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Transforming growth factor beta1 receptor II is downregulated by E1A in adenovirus-infected cells - PubMed

  • ️Tue Sep 11 2007

Transforming growth factor beta1 receptor II is downregulated by E1A in adenovirus-infected cells

Vera L Tarakanova et al. J Virol. 2003 Sep.

Abstract

Transforming growth factor beta1 (TGF-beta1) signaling is compromised in many tumors, thereby allowing the tumor to escape the growth-inhibitory and proapoptotic activities of the cytokine. Human adenoviruses interfere with a number of cellular pathways involved in cell cycle regulation and apoptosis, initially placing the cell in a "tumor-like" state by forcing quiescent cells into the cell cycle and also inhibiting apoptosis. We report that adenovirus-infected cells resemble tumor cells in that TGF-beta1 signaling is inhibited. The levels of TGF-beta1 receptor II (TbetaRII) in adenovirus-infected cells were decreased, and this decrease was mapped, by using virus mutants, to the E1A gene and to amino acids 2 to 36 and the C-terminal binding protein binding site in the E1A protein. The decrease in the TbetaRII protein was accompanied by a decrease in TbetaRII mRNA. The decrease in TbetaRII protein levels in adenovirus-infected cells was greater than the decrease in TbetaRII mRNA, suggesting that downregulation of the TbetaRII protein may occur through more than one mechanism. Surprisingly in this context, the half-lives of the TbetaRII protein in infected and uninfected cells were similar. TGF-beta1 signaling was compromised in cells infected with wild-type adenovirus, as measured with 3TP-lux, a TGF-beta-sensitive reporter plasmid expressing luciferase. Adenovirus mutants deficient in TbetaRII downregulation did not inhibit TGF-beta1 signaling. TGF-beta1 pretreatment reduced the relative abundance of adenovirus structural proteins in infected cells, an effect that was potentiated when cells were infected with mutants incapable of modulating the TGF-beta signaling pathway. These results raise the possibility that inhibition of TGF-beta signaling by E1A is a means by which adenovirus counters the antiviral defenses of the host.

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Figures

FIG. 1.
FIG. 1.

Adenovirus infection downregulates the TβRII protein. (A) A549 human lung adenocarcinoma cells were infected with rec700 at 50 PFU/cell. Some of the infected cells were maintained in 20 μg of AraC/ml. Cell lysates collected at 19 h p.i. were subjected to Western blotting using anti-TβRII antibodies (Santa Cruz Biotechnology). Upper and lower arrows, mature and immature forms of TβRII, respectively. (B) A549 cells were infected with rec700 at 10 PFU/cell. Cell lysates were collected at the indicated hours p.i. and analyzed by Western blotting. Equal protein amounts were loaded per lane.

FIG. 2.
FIG. 2.

The E1A 13S or 12S protein, including amino acids 2 to 36 and the CtBP binding site, is required to downregulate the TβRII protein in adenovirus-infected cells. A549 (A and B) or HepG2 (C and D) cells were infected with wild-type (wt) or mutant adenoviruses at 50 PFU/cell. Cell lysates were harvested at 24 h p.i. for the wild type and most mutants and at 48 h p.i. for dl313, 12Swt, dC-term, Ad/E3, 12S.2-36, 12S.928, and dl312. Cell lysates were subjected to Western blotting with anti-TβRII antibodies (Santa Cruz Biotechnology). (E) Schematic of the adenovirus (Ad) genome. The E1A proteins activate the transcription of adenovirus genes and deregulate the cell cycle by suppressing or activating cellular proteins and genes. E1B proteins suppress cellular apoptosis. E3 proteins confer a stealth function to the virus by inhibiting immune cell-mediated apoptosis. E4 proteins function in gene regulation, in part by facilitating degradation of p53; they are also required for viral mRNA transport from the nucleus. Virus DNA replication is necessary for late protein synthesis derived from the major late transcription unit. At about 24 h p.i., virions begin to assemble in the cell nucleus, and after 2 to 3 days cell lysis begins to occur, with the release of virions.

