QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 9 January 2007
doi: 10.1242/jcs.03331

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mebarek, S. Articles by Némoz, G. Search for Related Content PubMed PubMed Citation Articles by Mebarek, S. Articles by Némoz, G. Social Bookmarking                         What's this?

Inhibition of de novo ceramide synthesis upregulates phospholipase D and enhances myogenic differentiation

¹ INSERM, Unit 585, Villeurbanne, F-69621 France
² INSA-Lyon, Laboratoire de Physiopathologie des Lipides et Membranes, Villeurbanne, F-69621 France
³ IMBL, Villeurbanne, F-69621 France
⁴ Università di Roma-La Sapienza, Dipartimento di Istologia ed Embriologia Medica, Roma, Italy

View larger version (15K): [in this window] [in a new window]   Fig. 1. Ceramide levels in L6 cells induced to differentiate by treatment with Arg8-vasopressin. L6 cells were treated for the time indicated with 10-7 M AVP alone, 20 µM FB1 alone, in the presence of both compounds or left untreated. Lipids were extracted, and the ceramide mass was measured as described in the Materials and Methods using the DAG kinase method. The data are shown as percent of time 0 value (245±35 ng per mg protein, n=14). (A) Short-term (0-2 hours) AVP effects on ceramide levels. (B) Long-term (3 hours-8 days) AVP effects on ceramide levels. The data are the means ± s.e.m. of three to seven independent measurements. *Different from the time 0 value, P<0.05; **P<0.01.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Effects of ceramide on myogenin expression and nuclear accumulation. (A) Immunoblots of myogenin from L6 cells treated for 48 hours by AVP alone, or AVP in the presence of 20 µM FB1, or 100 nM myriocin, or 5-10 µM of short-chain ceramides or left untreated (control). The graph shows the videodensitometric quantitation of myogenin protein: the blots were reprobed for tubulin (data not shown), and myogenin amounts were normalized by tubulin. The means ± s.e.m. of four to nine determinations are shown. The absence of significant effects of FB1 or myriocin on myogenin expression in unstimulated cells was verified (data not shown). *Different from the + AVP value, P<0.05; **P<0.01. (B) Immunofluorescence analysis of myogenin expressed in L6 cells: the cells were left untreated (a, control), or were treated for 48 hours with AVP alone (b), or AVP in the presence of 20 µM FB1 (d), 100 nM myriocin (e) or 10 µM C6-ceramide (c). Nuclear myogenin was revealed by using a monoclonal anti-myogenin antibody and a fluorescein-conjugated secondary antibody. The total number of nuclei was evaluated on the phase contrast image. The mean percentage ± s.e.m. of myogenin-positive nuclei counted in four to six different fields (100 cells per field) from one representative experiment of three performed is shown in the graph. The absence of significant effects of FB1 or myriocin on myogenin nuclear accumulation in unstimulated cells was verified (data not shown). **Different from the + AVP value, P<0.002. Magnification 32x; bar, 50 µm. (C) Time-dependence of ceramide effects: 10 µM C6-ceramide was added at different times after the onset of AVP stimulation. Nuclear myogenin was analyzed by immunofluorescence as in B. The data are the means ± s.e.m. of three independent experiments.

View larger version (74K): [in this window] [in a new window]   Fig. 3. Effects of FB1 on the fusion of AVP-treated L6 cells. (A) L6 cells were treated without (control) or with 10-7 M AVP alone, or with the concomitant addition of 20 µM FB1 for 2 days. The cells were examined by phase contrast microscopy. Magnification, 32x; bar, 50 µm. Myogenic differentiation was evidenced by the formation of multinucleated myotubes. (B) The cells treated as above were examined at different times, and the fusion index was evaluated as detailed in the Materials and Methods.

View larger version (9K): [in this window] [in a new window]   Fig. 4. Effects of ceramide-modulating drugs on the activity of the late differentiation marker creatine kinase. L6 myoblasts were cultured for 6 days in 1% BSA medium in the absence (control) or presence of 10-7 M AVP alone, or with the addition of 20 µM fumonisin, or 100 nM myriocin, or 5 µM C6-ceramide or 10-20 µM PDMP. Creatine kinase-specific activity, expressed as the percentage of the + AVP value (1.7±0.25 OD units per minute per µg protein), was measured in cell homogenates. The mean ± s.e.m. of three measurements is shown. The absence of significant effects of FB1 or myriocin on creatine kinase activity of unstimulated cells was verified (data not shown). *Significantly different from the + AVP value, P<0.01. Three to five independent experiments gave similar results.

