The TWEAK receptor Fn14 is expressed by myoblasts, and TWEAK inhibits differentiation of C2C12 cells. (A) FACS showed that most human skeletal muscle myoblasts (HSMM, from Cambrex) expressed Fn14, as well as the lineage marker CD56, on the cell surface. (B) Myoblasts of the mouse C2C12 line also expressed Fn14 on the surface. (C–J) Myotube formation by C2C12 cells in low-serum differentiation medium (DM) was assessed after addition of the following reagents: (C) no addition, myotube formation was extensive, (D) 100 ng/ml murine recombinant TWEAK, myotube formation inhibited; (E) 100 ng/ml mTWEAK+10 μg/ml anti-TWEAK mAb ABG.11, normal myotubes; (F) 100ng/ml mTWEAK+10 μg/ml anti-Fn14 mAb P2D3, normal myotubes; (G) 5 ng/ml mTNF; decreased myotubes; (H) 5 ng/ml mTNF+10 μg/ml mTNFRI-Fc, normal myotubes; (I) 5 ng/ml mTWEAK+10 μg/ml mTNFRI-Fc to block TNFR1, decreased myotubes; and (J) 5 ng/ml mTNF+10 μg/ml anti-TWEAK ABG.11, decreased myotubes. Phase contrast at 5 days after switching to DM with indicated additions; images are representative of at least 10 independent experiments.

The TWEAK/Fn14 pathway regulates proliferation and differentiation of primary muscle myoblasts. TWEAK promoted the growth (A–C) and inhibited the differentiation (D, E) of primary myoblasts from wild-type mice. (A–C) After 3 days of proliferation in growth medium, cultures of primary myoblasts treated with 100 ng/ml Fc-TWEAK (B) contained ∼2 × more cells than untreated cultures (A) or cultures treated with both Fc-TWEAK and Fn14-Fc to block receptor function (C). (D, E) After 4 days in low-serum differentiation medium, untreated cultures of primary myoblasts formed multinucleate myotubes (D), whereas myotubes were rarely found in TWEAK-treated cultures (E). (F) Primary myoblasts isolated from Fn14-deficient mice produced fewer progeny than wild-type myoblasts in culture. Results from two independent experiments (exp. no. 1 and exp. no. 2) are shown. In each experiment, myoblasts were obtained from two individual wild-type (WT) and two individual Fn14^−/− (KO) mice. Cells were seeded at the same initial cell densities. (G, H) Phase-contrast images of myotubes formed by myoblasts isolated from WT and Fn14-deficient mice.

Fn14^−/− mice have delayed muscle regeneration. Cardiotoxin-treated muscles at indicated days after injury of wild-type (A–D) and Fn14^−/− (E–H) mice are shown. Detailed views of B and F are shown in (I) and (J) respectively to show regenerating, central nucleate myotubes, some of which are indicated by arrows. Images are representative of three independent experiments with seven mice per genotype in total. Scale bar: 200 μm (A–C, E–G), 100 μm (D, H). (K) The number of central nucleated myotubes in Fn14^−/− mice was significantly lower than in wild-type muscles at 5 and 7 days, but not 14 days, after cardiotoxin. Error bars=s.d.; ^**=P<0.01 by Student's t-test; n as indicated. (L) Myofibers that express embryonic myosin heavy chain (eMyHC), an additional marker of regenerating myotubes (dark brown stain), were more abundant in wild-type than in Fn14^−/− muscles at 4 days after injury.

A working model for regulation of muscle regeneration by the TWEAK/Fn14 pathway. Cardiotoxin-induced injury triggers release of soluble factors that recruit early inflammatory cells and induce Fn14 expression on muscle precursor cells. Secreted by infiltrating inflammatory cells, TWEAK engages Fn14 and promotes the proliferation and activation of myoblasts, which can in turn contribute to a robust inflammatory response by producing additional chemotactant. The TWEAK/Fn14 pathway therefore positively contributes to muscle regeneration by promoting cellular crosstalk between inflammatory cells and muscle precursor cells.

Human mesenchymal progenitor cells are a novel target cell type for TWEAK. (A) Human primary mesenchymal stem cells, skeletal muscle myoblasts, preadipocytes, chondrocytes and osteoblast precursors (Cambrex) were cultured according to the manufacturer's protocols. First-passage cells showed staining for TWEAK binding using Fc-TWEAK and for expression of Fn14 using the anti-hFn14 mAb ITEM-4. Anti-mouse and anti-human Fcs were used as negative controls, (B) NF-κB was activated in human mesenchymal stem cells (hMSCs) and osteoblast precursors (hOsteos) following 2 or 6 h of treatment with 100 ng/m TWEAK (Tw). Activation was measured using the TransAM NF-κB p65 activation assay system with cell lysates from normal and TNF-treated HeLa cells serving as negative and positive controls. The assays were carried out in triplicate and the data shown are representative of three independent experiments. (C) List of representative genes induced by TWEAK (100 ng/ml versus heat-inactivated TWEAK 100 ng/ml) in mesenchymal stem cells in low serum (LS: 0.2% FBS), moderate serum (MS: 2% FBS) and high serum (HS: 10% FBS). (D) List of some cell cycle-related genes induced by TWEAK (versus inactivated TWEAK) in mesenchymal stem cells cultured under low-serum conditions (0.2% FBS). Triplicate samples were analyzed for each condition and the fold changes were calculated using averages from triplicates. All fold changes reached statistical significance (P<0.01).

