PDF《differentiation》

2104 Apoptosis (2006) 11:2103C2113 as well as the ensuing dissipation of the mitochondrial membrane potential, is a critical component of the apoptotic process in most systems. The molecules directly responsible for this mitochondrial assault are the pro-apoptotic members of the Bcl2 family [21C23]. The signaling pathways through which particular pro-apoptotic Bcl2 family members are engaged are broadly categorized as either intrinsic or extrinsic . The intrinsic pathway is initiated through altered activity of either kinases or transcription factors while the extrinsic pathway is initiated by death ligand signaling [24]. Several of the proteins released from the mitochondria play either a direct or indirect role in caspase activation [22, 23]. Caspases are cysteine-aspartic acid speci?c proteases that play a crucial role during apoptosis [25]. These are categorized as either initiator caspases or executioner caspases. Activation of initiator caspase

9 is a result of the release of cytochrome C from the mitochondria while activation of initiator caspase

8 is a direct result of death ligand signaling [24, 26]. Herein, we report a role for caspase

8 activation during the apoptosis associated with skeletal myoblast differentiation. This ?nding prompted an investigation into the role of death ligand signaling and led us to discover that signaling by the death ligand TRAIL, through the TRAIL receptor DR5 and the adapter protein FADD, also plays a role in this apoptotic process. Further, we report that this death ligand pathway is engaged through increased expression of the TRAIL re- ceptor DR5 and decreased expression of FLIP, a caspase

8 antagonist. Materials and methods Cells and cell culture The growth and differentiation properties of 23A2 myoblasts have been reported previously [7, 19]. The 23A2 deriva- tives expressing dominant negative FADD (23A2:dnFADD myoblasts) and the 23A2 and C2C12 derivatives express- ing dominant negative DR5 (23A2:dnDR5 myoblasts and C2C12:dnDR5 myoblasts) were generated by transfecting myoblasts with

300 ng of the pcDNA3:AU1:dnFADD con- struct or

300 ng of the pcDNA3:AU1:dnDR5 construct using Lipofectamine Plus (GibcoBRL) as speci?ed by the manu- facturer. Brie?y, cells were plated at

105 in

100 mm dishes and transfected the next day with

300 ng of DNA. After

48 h, the culture was passaged and placed in selection me- dia containing G418 (750 ?g/ml). After

14 days of drug selection, individual G418 resistant colonies were isolated and propagated for further analysis. All cells were main- tained on gelatin coated plates in growth medium (GM), which consists of basal modi?ed Eagle'

s medium (BME), 10% fetal bovine serum (FBS),

100 I.U./ml penicillin and

100 ?g/ml streptomycin (1% P/S). Differentiation was in- duced by switching cells from GM to differentiation medium (DM), which consists of BME, 1% P/S and no FBS. Western blot analysis Cells were lysed as previously described. The protein con- centrations of all lysates were determined using Coomassie Protein Assay Reagent from Pierce as per manufacturer'

s in- structions. Following protein determination, lysates (200 ?g for AU1 detection and

100 ?g for Bid, FLAG, TRAIL, DR5 and FLIP detection) were denatured in sample buffer (?- nal concentration in lysates of 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, pH 6.8) and electrophoresed through denaturing polyacrylamide gels (15% for Bid and AU1 detection, 12% for FLIP detection, 10% for FLAG and TRAIL detection, and 8% for DR5 detection. Following SDS polyacrylamide gel electrophoresis (SDS-PAGE), samples were transferred electrophoretically for four amp hours to Hybond-P polyvinylidene di?uoride membranes in transfer buffer containing 80% methanol and