Also, these authors showed the involvement of ubiquitin-proteasome-dependent ERAD system in the constitutive degradation of mouse SCD1 endogenously expressed in adipocyte-differentiated NIH 3T3-L1 cells

Also, these authors showed the involvement of ubiquitin-proteasome-dependent ERAD system in the constitutive degradation of mouse SCD1 endogenously expressed in adipocyte-differentiated NIH 3T3-L1 cells. Hu et al., 2011). Apart from potentiating insulin resistance, increased ceramide generation has been shown to induce endoplasmic reticulum (ER) stress, which plays a fundamental role in the pathogenesis of several diseases such as diabetes, malignancy and neurodegenerative disorders (Salminen et al., 2010; Schonthal, 2012). A recent study has shown that fenretinide (N-(4-hydroxyphenyl)retinamide, NAV3 4HPR) a synthetic derivative of all-retinoic acid originally developed as a chemotherapeutic agent, improved insulin sensitivity in mouse liver and muscle mass cells by blocking the formation of ceramide due to its ability to inhibit dihydroceramide desaturase (Des1) (Bikman et al., 2012; Rahmaniyan et al., 2011). Fenretinide has been shown to activate the expression of alkaline ceramidase 2 (ACER2), an enzyme that catalyzes the hydrolysis of dihydroceramides to generate dihydrosphingosine (Mao et al., 2010). It also been shown to increase the activity of serine palmitoyl transferase (SPT), which catalyzes the first rate-limiting step in the synthesis of ceramides involving the condensation of L-serine with palmitate (Wang et al., 2001). The synthesis of ceramide from saturated fatty acids such as palmitate has been shown to increase the activity of SPT, while silencing the expression of SPT decreases palmitate-driven ceramide synthesis, and curbs lipid-induced insulin resistance (Watson et al., 2009). Interestingly, deleting expression has been shown to decrease ceramide synthesis by down-regulating SPT expression in mice skeletal muscle mass (Peter et al., 2009). Furthermore, EC-17 deficiency increased insulin sensitivity in mice, whereas increased SCD activity contributed to the insulin resistance in humans and animals (Dobrzyn et al., 2010; Garcia-Serrano et al., 2011; Gutierrez-Juarez et al., 2006; Peter et al., 2009; Rahman et al., 2003). Thus, it is possible that SCD could play an important role in mediating the effects of fenretinide on apoptosis and insulin signaling. However, the effect of fenretinide on SCD expression is not yet known. Retinal pigment epithelium (RPE) is usually a single layer of epithelial cells located between the light-sensing photoreceptor cells and the choriocapillaris. A normally functioning RPE is usually indispensable for vision, and any disruption or RPE cell death could hasten retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration (AMD) (Sparrow et al., 2010). Indeed fenretinide has been proposed as a treatment for the geographic atrophy form of AMD (Mata et al., 2012). We have shown earlier that fenretinide induces apoptosis in cultured human RPE cells (Samuel et al., 2006). We have also reported that SCD is usually expressed in RPE cells and that its expression is usually regulated by all-retinoic acid (Samuel et al., 2001; Samuel et al., 2002). The present work is undertaken to study the potential regulation of SCD during fenretinide-induced apoptosis in ARPE-19 cells, a human RPE cell collection. We show that fenretinide-induced ER stress decreased the SCD protein and enzymatic activity in RPE cells via an ubiquitin-dependent proteasomal pathway. Materials and Methods Materials Fenretinide, MG132, PSI, lactacystin, mono- and polyubiquitinated antibody, mouse anti-actin and anti–tubulin antibodies were obtained from Enzo Life Sciences, Inc. (Farmingdale, NY). D3-stearate and D3-palmitate were obtained from Cambridge EC-17 Isotope Laboratories, Inc. (Andover, MA). PYR41, inhibitor of ubiquitin activating enzyme E1, was from LifeSensors, Inc. (Malvern, PA). EC-17 Monoclonal anti-SCD antibody was obtained from Kamiya Biomedical Organization (Seattle, WA), and OriGene Technologies (Rockville, MD). Rabbit polyclonal BiP/GRP78 antibody was from Abcam EC-17 (Cambridge, MA). The enhanced chemiluminescence (ECL) detection system and peroxidase-conjugated anti-rabbit EC-17 and anti-mouse antibodies were from GE Healthcare Life Sciences (Piscataway, NJ). Cells and culture conditions Human retinal pigment epithelial cells (ARPE-19 cells) obtained from ATCC (Manassas, VA) were produced in Dulbecco’s altered Eagle’s medium (DMEM) containing nutrient combination F12 (Cellgro, Herdon, VA) supplemented with 5% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 g/ml) as explained previously (Samuel et al., 2001). Cells were seeded onto tissue culture plates at a density of 2105 cells/ml in total medium and allowed to grow overnight. The culture medium was replaced.