Cancer Res Treat.  2015 Jan;47(1):101-109. 10.4143/crt.2013.192.

TGF-beta Suppresses COX-2 Expression by Tristetraprolin-Mediated RNA Destabilization in A549 Human Lung Cancer Cells

• Kang S
• Min A
• Im SA
• Song SH
• Kim SG
• Kim HA
• Kim HJ
• Oh DY
• Jong HS
• Kim TY
• Bang YJ

• KMID: 2380395
• DOI: http://doi.org/10.4143/crt.2013.192

Abstract

PURPOSE
Overexpression of cyclooxygenase 2 (COX-2) is thought to promote survival of transformed cells. Transforming growth factor beta (TGF-beta) exerts anti-proliferative effects on a broad range of epithelial cells. In the current study, we investigated whether TGF-beta can regulate COX-2 expression in A549 human lung adenocarcinoma cells, which are TGF-beta-responsive and overexpress COX-2.
MATERIALS AND METHODS
Western blotting, Northern blotting, and mRNA stability assays were performed to demonstrate that COX-2 protein and mRNA expression were suppressed by TGF-beta. We also evaluated the effects of tristetraprolin (TTP) on COX-2 mRNA using RNA interference.
RESULTS
We demonstrated that COX-2 mRNA and protein expression were both significantly suppressed by TGF-beta. An actinomycin D chase experiment demonstrated that COX-2 mRNA was more rapidly degraded in the presence of TGF-beta, suggesting that TGF-beta-induced inhibition of COX-2 expression is achieved via decreased mRNA stability. We also found that TGF-beta rapidly and transiently induced the expression of TTP, a well-known mRNA destabilizing factor, before suppression of COX-2 mRNA expression was observed. Using RNA interference, we confirmed that increased TTP levels play a pivotal role in the destabilization of COX-2 mRNA by TGF-beta. Furthermore, we showed that Smad3 is essential to TTP-dependent down-regulation of COX-2 expression in response to TGF-beta.
CONCLUSION
The results of this study show that TGF-beta down-regulated COX-2 expression via mRNA destabilization mediated by Smad3/TTP in A549 cells.

Keyword

Cyclooxygenase 2; Transforming growth factor beta; Tristetraprolin; RNA stability; Smad3

MeSH Terms

Adenocarcinoma
Blotting, Northern
Blotting, Western
Cyclooxygenase 2
Dactinomycin
Down-Regulation
Epithelial Cells
Humans
Lung
Lung Neoplasms*
RNA Interference
RNA Stability
RNA*
RNA, Messenger
Transforming Growth Factor beta*
Tristetraprolin
Cyclooxygenase 2
Dactinomycin
RNA
RNA, Messenger
Transforming Growth Factor beta
Tristetraprolin

Figure

• 

  Fig. 1. Expression of cyclooxygenase 2 (COX-2) mRNA and protein is suppressed by transforming growth factor β (TGF-β) treatment in A549 cells. (A) COX-2 protein levels were spontaneously elevated after cell seeding. This increase was more marked with serum starvation. TGF-β blocked the induction of COX-2 expression in the presence or absence of serum. A549 cells were incubated with or without 5 ng/mL TGF-β in the presence or absence of serum. Total cellular proteins were isolated at the indicated times, after which Western blotting was performed with anti–COX-2 or anti– α-tubulin antibodies. (B) COX-2 mRNA expression was down-regulated by TGF-β. The cells were treated with 5 ng/mL TGF-β for 1.5 to 2 hours, after which the total RNA was isolated at the indicated times and subjected to Northern blot analysis (20 μg per lane) to measure COX-2 mRNA expression. (C) COX-2 protein levels were down-regulated by TGF-β in A549 cells. The cells were treated with 5 ng/mL TGF-β. Total cellular proteins were isolated at the indicated times and analyzed by Western blotting as described above.

• 

  Fig. 2. Transforming growth factor β (TGF-β) has no effect on cyclooxygenase 2 (COX-2) promoter activity, but affects COX-2 mRNA half-life. (A, B) Cells were transfected with a pGL2 basic control vector or the indicated reporter constructs, then treated with or without 5 ng/mL TGF-β for 6 hours. Levels of luciferase activity were measured as described in the Materials and Methods section. Data were normalized relative to β-galactosidase activity. No significant difference between the luciferase activities of TGF-β–treated and untreated cells was observed. (C) Cells were stimulated with TGF-β (5 ng/mL) for 1 hour, then treated with actinomycin D (5 μg/mL). The cells were harvested at the indicated times after actinomycin D was added. Total RNA was isolated, and real-time polymerase chain reaction was performed to measure the expression of COX-2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The levels of COX-2 mRNA were normalized relative to GAPDH mRNA and presented as a percentage of that found in the untreated control. NF-kB, nuclear factor kB; IL-6, interleukin 6; CRE, cAMP response element.

• 

  Fig. 3. Smad3 knockdown restores the transforming growth factor β (TGF-β)–mediated down-regulation of cyclooxygenase 2 (COX-2) expression. A549 cells were transiently transfected with 80 nM of Smad2- or Smad3-specific siRNA. After transfection, the cells were treated with vehicle or 5 ng/mL of TGF-β for 12 or 24 hours, after which cell lysates were obtained for Western blot analysis. Total cell proteins (70 μg) were subjected to Western blotting and the membranes were probed with the indicated antibodies.

• 

  Fig. 4. Tristetraprolin (TTP)-mediated cyclooxygenase 2 (COX-2) mRNA destabilization is induced by transforming growth factor β (TGF-β). (A, B) Total RNA was recovered from A549 cells treated with TGF-β (5 ng/mL) for different periods of time and Reverse transcribed. Reverse transcription–polymerase chain reaction (RT-PCR) and real-time PCR were then performed using primers specific for TTP, β-actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as described in the Materials and Methods section. The columns represent the mean of three independent experiments and are shown with error bars (±SE). *p < 0.001. (C) A549 cells were transfected with control or TTP-specific siRNA. After 24 hours of transfection, the cells were treated with TGF-β for 1 hour. RT-PCR and quantitative real-time PCR were used to measure the levels of TTP mRNA. Columns, the mean of three independent experiments; Bars, ±SE; *p < 0.001. (D) Cells transfected with control or TTP-specific siRNA were stimulated with or without TGF-β for 1 hour, after which actinomycin D (5 μg/mL) was added. Total RNA was isolated at different times, and COX-2 mRNA levels were quantified by real-time PCR. COX-2 mRNA expression was analyzed as described for Fig. 2C. (E) Cells transfected with control or TTP-specific siRNA were stimulated with TGF-β for the indicated times. Total cell proteins were then recovered and analyzed by Western blotting as described in the Materials and Methods.

Reference

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