Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells - PubMed (original) (raw)
Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells
E Felley-Bosco et al. Proc Natl Acad Sci U S A. 2000.
Abstract
To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway.
Figures
Figure 1
Levels of iNOS protein were reduced on ectopic expression of cav-1 in HT29 and DLD1 cells. iNOS and cav-1 protein levels were investigated by Western blot analysis. (A) The kinetics of cytokine-induced iNOS protein expression in HT29 and cav-1-expressing C13 cells is shown. Reduced levels of iNOS expression were apparent as early as 6 h after cytokine stimulation and remained low throughout the experiment up to 24 h after induction. Actin was used as control for protein loading. (B) Analysis of parental, mock-, and cav-1-transfected (C13) HT29 cells in the presence (+) or absence (−) of cytokines (cyt) and/or IPTG (150 μM). Extracellular signal-regulated kinase 1/2 (erk1/2) was used as control for protein loading in each lane. (C) iNOS and cav-1 protein levels were determined in DLD1-, mock-, and cav-1-transfected C4 cells after stimulation of the cells with cytokines. As above for HT29 cells, iNOS protein levels were substantially lower in cells ectopically expressing cav-1. Migration positions of molecular-mass marker proteins are indicated to the left of the individual panels.
Figure 2
Cav-1 expression did not alter iNOS mRNA levels induced by cytokines. iNOS mRNA levels were investigated by Northern blot analysis in parental, mock-, and cav-1-transfected (C13 and C16) HT29 cells in the presence (+) or absence (−) of cytokine (cyt) stimulation (15 h). No significant difference in iNOS mRNA levels was observed between the different cell lines. Labeling of the 18S RNA band is shown as a control for loading in each lane.
Figure 3
Cofractionation of cav-1 and iNOS in detergent-insoluble fractions. Mock- or cav-1-transfected (C13) HT29 cells were stimulated during 15 h with cytokines, lysed in 1% Triton X-100, and fractionated on a sucrose gradient. Samples from each fraction (1–12) of the sucrose gradient (plus pellet; fraction 13) were characterized by Western blot analysis. (A) Total protein distribution in a representative gradient is shown after Ponceau Red S staining (10 μl was loaded for each fraction). (B) Western blot analysis of iNOS and cav-1 expression. The abundance of iNOS in the light fraction was determined by comparing increasing volumes (10, 100, or 500 μl) of fraction 4 and fraction 10 (10 or 100 μl). PKCα was used as a control protein that was absent from cav-1-containing fractions. Nr, number.
Figure 4
In vitro binding of GST–cav-1 fusion proteins to iNOS. Cytosolic fractions (200 μg of protein) of cytokine-induced HT29 cells were precleared with GST-agarose beads, and incubated with immobilized GST or GST–cav-1 fusion proteins. After washing, bound proteins were eluted in Laemmli buffer and analyzed by Western blotting. Bound iNOS was detected in eluates from GST–caveolin (), and GST–caveolin () beads, but not from beads with immobilized GST, GST–caveolin (1–31), or GST–caveolin (). As a control, GST–cav-1 fusion proteins were visualized by a cav-1-specific antibody revealing that similar amounts of each fusion protein were immobilized in each case. Cav-1 fusion proteins of the expected size are indicated with arrows.
Figure 5
Cav-1-induced degradation of iNOS was mediated by the proteasome in detergent-insoluble fractions. Cells were stimulated for 15 h with cytokines before the addition of inhibitors for another 9 h. Degradation of iNOS protein was prevented by treatment of the cells with ALLN (10 μM) or lactacystin (lact.; 10 μM) but not E64D (50 μM) in both HT29 (A) and DLD1 (B) cells, independent of whether the parental, mock-, or cav-1-transfected (C13 or C4) cells were treated. (C) Mock- or cav-1-transfected (C13) HT29 cells were stimulated with cytokines, treated with ALLN, and fractionated on sucrose gradients as described.
References
- Moncada S, Palmer R M, Higgs E A. Pharmacol Rev. 1991;43:109–142. - PubMed
- Griffith O W, Stuehr D J. Annu Rev Physiol. 1995;57:707–736. - PubMed
- Nathan C, Xie Q W. Cell. 1994;78:915–918. - PubMed
- Marletta M A. Cell. 1994;78:927–930. - PubMed
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