The down-regulation of SREBP-1 and the up-regulation of SREBP-2 proteins led us to investigate whether the transcriptional levels could be affected in SM-enriched adipocytes

No substantial alterations ended up observed in the scenario of PPMP treatment method (Desk 5).Initial, the expression of SREBPs proteins was examined in SM-enriched adipocytes1312445-63-8 (Fig 4A and 4B). The nuclear fraction was immunoblotted with antibodies that recognize the energetic N-terminus of SREBP proteins. A sixty eight kDa band was detected in the nuclear portion and reduced in the case of SREBP-1 [-37% (P<0.01), -42% (P<0.01), -63% (P<0.005)] but increased in the case of SREBP-2 [71% (P<0.005), 42% (P<0.01), 40% (P<0.005)] by the SM treatments (Fig 4A). The down-regulation of SREBP-1 and the up-regulation of SREBP-2 proteins led us to investigate whether the transcriptional levels could be affected in SM-enriched adipocytes. First, the mRNA levels were estimated by conventional semi-quantitative RT-PCR (Fig 4B). Actin was selected as the housekeeping gene due to its stable expression across the treatments. The SREBP-1 mRNA levels were reduced by -12% (NS), -14% (P<0.05) and -23% (P<0.001),time-dependent response of membrane fluidity to sphingomyelin treatment. Cells were treated with SM-LA (15 M) or vehicle for the indicated incubation times. The cells were labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), and the fluorescence anisotropy of the probe was determined. The measurements were performed at 37 for 2 min immediately after the addition of the fluorescent probes. Each value is the meanEM of three independent experiments. P<0.05, P<0.005 and P<0.001 SM-treated cells compared with control cells. Fluorescence anisotropy Control SM-LA (2 h) SM-LA (24 h) SM-LA (48 h) SM-LA (72 h) sphingomyelin enrichment decreased membrane fluidity in 3T3-F442A adipocytes. 3T3-F442A adipocytes were treated with 15 M exogenous natural SM, 10 mM GSH, 20 M PPMP or vehicle for 24 h. The cells were labeled and the measurements were performed as indicated in Table 4. Each value is the meanEM of four independent experiments. P<0.0005 and P<0.0001 SM-, GSH-, and PPMP-treated cells compared with untreated cells. Fluorescence anisotropy Control SM-enriched cells SM-enriched cells SM-enriched cells SM-enriched cells SM-unmodulated cells whereas SREBP-2 mRNAs increased by 19% (NS), 22% (P<0.005) and 25% (P<0.01) with SM-PA, SM-SNA and SM-LA treatments, respectively. Additionally, the semi-quantitative PCR results were validated by real-time PCR, and the effects of GSH and PPMP were also investigated. The melting curves of the three genes revealed the absence of non-specific products. SM, GSH or PPMP treatments did not induce any significant change in the amounts of -actin mRNA. The data indicated that the regulation of SREBPs in adipocytes treated by N-SMase inhibitor or exogenous SM followed identical patterns. In cells treated with exogenous SMs, SREBP transcript expression significantly decreased by -2.5-fold for SREBP-1 (P<0.0001) but increased by 2.44-fold for SREBP-2 (P<0.0001) (Table 6). However, the changes in SREBP-1 were more noticeable after GSH treatment SREBP-1 gene expression decreased by approximately 5-fold. Interestingly, the changes were moderate in SM-unmodulated cells treated with PPMP. However, these changes were opposite those changes observed for the SM-enriched cells (Table 6) we noted a significant increase in SREBP-1 expression. To examine whether the changes in mRNA levels were due to the modulation of gene transcription or to changes in mRNA stability, we used the transcription inhibitor actinomycin D. 3T3-F442A adipocytes were treated with SM for 24 h, as described previously, followed by treatment with actinomycin D (5 g/ml). Total RNAs were subsequently extracted at