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First published online April 1, 2009
doi: 10.1242/10.1242/jcs.041665

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Necdin is expressed in cachectic skeletal muscle to protect fibers from tumor-induced wasting

¹ Division of Regenerative Medicine, San Raffaele Scientific Institute, 20132 Milan, Italy
² Department of Experimental Medicine, University of Milano-Bicocca, 20052 Monza, Italy
³ Department of Preclinical Sciences, LITA-Vialba, University of Milano, 20157 Milan, Italy
⁴ E. Medea Scientific Institute, 23842 Bosisio Parini, Italy

View larger version (17K): [in this window] [in a new window]   Fig. 1. Necdin inhibits colon carcinoma-induced muscle wasting. (A) Necdin expression measured by real-time PCR on TA muscle from PBS-treated (`C') or C26-treated animals sacrificed 6, 8, 10 and 12 days after tumor injection. Five animals were analyzed in parallel for each time. Results were normalized to levels of the GAPDH mRNA. Error bars represent s.e.m.; **P<0.005 vs PBS-treated mice. (B) Body weight of both PBS- and C26-treated animals was measured the day of tumor injection and every 2 days. Ten animals were analyzed in parallel for each group: wild-type mice (wt), MlcNec mice and Ndn–/– mice treated with C26. Results are expressed as % ± s.e.m. of weight obtained for PBS-treated animals of each group (PBS) (n=10 per group). The mean weight ± s.d. of all the PBS-treated animals was 21.60±0.52 g on the day of injection and 24.95±0.88 g 10 days later. *P<0.01 vs WT. (C) Glycemia measurement. Serum glucose level of PBS- and C26-treated animals was measured 8 days after tumor injection. Ten animals were analyzed in parallel for each C26-treated group: wild-type mice (wt), MlcNec mice and Ndn–/– mice. Error bars represent s.e.m.; *P<0.01 vs WT; +P<0.01 vs PBS treated mice. (D) TA, gastrocnemius (Gst) and quadriceps (Quad) muscles and liver and epydidimal fat (Ep.fat) were isolated from both PBS- and C26-treated animals 12 days after tumor injection and weighed. Ten animals were analyzed in parallel for each group: wild-type mice (wt), MlcNec mice and Ndn–/– mice. Error bars represent s.e.m.; *P<0.01 and **P<0.005 vs WT; +P<0.01 vs PBS-treated mice. PBS results in B-D are mean values from PBS-treated animals of all three genotypes.

View larger version (69K): [in this window] [in a new window]   Fig. 2. Necdin inhibits muscle protein catabolism induced by colon carcinoma. (A) Histology of TA muscle. Representative histological images of H&E (top two rows) or Azan-Mallory (bottom row) stained sections of TA muscles of mice sacrificed 12 days after PBS (top row) or tumor injection (middle and bottom rows). Ndn–/– C26-treated muscles show increased cell infiltrates, collagen accumulation and necrotic fibers (brown arrow). Centronucleated fibers can be seen in MlcNec C26-treated muscle (black arrows). Scale bar: 100 µm. (B) Distribution of cross-sectional area of TA fibers was analyzed on sections obtained from PBS- (red, blue and green) and C26-treated (yellow, light blue and light green) animals, 12 days after tumor injection (n=5). Ten H&E stained sections and a total of 300 fibers were measured for the different groups (WT mice, red and yellow lines; MlcNec, blue and light blue lines; Ndn–/– mice, green and light green lines). (C) Mean cross-section area (XSA) of TA muscle fibers from PBS- and C26-treated animals, 12 days after tumor injection (n=5). *P<0.05 and **P<0.01 vs WT mice; ++P<0.01 vs PBS-treated mice. (D) Expression of myosin heavy chain (MHC), myogenin, MyoD or glyceraldehyde phosphate dehydrogenase (GAPDH) in TA of animals sacrificed 12 days after PBS or C26 tumor cell injection. Images are representative of three different experiments with muscles isolated from five animals for each group of wild type (WT), MlcNec or Ndn–/– mice. (E) Graphs showing the mean values ± s.e.m. obtained for the ratio of protein bands:GAPDH band values of the experiments represented in Fig. 2D (n=3). Error bars represent s.e.m. *P<0.05 and **P<0.01 respectively vs WT; +P<0.01 vs PBS. (F) Embryonic myosin (eMyHC), atrogin-1 (Atg1) and MuRF1 expression was measured by real-time PCR on TA muscle from C26-treated animals sacrificed 10 days after tumor injection. Five animals were analyzed in parallel for each group: wild-type mice (WT), MlcNec mice and Ndn–/– mice. Results are normalized to levels of GAPDH RNA. Error bars represent s.e.m. **P<0.01 vs WT.