View larger version (57K): [in this window] [in a new window]   Fig. 5. Bacterial sphingomyelinase overexpression inhibits AVP-induced myogenic response. To induce the synthesis of endogenous ceramide, L6 cells were transfected with a vector allowing the expression of ER-targeted, GFP-tagged sphingomyelinase. Mock-transfection with a vector lacking the sphingomyelinase sequence was also performed. The transfected cells were cultured for 24 hours in 1% BSA medium in the absence (control) or presence of 10-7 M AVP, and nuclear myogenin immunofluorescence was assessed with a primary anti-myogenin monoclonal antibody and a secondary anti-mouse IgG-rhodamine conjugate. (A) Mock-transfected cells + AVP. (B) Sphingomyelinase-transfected cells + AVP. Bar, 20 µm. The graph shows the means ± s.e.m. of the percentages of myogenin-positive cells as calculated in the sole subsets of green fluorescent cells, in each condition (n=10 fields with 10-15 cells per field).

View larger version (22K): [in this window] [in a new window]   Fig. 6. Effect of PLD isoform selective depletion on myogenic differentiation. The cells were transfected with isoform-specific siRNAs. The cells were switched from proliferation medium (10% FBS) to differentiation medium (1% FBS + 10-7 M AVP), and myogenin immunofluorescence was assessed. The means ± s.e.m. of the percentages of myogenin-positive cells are shown in the graph (n=10 fields). Western blotting analyses were performed with PLD1- and PLD2-specific antibodies to assess the efficacy of siRNA silencing.

View larger version (24K): [in this window] [in a new window]   Fig. 7. Effect of ceramide-modulating drugs on PLD of L6 myoblasts. (A) PLD activity was assayed by evaluating phosphatidylbutanol formation in intact cells. The cells were labeled with [3H]palmitate, left untreated (control) or pretreated by 20 µM FB1 or 10 µM C6-ceramide for 3 hours. Butanol (1%) was added, with or without 10-7 M AVP, for the last 30 minutes before lipid extraction and analysis. Alternatively, the cells were pretreated by 10 µM C6-ceramide for 24 hours before labeling. PLD activity is expressed as the percentage of total phospholipid radioactivity present in phosphatidylbutanol. The means ± s.e.m. of three to five independent experiments are shown. **Different from the + AVP value, P0.001. (B) RT-PCR was performed with total RNA from myoblasts cultured for 3 hours in 1% BSA medium, in the absence (control) or presence of AVP, with or without different agents. Primers specific for either PLD1, PLD2 or β-actin transcripts were used. The graphs show the amounts of PLD1 and PLD2 amplification products normalized by the amounts of β-actin amplification product, as quantified by videodensitometry. The means ± s.e.m. of three experiments are shown for PLD1 (**different from + AVP, P0.01), and the means of two determinations differing by less than 7% are shown for PLD2.

View larger version (89K): [in this window] [in a new window]   Fig. 8. Ceramide impairs the PLD-dependent remodelling of cytoskeleton. (A) L6 cells were transfected with either the empty vector or the PLD1-carrying vector, and stimulated or not by 10-7 M AVP for 15 minutes. F-actin was stained with rhodamine-phalloidin, and the percentage of cells forming stress fibers (SF) was assessed. The means ± s.e.m. of 10 fields are shown. **Indicates a significant difference to the empty vector + AVP value, P<0.001. (B) The cells were pretreated for 3 hours with 10 µM C6-ceramide and/or 100 µM DiC8-PA in 1% BSA medium. They were then stimulated or not with 10-7 M AVP for 15 minutes, and SF formation was assessed as above. Only the cells that formed thick parallel and continuous SFs were considered positive. (C) Fluorescence microscopy of F-actin in the above experiment. AVP stimulation induced the formation of regularly arranged, parallel fibers. When treated by ceramide, the AVP-stimulated cells displayed a fainter actin labeling because of a decrease in the number and thickness of the fibers and the presence of incomplete fibers. An irregular organization of actin, with some radial foci, was also noticed. Conversely, in the presence of PA the cells displayed a dense array of parallel fibers.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter