Although apoptosis occurs during myogenesis its mechanism of initiation remains unknown. activation of ER stress signaling was sufficient for induction of apoptosis. Activation of caspase-12 was also detected in developing muscle mass of mouse embryos and gradually disappeared later. CHOP was Irsogladine also transiently induced. These results suggest that specific ER stress signaling transmitted by ATF6 prospects to naturally occurring apoptosis during muscle mass development. Introduction The central executioner molecules of apoptosis are a family of cysteine proteases the caspases which comprise multiple cascades (Degterev et al. 2003 All caspases are synthesized as precursor proteins procaspases which are activated Irsogladine by processing to ～20- (p20) and 10-kD (p10) mature fragments. Apoptosis is usually often associated with differentiation during development (Glücksmann 1951 However the Rabbit Polyclonal to PDGFB. cause of apoptosis and the mechanism of initiation of caspase activation during differentiation remain largely unknown. The present study focuses on the triggering of caspase activation during myoblast differentiation. Myoblast cells begin to exhibit considerable morphological changes when cultures are altered to low concentrations of mitogens. During this process myoblasts express muscle-specific proteins and fuse into multinucleated myotubes (Stockdale and Holtzer 1961 Nadal-Ginard 1978). Myoblast fusion is usually associated with apoptosis (Fidzianska and Goebel 1991 and activation of caspase-3 usually a downstream caspase was detected in a cell culture model (Dee et al. 2002 Fernando et al. 2002 The caspase initiator responsible for caspase-3 activation is usually unknown. Because caspase-12 is usually highly expressed in muscle mass of adult (Van de Craen et al. 1997 and embryonic mice (explained later) it might play a physiological role in muscle tissue. It is an ER stress-specific caspase functioning as the initiator caspase in response to ER stress that involves accumulation of misfolded proteins in the ER (Nakagawa et al. 2000 Due to the nature of apoptotic stimuli the effects of caspase-12 have been often discussed regarding pathological changes especially in relation to neurodegenerative disorders (Nakagawa et al. 2000 and prion diseases (Hetz et al. 2003 We previously found that caspase-12 initiates the ER stress-specific cascade including caspases-12 -9 and -3 in a myoblast cell collection C2C12 (Morishima et al. 2002 indicating that caspase-12 can function as an initiator caspase in myoblasts for implementation of apoptosis. ER stress-induced apoptosis is usually preceded by activation of a cytoprotective signaling cascade termed the unfolded protein response (UPR). The UPR alters transcriptional and translational programs to cope with accumulation of unfolded or misfolded proteins. Although protein synthesis is generally down-regulated in the UPR decreasing the load in the ER the UPR Irsogladine induces a group of specific proteins including BiP an ER-specific molecular chaperone and CHOP a transcription factor known to be up-regulated by ER stress (Zhang and Kaufman 2004 We demonstrate that both BiP and CHOP are induced in both Irsogladine apoptotic cells and differentiating myoblasts indicating that Irsogladine differentiation conditions elicit ER stress-specific signaling. Under such conditions activation of caspase-12 was detected Irsogladine suggesting a previously unidentified role of ER stress signaling in activating the ER-specific caspase cascade to induce naturally occurring apoptosis during development. Results and conversation Proliferating myoblasts undergo terminal differentiation in vitro under standard differentiation conditions (Stockdale and Holtzer 1961 Nadal-Ginard 1978 when cultures are changed to low concentrations of mitogens (differentiation medium [DM]). Approximately 15% of C2C12 cells died during the first 24 h of incubation in DM (Fig. 1 A); thereafter the percentage of lifeless cells gradually decreased over a week in parallel with myotube formation. Seven days after switch to DM myotubes were abundant and apoptosis experienced almost ceased (Fig. 1 A). On day 3 formation of myosin was obvious in multinucleated myotubes (Fig. 1 B). We collected apoptotic cells that were either floating or loosely.