU-638 and AGS were transfected with a pGL3-Mac Pro-2,377 and incubated with EGF for 24 hr followed by serum-starvation. EGF treatment also decreased the transcriptional activity of Mac-2BP in gastric cancer cells. Luciferase activities were determined as described under MATERIALS AND METHODS. The data shown are representative one of three independent experiments. ∗P<0.05 and †P<0.001 compared to the control sample.
Abbreviations: EGF, epidermal growth factor; Mac-2BP, Mac-2 binding protein; hTERT, human telomerase reverse transcriptase.
Fig. 3.
Comparative analysis of transcriptional activity using 5′ deletion mutants of Mac-2BP promoter. In this schematic, the transcription start site is indicated as +1. Promoter activities of the deletion mutants of Mac-2BP were measured by transient transfection into SNU-638 or AGS cells. And luciferase activities of reporter were normalized with pRL-TK in each cell line. pGL3-Mac Pro-2,377 transfected sample showed 50% decreased activity compared to pGL-Mac Pro-2,277 transfected cells. There is a putative STAT3 binding site (−2,366/-2,356) of Mac-2BP promoter region. The data shown are representative one of three independent experiments; bars, SD.
Abbreviations: Mac-2BP, Mac-2 binding protein; hTERT, human telomerase reverse transcriptase; STAT, signal transducer and activator of transcription.
Fig. 4.
STAT3 plays roles as a negative regulator of Mac-2BP expression. (A) HEK293 and SNU-638 cells were starved for 18 hr and was treated with EGF as time indicated. The cells were lysed and immunoblotted with STAT3 and alpha tubulin antibodies. STAT3 protein was induced by EGF signal in HEK293 or SNU-638 cells. (B) HEK293 cells were transfected with siRNA specific for STAT3 or hTERT. The cells were lysed and immunoblotted with STAT3, hTERT and alpha tubulin antibodies. The other cell lysates were used to measure the Mac-2BP by ELISA. STAT3 siRNA transfected cells showed to increase Mac-2BP level, but hTERT siRNA transfected cells decreased the Mac-2BP level.
Abbreviations: EGF, epidermal growth factor; Mac-2BP, Mac-2 binding protein; hTERT, human telomerase reverse transcriptase; STAT, signal transducer and activator of transcription.
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