Transfection of nonmuscle β- and γ-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

Abstract

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human β- and γ-actin genes. In γ-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human γ-actin expression resulted in a progressive down-regulation of mouse β- and γ-actin mRNAs. Transfection of the β-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse β-actin mRNA. In contrast, transfection of a β-actin gene carrying a single-point mutation (βsm) produced a feedback-regulatory response similar to that of the γ-actin gene. Expression of a β-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse γ-actin in both the γ- and βsm-actin transfectants. In contrast, a similar level of increase was observed for the mouse β-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse γ-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of β-actin may be distinct from that governing γ-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.