time points between 0 and 8 h. The mRNA levels of SREBP-1 and SREBP-2 were determined as previously described and normalized to -actin. Regardless of the studied gene, the degradation rate of mRNAs in SM-enriched cells did not significantly differ from that in control cells, suggesting that SM did not affect mRNA stability but regulated gene expression at the transcriptional level (data not shown).MAP kinase is a family of evolutionarily conserved serine/threonine kinases that includes ERK1/2, SAPK/JNK, and p38. In support of the strong linkage between the MAP kinasedependent pathway and SREBP-1 expression and to better understand the physiological implications of SM actions, we determined whether the phosphorylation of p44/42 ERK, JNK and p38 MAP kinases was affected by SM. We found that SM promoted the inhibition of ERK1/2 phosphorylation in a dose-dependent manner but had no effects on p38 or JNK (Fig 5A and 5B). SM caused the inhibition of the basal levels of phospho-ERK1 and phospho-ERK2 proteins in a dose-dependent manner, with 50% effective doses (ED50) of approximately 30 M and 40 M, respectively. Then, we measured the amounts of total and phosphorylated ERK proteins in cells incubated with or without natural SM, GSH and PPMP compounds. As shown in Fig 5C, the levels of phospho-ERK were reduced in SM-enriched cells parallel to a decrease in the total levels of the protein. The decreased levels of phospho-ERK1/2 were -13% (NS), -47% (P<0.005), -57% SREBP protein and mRNA expression in SM-enriched adipocytes. Cells were differentiated into adipocytes and treated with SMs as indicated in Fig 3. (A) Immunoblot analysis of nuclear SREBP proteins. Nuclear extracts were separated by SDS-PAGE (30 g of protein) and immunoblotted with SREBP-1 (H-160) and SREBP-2 (N-19) polyclonal antibodies raised against epitopes mapping at the N-terminus of SREBP. (B) Semi-quantitative RT-PCR analysis of SREBP-1 and SREBP-2 mRNA levels. Total RNAs were reversetranscribed and amplified by PCR as described in the Materials and methods. Amplified products were separated on an agarose gel and stained with ethidium bromide. Representative blots are shown under the histograms. The results are expressed as percentages of SM-untreated cells and are the meanEM of three independent experiments, performed in duplicate, triplicate or quadruplicate. P<0.05, P<0.01, P<0.005 and P<0.001 SM-treated cells compared with untreated cells.Real-time quantitative RT-PCR determination of SREBP-1 and SREBP-2 mRNA levels in SM-enriched adipocytes or unmodulated adipocytes. 3T3-F442A adipocytes were treated with 15 M natural SM (SM-LA, 24 h), 10 mM GSH (24 h), 20 M PPMP (24 h) or vehicle. The mRNA levels of the studied genes were determined. The results are expressed as-fold variations over respective controls (R) after normalization to -actin, as indicated in the Materials and methods. R values superior or equal to 2 were considered positive regulation of gene expression, whereas values lower than 0.5 indicated negative regulation. The results presented are the means of 3 independent experiments, which were performed twice each in duplicate. SREBP-1 R Control SM-enriched cells SM-enriched cells SM-unmodulated cells inhibition of ERK proteins, but not p38 MAPK and JNK, in SM-enriched adipocytes. Adipocytes were treated for 24 h with the indicated concentrations of SM-LA (A and B) or 30 M SM, 10 mM GSH, 20 M PPMP or vehicle (C and D). Cell lysates (40 g of proteins) were separated by SDS-PAGE and immunoblotted using antibodies recognizing (1) the phosphorylated (activated) forms of ERK (p44/42 Tyr204), p38 (Tyr180/Tyr182), JNK (Thr183/ Tyr185), and (2) total ERK, p38, and JNK proteins. (A,C) Representative blots of phosphorylated and total proteins are shown. (B) Quantitative variations in phospho-ERK/JNK/p38 