Transfection of Nonmuscle / 3-and ,-Actin Genes into Myoblasts Elicits Different Feedback Regulatory Responses from Endogenous Actin Genes

We have examined the role of feedbackregulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human ~and ~-actin genes. In ~-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human T-actin expression resulted in a progressive down-regulation of mouse/~and ~/-actin mRNAs. Transfection of the ~-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse/3-actin mRNA. In contrast, transfection of a/~-actin gene carrying a single-point mutation ~sm) produced a feedback-regulatory response similar to that of the 7-actin gene. Expression of a /~-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 downregulation of mouse ~,-actin in both the ~and/3smactin transfectants. In contrast, a similar level of increase was observed for the mouse/3-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse 3,-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of B-actin may be distinct from that governing 7-actin. We conclude that actin feedbackregulation provides a biochemical assay for differences between the two nonmuscle actin genes. M AMMALIAN nonmuscle cell cytoarchitecture is comprised of three major polymeric structures: microtubules, microfilaments, and intermediate filaments. Regulation of the synthesis and assembly of these polymers is likely to have an important role in the deterwination of cell morphology. In particular, it is to be expected that the regulation of cellular content of each of these polymers may contribute to the determination of cell size and shape. It is therefore important to determine whether the expression of structural protein genes is programmed to produce a fixed level of mRNA output. Such a mechanism could regulate cell morphology via the control of structural protein monomer supply. Alternatively, it might be expected that the function of architectural components is monitored by the cell such that gene expression of a structural protein can be feedbackregulated to satisfy cellular demand. The term autoregulation can be used to describe a system where it has definitely been established that the gene product directly regulates its own expression, however, in all other cases, the phenomenon will be described as feedback-regulation. The discovery of tubulin autoregulation has demonstrated that the cell can regulate monomer supply of tubulin in accord with cellular demand for microtubules (Ben-Ze'ev et al., 1979). Recent studies have suggested that the pool of unpolymerized ~/~-tubulin dimer can directly regulate the stability of/3-tubulin mRNA (Pachter et al., 1987). This mechanism allows the cell to set/3-tubulin mRNA levels and therefore monomer levels according to the cellular demand for incorporation of monomer into microtubules. While this might be taken to suggest that cellular microtubule levels are not set by tubulin gene expression, the recent work of Katz et al. (1990) suggests otherwise. Transfection studies in yeast suggest that /3-tubulin gene expression may determine microtubule levels and that c~-tubulin gene expression is feedback-reg~ted in response to that of/3-tubulin. Intermediate filament gene expression appears to be set in mammalian cells and not subject to feedback-regulation. Forced expression of a number of intermediate filament proreins both in cell culture and in transgenic mice has tidied to elicit any feedback-regulatory response from the endogenous intermediate filament genes (Monteiro et al., 1990). These results suggest that intermediate filament levels are largely controlled by programmed gene expression independent of cytoplasmic levels of the protein. Microfilaments, like microtubules, may be subject to feedback-regulation. It has been suggested that the microO The Rockefeller University Press, 0021-9525/92/05/787/11 $2.00 The Journal of Cell Biology, Volume 117, Number 4, May 1992 787-797 787 on Jne 6, 2017 D ow nladed fom Published May 15, 1992