Programme Contents

Protease-activated receptor mediated calcium signaling and cytokine production in cultured c2c12 skeletal muscle cells

Protease-activated receptors (PARs) are activated by proteases such as thrombin and tryptase, via proteolytic cleavage. In many cells, PAR activation results in a rise in intracellular Ca²⁺ through activation of G_q and phospholipase C, and store-mediated Ca²⁺ entry. This Ca²⁺ signal is thought to trigger the appropriate cellular response. PARs are thought to play a significant role in the inflammatory process, by triggering the release of cytokines from activated cells (Asokananthan et al., 2002). Skeletal muscle myotubes express PAR receptors, and increased intracellular Ca²⁺ has recently been shown to increase the production of inflammatory cytokines such as IL-6 in muscle cells (Keller et al., 2006). In this study, we examined the nature of PAR-mediated Ca²⁺ signaling in cultured skeletal muscle myotubes and determined whether PAR activation resulted in the release of the cytokine IL-6 from these cells.

C₂C₁₂ myoblasts were grown in Dulbecco’s modified Eagles medium with 20% foetal calf serum and equilibrated with 5% CO₂, 95% air at 37°C. Differentiation of myoblasts into myotubes was induced by lowering the serum concentration to 2%. PARs were activated with thrombin (activates PAR-1, 3 & 4 isoforms) and trysin (activates PAR-2 isoform. The intracellular Ca²⁺ concentration was measured using the fluorescent Ca²⁺ indicator fura-2. Measurements were undertaken using a Cairn Spectrophotometer. Experiments were undertaken at 22°C and IL-6 levels were detected by ELISA.

Exposure of the myotubes to thrombin resulted in a significant increase in intracellular Ca²⁺ (mean amplitude; 0.38 ± 0.03 μM, n = 13) that was larger in peak than electrically-induced Ca²⁺ transients elicited in similar C₂C₁₂ myotubes (mean amplitude; 0.20 ± 0.07 μM, n = 11). Pre-exposure to thrombin (10 U/ml) for one hour, 12 hours before experimentation resulted in 93% of myotubes responding to thrombin with a Ca²⁺ response. Experiments with specific PAR-activating peptides indicated that thrombin was activating Ca²⁺ signalling via PAR-1. In myotubes exposed to thrombin under Ca²⁺ free conditions, only 17% (n = 6) of myotubes displayed Ca²⁺ influx upon return of extracellular Ca²⁺. In addition, only 12% of cells (n = 17) responded with Ca²⁺ influx after the store was depleted with the sarcoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone, suggesting that in most myotubes, store-operated Ca²⁺ entry was absent. Exposure of myotubes to thrombin (5 U/ml) for 24 hours increased IL-6 levels to 140% of control levels (p = 0.01). Pre-exposure to thrombin (10 U/ml) for one hour, followed by a 24 hour exposure to thrombin (5 U/ml) 12 hours later, increased IL-6 levels to 188% of control levels (p = 0.01).

These results suggest that the Ca²⁺ signal associated withevoked by PAR-1 activation is predominantly associated with intracellular Ca²⁺ release, and that thrombin exposure results in the release of the IL-6, which may play a role in the muscle response to injury and inflammation.

Asokananthan N., Graham PT, Fink J, Knight DA, Bakker AJ, McWilliam AS, Thompson PJ & Stewart GA. (2002) Journal of Immunology, 168: 3577-85.

Keller C., Hellsten Y., Steensberg A. & Pedersen BK. (2006) Cytokine, 36: 141-7.