Death associated lepidopteran DALP, and its mammalian ortholog Hic-5, act as negative regulators of muscle differentiation
During muscle differentiation in vertebrates, myoblasts initially form in somites and then migrate to proper locations in the trunk and limbs. Once there, these cells are faced with one of three choices: differentiate into myotubes, arrest as satellite cells, or initiate apoptosis and die. The molecular mechanisms that regulate the decision of myoblasts to die are poorly understood. To gain insight into this process, we have cloned death-associated genes from the intersegmental muscles of the moth Manduca sexta, a model system for developmentally regulated muscle cell death. One of the genes isolated in this screen was DALP (Death Associated LIM Only Protein), a protein that shares 52% similarity at the protein level with mammalian Hic-5. Ectopic expression of DALP in the skeletal muscles of the fruit fly Drosophila caused atrophy and disorganization of the contractile apparatus. To determine the role of DALP/Hic-5 in mammalian myogenesis, we took advantage of the mouse myoblast cell line C2C12. Ectopic expression of either DALP or Hic-5 blocked the ability of myoblasts to differentiate following serum withdrawal. These cells failed to express muscle differentiation markers such as MyoD or myosin heavy chain. In addition, these cultures displayed greatly enhanced rates of cell death. Hic-5 expression is restricted to mononucleated and apoptotic C2C12 cells in serum-depleted medium. The effects of ectopic DALP or Hic-5 expression could be prevented by contact with wild type C2C12 cells or by ectopic expression of MyoD. Gene profiling experiment demonstrated that the ectopic expression of Hic-5 results in enhanced expression of pro-apoptotic Bcl-2 family members and of polyubiquitin. Taken together, these data suggest that Hic-5 acts upstream of MyoD and functions as a negative-regulator of myoblast differentiation and may facilitate the initiation of apoptosis. In separate studies, the functional roles of another death-associated molecule, m56, were studied in Ratl fibroblasts. Our data strongly support the hypothesis that m56 is a proteasome subunit and misexpressing m56 can sensitize Ratl cells to apoptotic stimuli.
0307: Molecular biology