Roles of proteasome, arachidonic acid, and oxytocin in bovine myoblast proliferation and differentiation

The overall objective of this dissertation project was to identify factors and mechanisms that control bovine myoblast proliferation, differentiation, and fusion. Three studies were conducted during this project. The objective of the first study was to determine the effect of oxytocin (OXT) on myoblast proliferation, differentiation and fusion. Treating primary bovine myoblasts in culture with 10 nM and 100 nM OXT for 24 h increased their proliferation rate by 7% (P < 0.05) and 10% (P < 0.05), respectively. Treating bovine myoblasts with either concentration of OXT for 48 h had no effect on their differentiation and fusion, as indicated by no changes in mRNA expression of selected myoblast differentiation markers and fusion index. The objective of the second study was to determine the effects of arachidonic acid (AA) and its major metabolites prostaglandin E2 (PGE2), PGF2a, and PGI2 on myoblast proliferation, differentiation and fusion. Treating myoblasts with 10 μM AA, 1 μM PGE2, 1 μM PGF2α, and 1 μM PGI2 for 24 h each increased the number of proliferating cells by 13%, 24%, 16%, and 16%, respectively, compared to the control (P < 0.05). At the same concentrations, AA, PGE2, and PGF2a stimulated myoblast differentiation and PGE2 improved myoblast fusion (P < 0.05). Treating myoblasts with AA and the cyclooxygenase (COX)-1 and COX-2 inhibitor indomethacin or the COX-2-specific inhibitor NS-398 reversed the stimulatory effect of AA on myoblast proliferation (P < 0.05). The objective of the third study was to determine the role of the proteasome in bovine myoblast differentiation and fusion. It was found that the proteasome activity increased (P < 0.05) during myoblast differentiation and fusion. Adding 5 μM lactacystin, a specific inhibitor of the proteasome, to the differentiation medium nearly completely blocked myoblast differentiation and fusion. Inhibitor of DNA-binding 1 (ID1) is known to inhibit myoblast differentiation and to be degraded by the proteasome in some cells. Both ID1 protein and mRNA expression were found to decrease during myoblast differentiation and fusion, and the decrease in ID1 protein but not ID1 mRNA was reversed (P < 0.05) by treating the cells with lactacystin. In summary, this project reveals that OXT and AA are stimulators of bovine myoblast proliferation and that AA is a stimulator of bovine myoblast differentiation. This project also indicates that the proteasome plays a positive role in bovine myoblast differentiation and fusion, and that it does so perhaps by reducing the accumulation of the ID1 protein.