View larger version (59K): [in this window] [in a new window]   Fig. 3. Necdin counteracts inhibition of myogenic differentiation induced by TNF and ROS. (A) Expression of MHC, myogenin, MyoD or glyceraldehyde phosphate dehydrogenase (GAPDH) in primary myoblasts isolated from wild-type (WT), transgenic (MlcNec) or null (Ndn–/–) newborn mice (myoblasts) or C2C12 cells transfected with a plasmid containing pIRES2-EGFP (EGFP) or pIRESEGF-necdin (EGFPNdn) (C2C12), and differentiated in the presence of culture medium from the fibroblast 3T3 cell line (3T3) or C26 colon carcinoma cells (C26). Images are representative of three reproducible experiments. Quantifications are shown in D. (B) Expression of sarcomeric myosin heavy chain by immunofluorescence on C2C12 cells transfected with pIRES2-EGFP (EGFP) or pIRESEGF-necdin (EGFPNdn) and differentiated in presence of culture medium of the fibroblast 3T3 cell line (3T3), C26 colon carcinoma cells, or 5 ng/ml TNF or 2 µM As2O3; nuclei are stained with Hoechst 33258. Scale bar: 200 µm. (C) Expression of MHC, Myogenin, MyoD or GAPDH in primary myoblasts isolated from WT, MlcNec or Ndn–/– newborn mice (myoblasts) or C2C12 cells transfected with a plasmid containing pIRES2-EGFP (EGFP) or pIRESEGF-necdin (EGFPNdn) (C2C12), and differentiated in presence of TNF (20 ng/ml myoblasts; 5 ng/ml, C2C12) or As2O3 (5 µM myoblasts; 2 µM C2C12). Images are representative of three reproducible experiments. Quantifications are shown in E. (D) Graphs of mean values ± s.e.m. obtained from the densitometric analysis of the gel band of the protein normalized on the GAPDH expression of the experiments shown in A (n=3). (E) Graphs of mean values ± s.e.m. obtained from the densitometric analysis of the gel band of the protein normalized on the GAPDH expression of the experiments shown in panel B (n=3).

View larger version (39K): [in this window] [in a new window]   Fig. 4. Necdin inhibits TNF cachectogenic signaling at different levels. (A) Expression of TNFR1 in primary myoblasts from wild-type (WT), MlcNec or Ndn–/– newborn mice measured by real-time PCR. Results are normalized to levels of the GAPDH RNA (n=3). Error bars represent s.e.m. (B) Flow cytometry analysis of TNFR1 receptor in primary myoblasts (left panel) or transfected C2C12 (right panel). Satellite cells, isolated from WT, MlcNec or Ndn–/– newborn mice, were analysed for TNFR1 expression (light blue, orange and light green solid lines, respectively). Controls were made using FITC-conjugated secondary antibody alone (blue, red and green dashed lines, respectively) (RFI: MlcNec, 29.9±0.4; WT, 44.23±1.1; Ndn–/–, 55.7±0.73; P<0.001 vs WT; n=3). C2C12 cells were transiently transfected with a plasmid containing pIRES2-EGFP (EGFP) or pIRESEGF-necdin (EGFPNdn) and analysed for TNFR1 expression after gating of transfected cells (light blue and red, respectively). Control sample was made using secondary antibody alone (blue and orange, respectively) (RFI: pIRESGFP, 19.4966±1.15; pIRESEGFP-necdin, 9.136±1.03; P<0.001 vs pIRESEGFP; n=3). (C) Primary myoblasts from wild type (WT), MlcNec or Ndn–/– newborn mice (myoblasts) or C2C12 cells transiently transfected with a plasmid containing pIRES2-EGFP (EGFP) or pIRESEGF-necdin (EGFPNdn) were differentiated in the absence (PBS) or in the presence of 5-20 ng/ml of TNF (TNF). Cells were analyzed for p53 expression and for the activation of caspase 9 and caspase 3 by western blot. Graphs show mean values ± s.e.m. obtained for ratio of protein:GAPDH band density values evaluated on the blot of the same experiments shown in D (n=3). In the case of caspase 9, only active caspase bands were analyzed. Error bars represent s.e.m. *P<0.05 and **P<0.01 vs WT; +P<0.01 vs 3T3 (A) or PBS (B,C). (D) Western blot images representative of three reproducible experiments quantified above in C. (E) p53 expression was measured by real-time PCR in TA muscle from C26-treated WT, MlcNec and Ndn–/– mice sacrificed 6, 8, 10 and 12 days after tumor injection (n=5) (left graph) or in primary myoblasts from wild type (WT), MlcNec or Ndn–/– newborn mice (myoblasts) (n=3) (right graph). Results are normalized to levels of the GAPDH RNA. Error bars represent s.e.m. *P<0.01 and **P<0.001 vs WT.

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