Fenretinide induces ubiquitin-dependent proteasomal degradation of stearoyl-CoA desaturase in human retinal pigment epithelial cells

Abstract

Stearoyl-CoA desaturase (SCD, SCD1), an endoplasmic reticulum (ER) resident protein and a rate-limiting enzyme in monounsaturated fatty acid biosynthesis, regulates cellular functions by controlling the ratio of saturated to monounsaturated fatty acids. Increase in SCD expression is strongly implicated in the proliferation and survival of cancer cells, whereas its decrease is known to impair proliferation, induce apoptosis, and restore insulin sensitivity. We examined whether fenretinide, (N-(4-hydroxyphenyl)retinamide, 4HPR), which induces apoptosis in cancer cells and recently shown to improve insulin sensitivity, can modulate the expression of SCD. We observed that fenretinide decreased SCD protein and enzymatic activity in the ARPE-19 human retinal pigment epithelial cell line. Increased expression of BiP/GRP78, ATF4, and GADD153 implicated ER stress. Tunicamycin and thapsigargin, compounds known to induce ER stress, also decreased the SCD protein. This decrease was completely blocked by the proteasome inhibitor MG132. In addition, PYR41, an inhibitor of ubiquitin activating enzyme E1, blocked the fenretinide-mediated decrease in SCD. Immunoprecipitation analysis using anti-ubiquitin and anti-SCD antibodies and the blocking of SCD loss by PYR41 inhibition of ubiquitination further corroborate that fenretinide mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. Therefore, the effect of fenretinide on SCD should be considered in its potential therapeutic role against cancer, type-2 diabetes, and retinal diseases.

Fig. 1. SCD protein is decreased during fenretinide induced apoptosis in human RPE cells

ARPE-19 cells were treated with indicated concentrations of fenretinide (4HPR) for 24 h. Panel A, Phase-contrast microscopic analysis. Panel B, Fenretinide induced apoptosis. Mono-and oligonucleasomes formation was analyzed by ELISA. Panel C, Fenretinide treatment decreases SCD protein. Cell lysates were analyzed by western blotting using antibody against SCD. α-Tubulin shows that the amount of protein used in different samples were similar. *P < 0.01 compared to control. The values are mean ± SD, n=4.

Fig. 2. SCD enzyme activity is decreased by fenretinide in ARPE-19 cells

ARPE-19 cells were treated with or without fenretinide (4HPR) for 16 h before incubating with 50 μM D3-stearate or 50 µM D3-palmitate for 5h. Lipids were extracted then analyzed by LCMS. Panel A, Extracted ion chromatograms of D3-oleic acid, m/z 284.3. *The peak denotes the 13C isotope of naturally occurring stearic acid in the cell. Panel B, Extracted ion chromatograms of D3-palmitoleic acid, m/z 256.4. *The peak denotes the 13C isotope of naturally occurring palmitic acid in the cell. Panel C, The histogram shows the effect of fenretinide on the ratio of unsaturated fatty acid to saturated fatty acid.

Fig. 3. Proteasome inhibitors blocks fenretinide-induced decrease in SCD protein

ARPE-19 cells were treated with fenretinide (4HPR) in the presence or absence of proteasome inhibitors MG132 and PSI, and analyzed by western blotting using SCD antibody. α-Tubulin expression shows that the amount of protein used in different samples were similar.

Fig. 4. Fenretinide induces ER stress in ARPE-19 cells

ARPE-19 cells were treated with indicated concentrations of fenretinide (4HPR) for 24 h in the presence or absence of MG132. Panel A, RT-PCR analysis of ATF4 expression. Panel B, RT-PCR analysis of GADD153 expression. Panel C, Fenretinide treatment increases BiP/GRP78 protein expression when analyzed by western blotting. α-Tubulin expression shows that the amount of protein used in different samples were similar. *P < 0.01 compared to control. The values are mean ± SD, n=4.

Fig. 5. Endoplasmic reticulum stress inducers decrease SCD protein

ARPE-19 cells were treated with tunicamycin and thapsigargin in the presence or absence of MG132, and SCD protein was analyzed by western blotting. The decrease in SCD protein induced by tunicamycin (Tun) and thapsigargin (Thaps) was blocked by MG132. α-Tubulin expression shows that the amount of protein used in different samples are similar.

Fig. 6. Fenretinide induces the accumulation of polyubiquitinated proteins

ARPE-19 cells were treated with fenretinide (4HPR) in the presence or absence of MG132. The cell lysates were used for ubiquitin enrichment as described under Materials and Methods, and then analyzed by western blotting using anti-ubiquitin antibody. Panel A, Fenretinide induced the accumulation of polyubiquitinated proteins in the presence of MG132. Cell lysate from epoxomicin-treated HeLa cells was used as a positive control (P.C). Panel B, Polyubiquitinylated SCD accumulates in the presence of MG132. ARPE-19 cells were treated with fenretinide (4HPR) in the presence or absence of MG132, and the cells were solubilized under denaturing conditions with a buffer containing SDS (see Materials and Methods) and subjected to immunoprecipitation using mouse monoclonal anti-SCD antibody. The immunoprecipitates were then analyzed by western blotting using rabbit polyclonal anti-mono- and polyubiquitin antibody.

Fig. 7. Fenretinide induces ubiquitination of SCD protein in ARPE-19 cells

ARPE-19 cells were treated with fenretinide (4HPR) in the presence or absence of MG132. The cell lysates prepared under non-denaturing conditions were used for the immunoprecipitation analysis using biotinylated mouse monoclonal SCD antibody. The immunoprecipitates were then analyzed by western blotting using anti-mono- and polyubiquitin antibody. Panel A, Western blot analysis of mono-and polyubiquitinated proteins in the cell lysates after immunoprecipitating with biotinylated mouse monoclonal anti-SCD antibody. Panel B, Western blot analysis of SCD in cell lysate prior to immunoprecipitation with anti-SCD mouse monoclonal antibody. α-Tubulin expression shows that the amount of protein used in different samples were similar.

Fig. 8. Fenretinide induces SCD protein degradation by the ubiquitin-proteasome system

PYR41, an inhibitor of ubiquitin activating enzyme E1, inhibited fenretinide-induced degradation of SCD. ARPE-19 cells were treated with fenretinide (4HPR) in the presence or absence of PYR41, and then analyzed by western blotting using antibody specific to SCD. α-Tubulin expression shows that the amount of protein used in different samples were similar.