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An anti-sense c-erbA clone inhibits thyroid hormone-induced expression from the alpha-myosin heavy chain promoter
B E Markham1, R W Tsika, J J Bahl
1University Heart Center, University of Arizona, Tucson 85724.
Abstract:
The effects of thyroid hormone on expression of cardiac myosin heavy chain genes generally are thought to be mediated by nuclear 3,5,3'-triiodo-L-thyronine (T3) receptors that have been identified as the products of the protooncogene, c-erbA. This hypothesis has been tested by transfection of cardiomyocytes in primary culture with a plasmid, pRSVhEACAT-, expressing anti-sense c-erbA mRNA. Because only a low percentage of cells (20%) could be transfected in primary culture an alpha-myosin heavy chain-chloramphenicol acetyltransferase fusion construct was used as a reporter gene. The results indicate that the anti-sense plasmid almost completely blocks T3-induced activity of the reporter gene (less than 1% control) while transfection of a similar amount of the sense construct, pRSVhEACAT+, has no effect. When the c-erbA plasmids were cotransfected with constructs containing T3-independent promoters, no effects on expression were observed. The combined use of an anti-sense construct and a report gene provides a means of studying the role of c-erbA products in intracellular signal transduction even in differentiated, nondividing cells like those of the heart.
Insights
Thyroid hormone
Area of Science:
- Molecular Biology
- Cardiovascular Physiology
- Endocrinology
Background:
- Thyroid hormone regulates cardiac gene expression via nuclear receptors.
- These receptors are products of the protooncogene c-erbA.
- Understanding this mechanism is crucial for cardiac function.
Purpose of the Study:
- To investigate the role of c-erbA gene products in mediating thyroid hormone effects on cardiac myosin heavy chain gene expression.
- To validate the use of anti-sense RNA technology in studying gene function in cardiomyocytes.
Main Methods:
- Transfection of primary cultured cardiomyocytes with an anti-sense c-erbA expression plasmid.
- Utilizing an alpha-myosin heavy chain-chloramphenicol acetyltransferase fusion construct as a reporter gene.
- Assessing reporter gene activity following transfection and hormone treatment.
Main Results:
- The anti-sense c-erbA plasmid significantly inhibited thyroid hormone-induced reporter gene activity (less than 1% of control).
- A sense c-erbA construct did not affect reporter gene activity.
- No effect was observed when c-erbA plasmids were cotransfected with T3-independent promoters.
Conclusions:
- c-erbA gene products are essential mediators of thyroid hormone action on cardiac myosin heavy chain gene expression.
- Anti-sense RNA technology combined with reporter gene constructs is effective for studying intracellular signal transduction in differentiated cardiac cells.
- This approach allows for the investigation of c-erbA's role in non-dividing cells.