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. 2004 Feb;78(4):1697-705.
doi: 10.1128/jvi.78.4.1697-1705.2004.

Latent membrane protein 2A inhibits transforming growth factor-beta 1-induced apoptosis through the phosphatidylinositol 3-kinase/Akt pathway

Makoto Fukuda  1 , Richard Longnecker
Affiliations

Latent membrane protein 2A inhibits transforming growth factor-beta 1-induced apoptosis through the phosphatidylinositol 3-kinase/Akt pathway

Makoto Fukuda et al. J Virol. 2004 Feb.

Abstract

Latent membrane protein 2A (LMP2A) blocks B-cell receptor signal transduction in vitro by binding the Syk and Lyn protein tyrosine kinases. As well as blocking B-cell signal transduction, LMP2A has been shown to activate the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, which acts as a survival signal in both B cells and epithelial cells. Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine that plays important roles in regulating cell growth and differentiation in many biological systems. The loss of the growth-inhibitory response to the TGF-beta 1 signal is found in many cancers and is widely thought to promote tumor development. In this study, we found that LMP2A induced the phosphorylation of Akt (serine 473) in Burkitt's lymphoma cell line Ramos and in gastric carcinoma cell line HSC-39 and partially enhanced cell viability following TGF-beta 1 treatment. In addition, LMP2A partially inhibited TGF-beta 1-induced DNA fragmentation and cleavage of poly(ADP-ribose) polymerase (PARP). In the presence of LY294002, an inhibitor of PI3-K, the LMP2A-mediated inhibitory effects on TGF-beta 1-induced DNA fragmentation and cleavage of PARP were alleviated. Furthermore, LMP2A did not alter the levels of expression of type I and type II TGF-beta 1 receptors. Taken together, these results suggest that LMP2A may inhibit TGF-beta 1-mediated apoptosis through activation of the PI3-K/Akt pathway.

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Figures

FIG. 1.
FIG. 1.
LMP2A induces the phosphorylation of Akt in Ramos and HSC-39 cells. (A) Ramos and HSC-39 cells were stably retrovirally transduced with LMP2A and vector control expression constructs. After 24 h of serum starvation, whole-cell extracts were separated by SDS-polyacrylamide gel electrophoresis. The levels of expression of LMP2A (54 kDa) and phosphorylated Akt (serine 473) (P-Akt) (60 kDa) in parental (P), vector control (V), and LMP2A-expressing Ramos and HSC-39 cells were determined by immunoblotting. LCL, lymphoblastoid cell line. LCL was used as the positive control for LMP2A and Akt phosphorylation. (B) Effect of serum starvation on the phosphorylation of Akt in LMP2A-expressing Ramos and HSC-39 cells. Parental (P), vector control (V), and LMP2A-expressing Ramos and HSC-39 cells were switched to RPMI 1640 medium without FBS (−), harvested at the indicated times, and analyzed by immunoblotting for P-Akt. The lower panels show equal loading of proteins and the expression of total Akt (T-Akt) (60 kDa).
FIG. 2.
FIG. 2.
LMP2A enhances Ramos and HSC-39 cell viability in the presence of TGF-β1 treatment. Parental (P), vector control (V), and LMP2A-expressing Ramos (A) and HSC-39 (B) cells were seeded at 3 × 104 cells/100 μl per well with serum-free medium. Following 1 h of serum starvation, the cells were left untreated (control) or were treated with 10 ng of TGF-β1/ml for 24 or 48 h. Cell viability was measured with an MTT assay. Results are the means and standard deviations for five experiments.
FIG. 3.
FIG. 3.
Determination of expression of TGF-β receptor type I and receptor type II in LMP2A-expressing Ramos and HSC-39 cells. (A) Immunoblot analysis. Parental (P), vector control (V), and LMP2A-expressing Ramos and HSC-39 cells were analyzed by immunoblotting. The positions of TGF-β receptor type I (53-kDa) (R I) and type II (70-kDa) (R II) bands are indicated at the right. The amount of protein loaded in each lane was assessed by rehybridization of the filter with a specific antibody for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B) Flow cytometric analysis. The broken line represents a negative control. The solid line represents cells stained with anti-TGF-β receptor type I or type II antibody and FITC-conjugated secondary antibody. The data are representative of three experiments.
FIG. 4.
FIG. 4.
LMP2A inhibits TGF-β1-induced DNA fragmentation in Ramos and HSC-39 cells. Parental (P), vector control (V), and LMP2A-expressing Ramos (A) and HSC-39 (B) cells (106/ml) were cultured for 1 h with or without serum. Cells were preincubated for 1 h without or with zVAD-fmk (50 μM) before incubation without or with TGF-β1 (10 ng/ml). After 24 h of incubation, cells were analyzed for DNA content by propidium iodide (PI) staining and flow cytometry. Gates used to ascertain cell cycle distribution and the percentage of cells with sub-G1 (<G1) and G2/M DNA contents are shown. The data are representative of three experiments.
FIG. 5.
FIG. 5.
LMP2A inhibits TGF-β1-induced cleavage of PARP. Parental (P), vector control (V), and LMP2A-expressing Ramos (A) and HSC-39 (B) cells (106/ml) were cultured for 1 h with or without serum. Cells were preincubated for 1 h without or with zVAD-fmk (50 μM) before incubation without or with TGF-β1 (10 ng/ml). After 24 h of incubation, PARP cleavage was analyzed by immunoblotting with a specific anti-PARP antibody. The full-length 113-kDa and cleaved 89-kDa (fragment) PARP proteins are indicated at the right. The amount of protein loaded in each lane was assessed by rehybridization of the filter with a specific antibody for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
FIG. 6.
FIG. 6.
Effect of LY294002 on Akt phosphorylation in LMP2A-expressing Ramos and HSC-39 cells. LMP2A-expressing Ramos (A) and HSC-39 (B) cells were cultured for 1 h without serum. Cells were incubated for 1 h with LY294002 (50 μM). Levels of P-Akt (60 kDa) were determined by immunoblotting. The lower panels show equal loading of proteins and the expression of total Akt (T-Akt) (60 kDa).
FIG. 7.
FIG. 7.
LY294002, a specific inhibitor of PI3-K, alleviates LMP2A-mediated cell viability and antiapoptotic effects. (A) Cell viability. LMP2A-expressing Ramos and HSC-39 cells (3 × 104/100 μl) were switched to RPMI 1640 medium without FBS. Cells were preincubated for 1 h without or with LY294002 (50 μM) before incubation without or with TGF-β1 (10 ng/ml). After 24 or 48 h of incubation, cell viability was measured with an MTT assay. Results are the means and standard deviations for five experiments. (B) DNA fragmentation. Cells (106/ml) were switched to RPMI 1640 medium without FBS. Cells were preincubated for 1 h without or with LY294002 (50 μM) before incubation without or with TGF-β1 (10 ng/ml). After 24 h of incubation, cell cycle analyses were performed as described in the legend to Fig. 4. PI, propidium iodide. (C) Evaluation of cleavage of PARP. Cells (106/ml) were switched to RPMI 1640 medium without FBS. Cells were preincubated for 1 h without or with LY294002 (50 μM) before incubation without or with TGF-β1 (10 ng/ml). After 24 h of incubation, PARP cleavage was analyzed as described in the legend to Fig. 5. Control cells were not treated with various agents. The amount of protein loaded in each lane was assessed by rehybridization of the filter with a specific antibody for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
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