טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGredinger Eran
SubjectInvolvement of Mitogen Activated Protein Kinase (MAPK)
Signal Transduction Pathways in Muscle
Differentiation
DepartmentDepartment of Medicine
Supervisor Professor Eyal Bengal


Abstract

Differentiation of muscle cells is regulated by extracellular growth factors . Some of these growth factors induce mitogen activated protein kinase ( MAPK ) cascades within muscle cells. In this work we show that the activity of extracellular regulated kinase (ERK) is low in myoblasts and is induced with the onset of terminal differentiation. By transfection of alleles that activate or suppress the ERK -MAPK pathway and by adding the specific inhibitor of this pathway-PD098059 to cells, we show that the ERK pathway promotes muscle differentiation via the expression and activity of the MyoD protein. Our studies also suggest that the ERK-MAPK pathway is involved in fusion and survival of myotubes. 

In the second part we show that the activity of P38 - MAPK is induced early in the differentiation of myoblasts. Addition of a specific P38 inhibitor - SB203580 to myoblasts blocked their fusion to multinucleated myotubes and prevented the expression of MyoD and MEF2 family members. The expression of MKK6, a direct activator of P38 , enhanced the activity of MyoD in converting 10T1/2 fibroblasts cells to muscle . In this part of the work we show that the involvement of P38 in MyoD activity is mediated via its co-activator MEF2C. Our study suggests that MEF2C proteins and MEF2 binding sites are necessary for the P38 - MAPK pathway to regulate the transcription of a muscle creatine kinase reporter gene. The results in this part indicate that the P38 - MAPK pathway promotes skeletal muscle differentiation via the activation of MEF2C transcription factor.

In the third part of the work we generated stable myoblast cell lines expressing the different forms of Rac1. We found that a constitutively active form of Rac1 (Act-Rac1) induced while dominant negative Rac1 ( DN-Rac1 ) inhibited the activity of MyoD in promoting muscle differentiation. MyoD is not active in proliferating myoblasts, therefore, we analyzed whether Rac1 affected the withdrawal of myoblasts from the cell cycle. Our studies indicate that in cells expressing Act-Rac1 the ratio between hyperphosphorylated to underphosphorylated PRb remains constant under media conditions that promotes withdrawal from the cell cycle. Conversely, all PRb protein turnes to be underphosphorylated in cells expressing DN-Rac1 even under conditions that promotes cell growth. We observed that cells expressing the DN-Rac1 exit the cell cycle faster than control cells or cells expressing Act-Rac1. Our study indicates that improper and untimely activation of Rac1 in myoblasts interferes with the exit from the cell cycle and subsequent differentiation.