TWEAK blocks the myogenic program and prevents cell cycle arrest in C2C12 cells. (A) TWEAK induced expression level changes in many genes important for cell cycle control and myogenic differentiation,for example, the muscle-specific transcription factors myogenin and MyoD. Fold changes and s.d.s were calculated using data from duplicate samples that passed statistical tests. (B) C2C12 cells continued to proliferate in low-serum DM when treated with TWEAK as shown by the much larger percentage of cells that incorporated BrdU in TWEAK-treated than in untreated cultures. Cells were analyzed after culture in DM for 3 days with or without 100 ng/ml of Fc-TWEAK and after 12 h BrdU labeling. (C) Myogenin protein was much lower after TWEAK treatment as shown by immunoblots of C2C12 cells in DM in the absence (none) or presence of Fc-hTWEAK (hTweak, 100 ng/ml) or hTNFa (10 ng/ml). (D) Staining of actin filaments by fluorescent phalloidin (red) was decreased after TWEAK treatment of C2C12 cells cultured in DM for 5 days with or without 100 ng/ml of Fc-TWEAK as indicated. Nuclei were identified by DAPI stain (blue).

TWEAK and Fn14 mRNAs increased upon injury, but increased TWEAK persists longer in Fn14^−/− than in wild-type muscles and is produced by macrophages. (A–L) In situ hybridzation of TWEAK (A–F) and Fn14 (G–L) mRNAs in control (no inj) and cardiotoxin-treated wild-type and Fn14^−/− muscles at 3 or 5 days post injection as indicated. Scale bar in (A) represents 100 μm. (M) At day 3 after cardiotoxin treatment, quantification of grain density showed that TWEAK mRNA was significantly (P<0.01, n=4) increased several-fold in both wild-type (gray bars) and Fn14^−/− (blue bars) muscles. At day 5, in constrast, TWEAK persisted at a high level in Fn14^−/− muscles, but returned to near the initial level in wild-type muscles. Fn14 mRNA was also increased by injury at days 3 and 5 in wild-type muscles (green bars, right panel). Error bars=s.d. No Fn14 signal above background was found in Fn14^−/− muscles (not shown). (N) Macrophages are the major source of TWEAK expression in regenerating muscles. At 3 days after cardiotoxin, TA muscles (n=3) were dissociated into single-cell suspensions and three cell preparations were made: (i) cells positive for the macrophage marker Mac-1⁺ were purified using Mac-1 magnetic beads (macrophages, blue bars); (ii) myogenic cells were purified on a Percoll gradient (myoblasts, red bars); and (iii) Mac-1-negative cells, which were cells that did not bind to the Mac-1 beads (flow-through, green bars). RNA was isolated from each cell fraction and quantitative PCR was used to measure the amount of TWEAK mRNA in each cell type using an equal amount of input RNA and normalized versus the amount of GAPDH mRNA. TWEAK mRNA expression was significantly higher (^*P<0.05, n=3) in macrophages than in myoblasts or flow-through cells. Error bars=s.d. (O) mRNA levels of TNF/TNFRs and EDA/XEDAR with and without cardiotoxin in wild-type and Fn14^−/− mice. At 3 days after cardiotoxin (CTX), total RNA was isolated from TA muscles (n=3) and quantitative PCR was used to measure the mRNA levels for TNF, TNFR1, TNFR2, EDA and XEDAR. Statistical significance was not achieved between wild-type and Fn14-deficient mice.

Altered inflammatory response in Fn14^−/− muscles in response to cardiotoxin (CTX) injury. (A) FACS based on myeloid markers Mac-1 and Gr-1 showed that inflammatory cells were more abundant in wild-type than in Fn14^−/− muscles at 2 and 3 days post injury. Slightly more cells were consistently recovered from wild-type than Fn14^−/− mice at all time points; the same number of cells (10 000) are shown in each dot plot. For each genotype, cells from two mice were pooled for each time point and the results shown are representative of three independent experiments. (B–E) F4/80 expression (brown staining) of muscle tissue cross-sections shows higher numbers of inflammatory cells in wild-type (B) than in Fn14^−/− (C) muscles on day 3 following cardiotoxin. Detailed views of (B) and (C) are shown in (D) and (E), respectively. Scale bar: 100 μm (B, C), 50 μm (D, E). (F) mRNA levels of MCP-1, MCP-3 and RANTES with and without cardiotoxin injection in wild-type and Fn14-deficient mice. At 1 and 3 days after cardiotoxin, total RNA was isolated from TA muscles (n=3) and quantitative PCR was used to quantify mRNAs. Asterisk indicates that the difference for MCP-3 expression at day 1 reached statistical significance (P<0.05, by Student's t-test).