amounts relative to total proteins. (D) Quantitative variations in total and phospho-ERK amounts: the blots of p42 and p44 ERKs shown in (C) were combined to quantify the amounts of total ERK1/2 proteins. The results are expressed as percentages of control cells and are the meanEM of four independent experiments, which were each performed in duplicate. P<0.05, P<0.01 and P<0.005 SM-, GSH-, and PPMPtreated cells compared with untreated cells.MEK1/2 selective inhibitor (PD98059) and SM reduce the phosphorylation of ERK and SREBP-1. Cells were treated 24 h with 50 M PD98059 with or without 30 M SM-LA. Cell lysates were separated by SDS-PAGE and immunoblotted with affinity-purified: (1) monoclonal antibody raised against a sequence containing phosphorylated Tyr204 of ERK1/2 and polyclonal antibody raised against a peptide mapping subdomain X1 of ERK and (2) with polyclonal antibodies raised against SREBP-1 or SREBP-2, as indicated in Fig 4. Representative blots (A) and quantitative variations (B) are shown. The results are expressed as the percentages of maximum and are the meanEM of four (ERK and SREBP-1) and six (SREBP-2) independent experiments, which were each performed in duplicate. P<0.05, P<0.01 and P<0.005 SM/ PD98059-treated cells compared with control cells.To determine whether ERK is required for the SM action, the role of PD98059, which is a selective inhibitor of MEK1/2 (the direct upstream kinase activator of ERK1/2) that inhibits ERK1/ 2 phosphorylation, was examined (Fig 6). PD98059 strongly inhibited the phosphorylation of ERK1/2 proteins (-71%, P<0.005) and significantly diminished the levels of the p68 SREBP-1 proteins (-34%, P<0.01) in SM-untreated cells. In SM-treated cells, the reduction in ERK phosphorylation by PD98059 was maintained (-84%, P<0.005), and the reduction in the expression of the mature SREBP-1 protein was reinforced (-55%, P<0.01). PD98059 is known to inhibit of Ras, Raf, MEK(A and B)and KSR (C and D) by SM in 3T3-F442A adipocytes. Cells were treated for 24 h with the indicated concentrations of SM-LA. Cell lysates (300 g of protein) were separated by SDS-PAGE and immunoblotted with antibodies recognizing (1) the phosphorylated (activated) forms of Raf-1 (Tyr340/341), MEK-1/2 (Ser218/222) and KSR-1 (Ser392) and (2) total pan-Ras (N/H/K-Ras), Raf-1, MEK1/2 and KSR-1. Quantitative variations in phosphorytated amounts relative to total proteins are shown. The Ras activity was determined by ELISA (60 g of cell lysate protein): active GTP-bound state of Ras is detected and measured quantitatively through the addition of a monoclonal anti-Ras antibody that detects K-, H-, N- Ras isoforms. GTP-Ras is normalized to the total-Ras. The results are expressed as percentages of SM-untreated cells and are the meanEM of four independent experiments, which were each performed twice. P<0.05, P<0.01 and P<0.005 SM-treated cells compared with untreated cells.SREBP-1 expression however, SREBP-2 seems to be up-regulated by PD98059 in control and SM-enriched 3T3-F442A cells, with expression levels of 54% (P = 0.031), 40% (P = 0.015) and 72% (P = 0.003) when these cells were treated with PD98059, SM and SM+PD98059, respectively.To elucidate whether Ras/Raf/MEK are required for ERK inhibition by membrane SMs, cells were treated with various concentrations of SM-LA (040 M), and anti-phosphoMEK1/2, anti-phosphoRaf-1 and anti-Ras immunoprecipitates were analyzed (Fig 7A and 7B). SM reduced the levels of phosphorylated proteins as well as the total proteins in a dose-dependent manner. Remarkably, SM dramatically reduced Ras protein levels the maximal inhibition reached -80% (P<0.005) with 120 M of SM. For the same concentration of SM, the ratio of phospho/total-Raf-1 and phospho/total-MEK1/2 protein levels decreased by -44% (P<0.005) and -33% (P<0.01), respectively. Ras