Roles Of FADD In Skeletal Muscle Regeneration And Cell Cycle Regulation

FADD(Fas-associated protein with death domain)was first found as a adaptor protein which transduced the apoptotic signal from death receptors in 1995.Since then FADD has also been found involved in many other non-apoptotic processes such as cell proliferation,cell cycle regulation,tumorigenesis,inflammation,innate immunity,embryonic development,autophagy,etc.And more and more researches have indicated that these non-apoptotic functions of FADD are independent on death receptors,but dependent on the subcelluar localization and the phosphorylated status of FADD,which makes FADD an indispensable and unique regulator of these processes.This thesis is dedicated to the roles of FADD in skeletal muscle regeneration and cell cycle regulation.Adult mammalian skeletal muscle is a stable tissue with little turnover of nuclei.Minor lesions inflicted by day-to-day wear and tear elicit only a slow turnover of its constituent multinucleated muscle fibers.Nonetheless,mammalian skeletal muscle has the ability to complete a rapid and extensive regeneration in response to severe damage.Skeletal muscle regeneration mimics embryonic muscle development in several ways:first the muscle stem cells,also known as satellite cells,are activated and start to proliferate followed by theirs lineage commitment and differentiation;then myofibers are formed by the fusion of myoblasts,which combined with connection tissue,neurons and blood vessels form the contractile skeletal muscle tissue.Notch signaling pathway is involved in the activation and proliferation of satellite cells,however the differentiation and fusion of the myoblasts are mainly controlled by Wnt signaling pathway.Temporal switch from Notch signaling pathway to Wnt signaling pathway is critical for an effective regeneration,and enhanced activation of Notch can inhibit Wnt.We noticed that mice bearing a constitutively phosphoryl-mimicking mutation of FADD(FADD-D)display a defection of skeletal muscle regeneration.Further studied showed that this defection was induced by the inhibition of the differentiation of the myoblasts,which indicates an enhanced Notch activation.Protein kinase C(PKC)belongs to AGC kinase family,which is activated by growth factors-generated second messengers and conveys the extracellular signals to initiate a cascade of cellular events.Evidence from diverse cell types has demonstrated PKC manipulates cell cycle progression to control proliferation and differentiation.It has been validated that FADD negatively regulates PKCa through dephosphorylation of PKCa,which is antagonized by FADD-D,hence the enhanced activation of PKCa in FADD-D mice.Inhibition of PKCa can recover skeletal muscle regeneration in FADD-D mice,furthermore Notch is stabilized by PKCa.Therefore we presume that FADD regulates Notch through PKCa.In addition to Notch,previous studies have indicated that fibroblast growth factors(FGF)family also promotes myoblasts activation and proliferation,which in turn promotes muscle regeneration.It's still not clear whether these proliferative signaling pathways are integrated in the proliferative myoblast or not.In the present study,we have showed that bFGF promotes the interaction between PKCa and Notch-1,and also promotes phosphorylation of PKCa.Therefore it's highly possible that bFGF signaling pathway and Notch signaling pathway crosstalk through PKCa.Taken together the conclusion we would like to present is that Notch signaling pathway is activated by bFGF signaling pathway through phosphorylation of PKCa,which is terminated by dephosphorylation of PKCa by FADD.These datas identify a novel mechanism for the temporal regulation of Notch signaling during myogenic lineage progression.As an apoptotic adaptor protein,it has been reported that FADD involves in the regulation of cell cycle.Therefore we checked the cell cycle status of FADD-/-cell.It turned out that the cell cycle of FADD-/-cell was arrested in G2/M phase,in consistent with which,p-Y15-cdc2 and cyclin B1 are abnormally expressed in FADD-/-cell,hence the defective of mitosis and the appearance of aneuploidy of FADD-/-cell.In addition,FADD-/-cell also display a phenotype of senescence.It has been demonstrated that activation of p53/p21 pathway and p16 can lead cells to senescence,and the protein level of these three proteins are substantiallyelevated in FADD-/-cell.Further studies showed that ROS level was also elevated in FADD-/-cell.It has been showed that senescence can be induced in cells with an increased generation of ROS through p53 and p16,in addition,p53/p21 pathway also involves in G2/M transition.Therefore,it's highly possible that p53/p21 pathway is activated by increased ROS generation in FADD-/-cell.Taken together we presume that p53/p21 pathway is activated by increased ROS generation in FADD-/-cell,which in turn induce G2/M arrest and senescence.