Inhibition of thioredoxin reductase 1 by caveolin 1 promotes stress-induced premature senescence

Thioredoxin reductase 1 (TrxR1) is an important antioxidant enzyme that controls cellular redox homeostasis. By using a proteomic-based approach, here we identify TrxR1 as a caveolar membrane-resident protein. We show that caveolin 1, the structural protein component of caveolae, is a TrxR1-binding protein by demonstrating that the scaffolding domain of caveolin 1 (amino acids 82–101) binds directly to the caveolin-binding motif (CBM) of TrxR1 (amino acids 454–463). We also show that overexpression of caveolin 1 inhibits TrxR activity, whereas a lack of caveolin 1 activates TrxR, both in vitro and in vivo. Expression of a peptide corresponding to the caveolin 1 scaffolding domain is sufficient to inhibit TrxR activity. A TrxR1 mutant lacking the CBM, which fails to localize to caveolae and bind to caveolin 1, is constitutively active and inhibits oxidative-stress-mediated activation of the p53/p21Waf1/Cip1 pathway and induction of premature senescence. Finally, we show that caveolin 1 expression inhibits TrxR1-mediated cell transformation. Thus, caveolin 1 links free radicals to activation of the p53/p21Waf1/Cip1 pathway and induction of cellular senescence by acting as an endogenous inhibitor of TrxR1

Ku70 binds to the scaffolding domain of caveolin-1.

<p>(<b>A</b>) The consensus caveolin binding domain (CBD) is shown together with the caveolin binding domain of Ku70 (amino acids 471 to 478) and a mutant form of Ku70’s CBD in which aromatic residues were mutated to alanines. (<b>B</b>) Ponceau S staining of GST alone and GST-caveolin-1 fusion proteins. (<b>C</b>) GST-caveolin-1 fusion protein pull-down assays were performed using cell lysates from HCT116 cells transiently transfected with wild type Ku70-HA. A blot that is representative of two independent experiments is shown.</p

Chemotherapeutic drugs promote the Ku70-Cav-1 interaction.

<p>(<b>A</b>) HCT116 cells were treated with either etoposide or 5-FU for 24 hours. Untreated cells were used as control. Cell lysates were then immunoprecipitated with an antibody probe specific for caveolin-1 and immunoprecipitates subjected to immunoblotting analysis with anti-Ku70, anti-Bax and anti-caveolin-1 IgGs. (<b>B</b>) HCT116 colon cancer cells were transfected with HA-tagged wild type Ku70. One day after transfection, cells were treated with 5-FU for 24 hours. Untreated cells were used as control. Cells were then subjected to immunofluorescence analysis using antibody probes specific for the HA tag (green) and caveolin-1 (red). Nuclei were detected by DAPI staining (Blue). Yellow staining in the merged images shows co-localization between caveolin-1 and Ku70 only after 5-FU treatment (see arrows). Representative images are shown.</p

Knockdown of caveolin-1 protein expression sensitizes HCT116 colon cancer cells to apoptosis.

<p>HCT116 colon cancer cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. (<b>A</b>) Endogenous caveolin-1 expression was determined by immunoblotting analysis 24, 48 and 96 hours after transfection using an antibody probe specific for caveolin-1. Immunoblotting with anti-β-actin IgGs was done to show equal loading. (<b>B–D</b>) One day after transfection, HCT116 cells were treated with different concentrations of 5-FU for 48 hours. Untreated cells were used as control. In (<b>B</b>), cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. Values represent mean ± SEM; *<i>P</i><0.001; ^#<i>P</i><0.005. In (<b>C</b>), cells were subjected to Annexin V staining and flow cytometry. Values represent mean. In (<b>D</b>), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3 and PARP. Immunoblotting with anti-β-actin IgGs was done to show equal loading.</p

Φ→A Ku70 fails to interact with caveolin-1, to protect against chemotherapeutic drug-induced apoptosis and to limit the 5-FU-induced dissociation of Bax from Ku70.

<p>HCT116 cells were transfected with either wild type Ku70-HA (WT Ku70) or Φ→A Ku70-HA (Φ→A Ku70) and treated with different concentrations of either 5-FU (for 24 h in A and E; for 48 h in C and D) or etoposide (for 24 h in C and D). Untreated cells were used as control. In (A), cell lysates were immunoprecipitated with an antibody probe specific for caveolin-1 and immunoprecipitates subjected to immunoblotting analysis with anti-HA, anti-Bax and anti-caveolin-1 IgGs. In (B), total expression of WT Ku70-HA and Φ→A Ku70-HA was detected by immunoblotting analysis with anti-HA IgGs before drug treatment. In (C), cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. Values represent mean ± SEM; *<i>P</i><0.001. In (D), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3. Immunoblotting with anti-β-actin IgGs was done to show equal loading. In (E), cell lysates were immunoprecipitated with and antibody probe specific for Bax and immunoprecipitates subjected to Western blotting analysis with anti-HA and anti-Bax IgGs.</p

Caveolin-1 promotes long-term survival and inhibits apoptosis of colon cancer cells after treatment with chemotherapeutic drugs.

<p>HCT116 (A, B, E and F) and HT29 (C and D) colon cancer cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. One day after transfection, cells were treated with different concentrations of 5-FU for 48 h (A–D) or etoposide for 24 h (E and F). Untreated cells were used as control. In (A and B), cells were cultured for 7 days and stained with crystal violet. A crystal violet staining that is representative of three independent experiments is shown in (A), quantification of the number of clones after crystal violet staining is shown in (B). Values represent mean ± SEM; *<i>P</i><0.001; ^#<i>P</i><0.005. In (C and E), cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. Values represent mean ± SEM; *<i>P</i><0.001; ^#<i>P</i><0.005. In (D and F), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3 and PARP. Immunoblotting with anti-β-actin IgGs was done to show equal loading.</p