STAT3 is involved in phosphatidic acid-induced Bcl-2 expression in HeLa cells

Abstract

Phosphatidic acid (PA), the product of a PLD-mediated reaction, is a lipid second messenger that participates in various intracellular signaling events and is known to regulate a growing list of signaling proteins. We found that Bcl-2 was upregulated by PA treatment in HeLa cells. However, how PA upregulates Bcl-2 expression has not yet been studied. In this study, we tried to discover the mechanisms of Bcl-2 up-regulation by PA treatment in HeLa cells. Treatment with PA resulted in significantly increased expression of Bcl-2 in HeLa cells. Moreover, PA-induced Bcl-2 expression was blocked by mepacrine, an inhibitor of PLA2, but not by propranolol, an inhibitor of PA phospholyhydrolase (PAP). Treatment of 1,2-dipalmitoryl-sn-glycero-3- phosphate (DPPA) also increased Bcl-2 expression. These results indicate that Bcl-2 expression is mediated by lysophosphatidic acid (LPA), not by arachidonic acid (AA). Thereafter, we used MEK1/2 inhibitor, PD98059 to investigate the relationship between ERK1/2 MAPK and PA-induced Bcl-2 expression. PA-induced Bcl-2 expression was decreased when ERK1/2 was inhibited by PD98059. The transcription factor such as STAT3 which is controlled by ERK1/2 MAPK was increased along with Bcl-2 expression when the cells were treated with PA. Furthermore, STAT3 siRNA treatments inhibited PA-induced Bcl-2 expression, suggesting that STAT3 (Ser727) is involved in PA-induced Bcl-2 expression. Taken together, these findings indicate that PA acts as an important mediator for increasing Bcl-2 expression through STAT3 (Ser727) activation via the ERK1/2 MAPK pathway.

Figure 1

Effects of PA on the Bcl-2 expression in HeLa cells. PA increased Bcl-2 expression in both a dose- and time-dependent manner. (A) HeLa cells were cultured in DMEM containing 10% FBS; HeLa cells were further incubated without FBS for 18 h and treated with different concentrations of PA for 3 h. (B) Cells treated with 50 µM PA for the indicated time. The relative quantities of each protein band, normalized to control cells, were quantified using Quantity One software (Bio-Rad).

Figure 2

Effects of propranolol, mepacrine, and DPPA pretreatment on the expression of Bcl-2 in HeLa cells. (A) HeLa cells were pretreated with 50 µM propranolol for 30 min before being treated with 50 µM PA for 30 min for RT-PCR, and for 3 h for Western blotting. (B) HeLa cells were pretreated with 50 µM mepacrine for 30 min before 50 µM PA treatmemt for 30 min for RT-PCR, and for 3 h for Western blotting, respectively. (C) HeLa cells were cultured for 1 h with 5 and 10 µM DPPA after starvation for 18 h, respectively. The upper panel represents mRNA expression and lower panel represents protein. The cells were harvested, lysised and subjected to RT-PCR and Western blotting as described in the Methods. The relative quantities of each protein band, normalized to control cells, were quantified using Quantity One software (Bio-Rad).

Figure 3

Effects of a PLA₂ inhibitor, mepacrine, and MEK inhibitor, PD98059, on PA-induced ERK1/2, STAT3 (Ser⁷²⁷) phosphorylation, and Bcl-2 expression in HeLa cells. (A) RNA was extracted from cells pretreated with 50 µM mepacrine for 30 min, followed by stimulation with 50 µM PA for 30 min and Bcl-2 mRNA was amplified by RT-PCR. HeLa cells were pretreated with 50 µM mepacrine for 30 min, followed by stimulation with 50 µM PA for 15 min. Cell lysates were analyzed by immunoblotting with anti-ERK1/2, anti-STAT3, and phospho-ERK1/2, phospho-STAT3 (Ser⁷²⁷) antibodies. In the case of Bcl-2 Western blotting, cells were pretreated with 50 µM mepacrine for 30 min, followed by stimulation with 50 µM PA for 3 h. Cell lysates were analyzed by immunoblotting with Bcl-2 antibody. (B) RNA was extracted from cells treated with 50 µM PD98059 for 1 h, followed by stimulation with 50 µM PA for 30 min and Bcl-2 mRNA was amplified by RT-PCR. HeLa cells were pretreated with 50 µM PD98059 for 1 h, followed by stimulation with 50 µM PA for 15 min. Cell lysates were analyzed by immunoblotting with anti-ERK1/2, STAT3, and phospho-ERK1/2, STAT3 (Ser⁷²⁷) antibodies. In the case of Bcl-2 Western blotting, cells were pretreated with 50 µM PD98059 for 1 h, followed by stimulation with 50 µM PA for 3 h. Cell lysates were analyzed by immunoblotting with Bcl-2 antibody. The relative quantities of each protein band, normalized to control cells, were quantified using Quantity One software (Bio-Rad).

Figure 4

Effects of STAT3 siRNA on the expression of Bcl-2 in HeLa cells. HeLa cells were transiently transfected with 100 nM STAT3 siRNA or scramble siRNA for 72 h and then stimulated with 50 µM PA for 15 min (for p-STAT3/STAT3 blots) or 3 h (for Bcl-2 blots). Expression level of p-STAT3 (Ser⁷²⁷), STAT3, and Bcl-2 were determined by Western blot analysis. The relative quantities of each protein band, normalized to control cells, were quantified using Quantity One software (Bio-Rad).

Figure 5

A proposed model for the signaling pathway of PA-induced Bcl-2 expression. We diagramed a mechanism of up-regulation of Bcl-2 expression induced by PA. PA can be converted to LPA by PLA₂, but does not pass through the DAG pathway by PAP. Subsequently, LPA acts as an important signal molecule to upregulate Bcl-2 expression. PA leads to activation of downstream kinases, ERK1/2, which are responsible for the phosphorylation of STAT3 (Ser⁷²⁷).