טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAlter Joel
SubjectStress Pathways Maintaining Undifferentiated Reserve Cells
in the Skeletal Muscle Lineage
DepartmentDepartment of Medicine
Supervisor Professor Eyal Bengal
Full Thesis textFull thesis text - English Version


Abstract

The proinflammatory cytokine, TNFα plays a major role in muscle wasting occurring in chronic diseases and muscular dystrophies. TNFα perturbs muscle regeneration by preventing satellite cell differentiation. In the present study, the role of c-Jun N-terminal kinase (JNK) was investigated in differentiating myoblast cell lines. Addition of TNFα to C2 myoblasts induced immediate and delayed phases of JNK activity. The delayed phase is associated with myoblast proliferation. Inhibition of JNK activity prevented proliferation and restored differentiation to TNFα-treated myoblasts. Studies with cell lines expressing MyoD:ER chimera and lacking JNK1 or JNK2 genes indicate that JNK1 activity mediates the effects of TNFα on myoblast proliferation and differentiation. TNFα does not induce proliferation or inhibit differentiation of JNK1-null myoblasts. However, differentiation of JNK1-null myoblasts is inhibited when they are grown in conditioned medium derived from cell lines affected by TNFα. We investigated the induced synthesis of several candidate growth factors and cytokines following treatment with TNFα. Expression of IL-6 and leukemia inhibitory factor (LIF) was induced by TNFα in wild-type and JNK2-null myoblasts. However, LIF expression was not induced by TNFα in JNK1-null myoblasts. Addition of LIF to the growth medium of JNK1-null myoblasts prevented their differentiation. Moreover, LIF-neutralizing antibodies added to the medium of C2 myoblasts prevented inhibition of differentiation mediated by TNFα. Hence, TNFα promotes myoblast proliferation through JNK1 and prevents myoblast differentiation through JNK1-mediated secretion of LIF.

When myoblasts or satellite cells differentiate the expression of MyoD is excluded from a subset of cells that do not differentiate and become reserve cells. Yet, it remains unclear which processes are involved in downregulation of MyoD expression and the maintenance of a reserve cell population. We identified that the phosphorylation or expression of several stress response proteins is transiently induced during differentiation of myoblasts. The phosphorylation of eIF2α occurred before the induced expression of CHOP and ATF3 transcription factors and was intrinsic to the differentiation process. We find that ATF3 expression protects differentiating myoblasts from cell death while the expression of CHOP prevents differentiation. Interestingly, cells expressing CHOP did not express the MyoD protein. Further investigation indicated that CHOP functioned as a transcription repressor that directly reduced MyoD transcripts. CHOP associated with upstream regulatory regions of the MyoD gene, and its activity reduced histone acetylation at specific regions. Therefore, stress-induced CHOP activation in a subset of myoblasts promotes their withdrawal from the myogenic program through the direct repression of MyoD transcription.