FIG. 3.
FIG. 3.

E1A is required to reduce TβRII mRNA levels in adenovirus-infected cells. HepG2 cells were infected with wild-type (wt) or mutant adenoviruses at 50 PFU/cell and maintained in the presence of AraC throughout the infection. Total RNA was isolated at 19 (mock, rec700, and pm975) and 43 h p.i. (12Swt, 12S.2-36, 12S.928, dl312, and dCtBP). An RNase protection assay was performed using 30 and 6 μg of total RNA for detection of TβRII and GAPDH mRNA, respectively. Lanes a and b (A and B), full-length GAPDH and TβRII probes, respectively, hybridized with yeast (Torula sp.) total RNA without subsequent RNase treatment; lanes c (A and B), both probes hybridized with yeast total RNA and treated with RNase. Radioactively labeled DNA markers (A, lane n) are 300, 200, and 100 bp. RNase-protected TβRII and GAPDH mRNA fragments were quantified by densitometry (bar graphs). The densities of TβRII fragments normalized against the density of GAPDH fragments are presented under the corresponding data.

FIG. 4.
FIG. 4.

Stability of the TβRII protein as determined by pulse-chase analysis in adenovirus-infected cells. (A and D) In two independent experiments, A549 cells were mock infected or infected with rec700 at 50 PFU/cell and maintained in the presence of AraC throughout the infection. At 19 h p.i., cells were pulsed with [35S]methionine-cysteine for 15 min and chased in 10% FCS-supplemented nonradioactive medium for the indicated time periods. Immunoprecipitated, radioactively labeled TβRII was quantified by phosphorimager at each time point of chase (B and E, respectively). (C) Calculations of the TβRII half-life (t1/2).

FIG. 5.
FIG. 5.

E1A inhibits TGF-β-induced signal transduction in adenovirus-infected cells as determined by using the 3TP-lux reporter plasmid. (A) HepG2 cells were transfected with 3TP-lux and pCMV-βGal plasmids. After 6 h of transfection, cells were infected with rec700 at 50 PFU/cell or mock infected. Infections were maintained in the presence of AraC. Cells were treated with TGF-β1 from 12 to 26 h p.i.; subsequently, cells were lysed and luciferase and β-Gal activities were measured. Luciferase values were normalized against β-Gal activity for each sample. Each experimental condition was done in triplicate, and the average values are shown. (B) HepG2 cells were transfected with 3TP-lux and CMV β-Gal plasmids and an empty vector or a plasmid expressing either the 13S or 12S isoform of E1A. At 18 h posttransfection, cells were treated with recombinant TGF-β1 (3 ng/ml) for 8 h. (C) HepG2 cells were treated as described for panel A. Cells were maintained from 12 to 48 h p.i. in the presence of 5 ng of TGF-β1/ml. (D) A549 cells were infected with 50 PFU/cell of rec700 or mock infected and maintained in the presence of AraC. At 20 (lane d) or 24 (lane e) h p.i. cells were mock treated or treated with 5 ng of TGF-β1/ml for 20 min. Levels of phospho-Smad 2 (arrow) were determined by Western analysis. Molecular weight marker positions are shown.

FIG. 6.
FIG. 6.

TGF-β1 suppresses adenovirus late protein synthesis and virus yields in infected cells. A549 cells were maintained in 10 or 0.2% FCS or 0.2% FCS plus 5 ng of TGF-β1/ml as indicated throughout the experiment. Following 3 days of pretreatment, cells were infected with adenovirus mutants at 10 PFU/cell. At 24 h p.i., cell lysates were collected and analyzed by Western blotting using anti-Ad5 (A) and anti-E1A (B) antibodies. Equal protein concentrations were loaded in all lanes. The signal from every lane was measured and quantified with FluorChem software (Alpha Innotech Corporation). (C) Ratio of the signal in serum-starved cells to that in cytokine-treated cells for each adenovirus mutant. (D) A549 cells were treated and infected as for panels A and B. Cell lysates and supernatants were collected, and total virus yields at the indicated times postinfection were determined.

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