J Korean Med Sci.  2008 Oct;23(5):870-876. 10.3346/jkms.2008.23.5.870.

Overexpression of USF Increases TGF-beta1 Protein Levels, But G1 Phase Arrest was not Induced in FRTL-5 Cells

• Kim KS
• Jung HS
• Chung YJ
• Jung TS
• Jang HW
• Lee MS
• Kim KW
• Chung JH

• KMID: 1783075
• DOI: http://doi.org/10.3346/jkms.2008.23.5.870

Abstract

Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of cellular growth and proliferation by G1 phase arrest or apoptosis. We investigated the association of TGF-beta1 with the anti-proliferative effect of upstream stimulatory factor (USF) in Fischer rat thyroid cell line (FRTL-5) cells. [Methyl-(3)H] thymidine uptake was measured after treatment of FRTL-5 cells with TGF-beta1 to identify its anti-proliferative effect. USF-1 and USF-2 proteins were in vitro translated, and an electrophoretic mobility shift assay was performed to identify the interaction between USF and the TGF-beta1 promoter. FRTL-5 cells were transfected with USF cDNA, and then the expression of TGF-beta1 was examined with Northern and Western blotting. The cell cycle-regulating proteins associated with TGF-beta1 were also measured. TGF-beta1 significantly inhibited [methyl-(3)H] thymidine uptake in FRTL-5 cells. Two specific binding sites for USF were found in the TGF-beta1 promoter: -1,846~-1,841 (CACATG) and -621~-616 (CATGTG). Overexpression of USF increased both the mRNA levels and protein levels of TGF-beta1. However, the expression of cyclin D1, CDK4, cyclin E, and CDK2, and the phosphorylation of retinoblastoma protein remained unchanged. Overexpression of USF in FRTL-5 cells increased the expression of TGF-beta10 through specific binding to TGF-beta1 promoter. However, the USF-induced expression of TGF-beta1 did not cause G1 arrest.

Keyword

Upstream Stimulatory Factor; Transforming Growth Factor beta1; FRTL-5 Cells

MeSH Terms

Animals
*Apoptosis
Binding Sites
Cell Cycle
Cell Line
G1 Phase
*Gene Expression Regulation
Promoter Regions, Genetic
Protein Biosynthesis
Rats
Thymidine/chemistry
Transfection
Transforming Growth Factor beta1/metabolism
Upstream Stimulatory Factors/*metabolism

Figure

• 

  Fig. 1 Inhibition of cellular proliferation by TGF-β1 in FRTL-5 cells. FRTL-5 cells were cultured with 5 ng/mL of TGF-β1, and thymidine uptake of the cells was measured. The values of thymidine uptake were analyzed using Student's t-test, and were expressed as mean±standard deviation. Treatment with TGF-β1 significantly decreased thymidine uptake by 60-70% in FRTL-5 cells. *p<0.05 vs. no TGF-β1 treatment.

• 

  Fig. 2 Electrophoretic mobility shift assay (EMSA) of the complexes between the USF protein and the E-box sequences in the TGF-β1 promoter. The in vitro translated USF-1 and USF-2 were confirmed by Western blotting, and this is illustrated in a box located in the right upper side of the figure. Four types of E-boxes (E1-E4) existed in the TGF-β1 promoter, and each was tested to determine whether they would bind to USF-1 and USF-2 by using EMSA (black arrows). Among them, the affinities to E2 and E3 were much stronger than the affinities shown to E1 and E4. Cold competitors eliminated the DNA-protein complexes, and a specific anti-USF-1 or anti-USF-2 antibody shifted these complexes to an upper position to cause 'super-shifts' (white arrows).

• 

  Fig. 3 Electrophoretic mobility shift assay (EMSA) of the complexes between USF and the E2/E3 of the different loci in the TGF-β1 promoter. There were two loci of CACATG (E2) and CATGTG (E3), respectively: -1,846~-1,841 (E2) and -3,514~-3,509 (E2'), and -621~-616 (E3) and -1,563~-1,558 (E3'). Specific sites for USF binding were identified by performing EMSA with four probes (T2, T2', T3, and T3') that contained each E-box. The specific sites were revealed as -1,846~-1,841 and -621~-616. Black arrows indicate the DNA-protein complexes, and the white arrows indicate their super-shifts according to the specific antibodies.

• 

  Fig. 4 Effects of USF-1 and USF-2 overexpression on the expression of TGF-β1 in the FRTL-5 cells. Total RNA and protein were isolated from FRTL-5 cells that were transiently overexpressed with USF. Northern (left panel) and Western blotting (right panel) were performed to evaluate the mRNA and protein levels of USF and TGF-β1. Expression of TGF-β1 was induced by the overexpression of USF-1 and USF-2. Three sets of experiments were repeated. GAPDH was used as the internal control for Northern blotting, and tubulin was used as the internal control for Western blotting.

• 

  Fig. 5 Effects of USF-1 and USF-2 overexpression on the expression of TGF-β1-related proteins in FRTL-5 cells. Total protein was isolated from FRTL-5 cells that transiently overexpressed USF, and Western blotting was then performed. The p27 levels were increased by USF overexpression, but the cyclin D1, CDK4, cyclin E, and CDK2 levels were not changed. Accordingly, dephosphorylation of retinoblastoma (Rb) protein was not observed. Three sets of experiments were performed, and tubulin was used as the internal control. ppRb, hyperphosphorylated Rb; pRb, hypophosphorylated Rb.

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