proteins function as GDP/GTP-regulated binary switches in signal transduction cascades. Ras activity was determined by ELISA by detecting the active Ras (GTP bound state) relative to the total-Ras proteins. We observed a significant decrease in the activation of Ras by SM-LA (040 M), reaching a maximal inhibition value of -72% (P<0.01) with 60 M of SM (Fig 7B). These decreases are consistent with our findings for ERK1/2 regulation by SM.The next set of experiments examined the effects of SM on KSR, the Kinase Suppressor of Ras. KSR is a molecular scaffold that interacts with the components of the Raf/MEK/ERK kinase cascade and that positively regulates ERK signaling. KSR was defined in genetic screens as downstream of Ras and either upstream or parallel to Raf-1 [42]. KSR facilitates the phosphorylation of MEK by RAF. In mammalian cells, ceramide-activated protein kinase (CAPK) has been considered the counterpart of KSR [43], implicating Raf-1 as a ceramide-activated kinase. Cells were treated with increasing concentrations of SM-LA for 24 h. Whole-cell extracts were immunoblotted using a KSR-1 and phospho-KSR-1 antibodies. SM treatment caused decreases in the phosphorylated and total levels of KSR-1 protein in a dose-dependent manner (Fig 7C and 7D).Another distinctive characteristic of the SM-enriched cells could be altered expression of the caveolin gene. Based on several important observations, one of the most attractive candidates that links the Ras-p42/44 MAP kinase signaling to SREBP is caveolin [19,20]. Caveolins are cholesterol-binding integral membrane proteins that are distributed primarily at the plasma membrane but also in the Golgi complex. These proteins exist as three distinct isoforms, where caveolin 1 (Cav-1) and 2 (Cav-2) are strictly co-localized within the plasma membrane [44]. Therefore, we were interested in elucidating any possible relation between Cav-1 and Cav-2 and SM. First, cells were treated for 24 h with SM-LA (30 M), GSH (10 mM), PPMP (20 M) or vehicle, and the expression levels of Cav-1 and Cav-2 mRNA were examined. Cav-1 and Cav-2 mRNA were enhanced in the presence of exogenous SM-LA (4.01 and 2.35 fold) or GSH (5.37 and 4.64 fold), respectively (Table 7), whereas no changes were observed in PPMPtreated cells. Caveolins mRNA stability were also assessed, as previously described, using the transcription inhibitor actinomycin D. The degradation rate in SM-enriched cells did not significantly differ from that in control cells, suggesting that SM regulates gene expression at the transcriptional level (data not shown). Caveolins were significantly more abundant in the case of SM-LA treatment (Fig 8). The increases for Cav-1 and Cav-2 were respectively 2.1- and 1.7-fold for mRNA, and 2.2- and 1.7-fold for the cellular protein. In total membranes, the increases were: 2.0- and 1.8-fold respectively. Interestingly, the amounts of caveolins proteins were markedly higher (3.3- and 2.7-fold, respectively) in the plasma membranes of SM-enriched adipocytes.Real-time RT-PCR determination of caveolins mRNA levels in SM-enriched adipocytes or unmodulated adipocytes. 3T3-F442A adipocytes were treated with 30 M exogenous natural SM (SM-LA, 24 h), 10 mM GSH (24 h), 20 M PPMP (24 h) or vehicle. 2859375The results are expressed as-fold variation over respective controls (R) after normalization to -actin, as indicated in the Materials and methods. R values greater than or equal to 2 were considered positive regulation of gene expression, whereas values lower than 0.5 indicated negative regulation. The results presented are the means of 3 independent experiments, which were performed twice each in duplicate. Cav-1 R Control SM-enriched cells SM-enriched cells SM-unmodulated cells effects of exogenous sphingomyelins on caveolin expression.