טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZetser Anna
SubjectInvolvement of MAP Kinase Signal Transduction Pathways in
the Differentiation of Skeletal Muscle
DepartmentDepartment of Medicine
Supervisors Professor Dale Frank
Professor Eyal Bengal


Abstract

The development of skeletal muscle cells can be devided into two main stages: determination and differentiation.  During the determination stage, pluropotent mesodermal cells begin to express the MyoD or Myf5 transcription factors.  These proteins are not functional in myoblasts that continue to proliferate until they receive a signal to differentiate.  During the differentiation stage MyoD and Myf5 are activated and cause myoblasts to withdraw from the cell cycle.  MyoD or Myf5 activates the expression of additional downstream transcription factors, members of myocyte enhancer factor 2 (MEF2) family and myogenin.  These in turn induce the expression of muscle specific genes that dictate the properties of the muscle phenotype.  The combined activity of MyoD and MEF2 members is critical for the induction of the differentiation process.

      In the first part we studied the involvement of the p38 MAPK in the in vitro differentiation of myoblasts. We found that the activity of p38 MAPK was induced early in the differentiation of L8 cells.  Addition of specific p38 inhibitor - SB203580, to myoblasts blocked their fusion to multinucleated myotubes and prevented the expression of MyoD, MEF2  and muscle specific genes like myosin light chain.

The expression of MKK6, a direct activator of p38, enhanced the activity of MyoD in converting 10T1/2 fibroblasts to muscle cells.  We demonstrated that MEF2 DNA binding sites and MEF2C proteins were necessary for the p38 MAPK pathway to regulate the transcription of muscle creatine kinase reporter gene.  We conclude that the activity of p38 MAPK is induced during muscle differentiation and participates in phosphorylation and activation of the MEF2C transcription factor. 

In the second part of the work we investigated the involvement of Extracellular Regulated Protein Kinase (ERK MAPK) pathway in the differentiation of skeletal muscle in vivo, in Xenopus laevis.  In Xenopus laevis, muscle differentiates from cells located in the dorsal-lateral region of the marginal zone.  Initially, this region is not specified for muscle and it will not differentiate as muscle if removed from the embryo prior to gastrulation for explant culture.  During gastrulation, "dorsalizing" signals such as noggin, chordin and follistatin (Bone Morphogenetic Protein (BMP) antagonist proteins) are secreted from the adjacent dorsal marginal zone, the Spemann organizer region.  Dorsalization of the ventrally fated DLMZ to muscle requires inhibition of endogenous BMP signaling in the DLMZ region by these antagonist molecules. 

In the present study, we have examined the role of ERK signaling in the muscle specification process in Xenopus.  We found that inhibition of BMP activity stimulated ERK activity in dorsalized VMZ explants.  From gastrula to neurula stages, there is a sharp increase in ERK activity in DLMZ explants compared to VMZ explants. Dorsalized-VMZ and DLMZ explants treated with ERK inhibitors did not differentiate as muscle and maintained ventral mesoderm identity.  Co-ectopic expression of activated MAP kinase kinase (MEK) and XMyoD RNAs induced significant cell-movement elongations and terminal differentiation in VMZ explants; muscle actin gene expression was highly induced in these explants. In VMZ explants, co-ectopic expression of activated MEK and XMyoD stimulated the ability of XMyoD to transcriptionally activate an E-box dependent reporter gene; XMyoD protein appeared more stable and more localized to the nucleus in these co-injected explants.  These results suggest that BMP antagonists activate an ERK cascade in the DLMZ, and ERK activation is required to convert MyoD into a terminal differentiation factor.