Transcription of the Rat Sarcoplasmic Reticulum Ca Adenosine Triphosphatase Gene Is Increased by 3,5,3*- Triiodothyronine Receptor Isoform-Specific Interactions with the Myocyte-Specific Enhancer Factor-2a*

Thyroid hormone (T3) increases the transcription of the sarcoplasmic reticulumCa adenosine triphosphatase (ATPase) gene (SERCA 2) through three thyroid hormone response elements. The existence of repetitive cis elements with different configurations is likely to serve specific functions such as interactions with nuclear transcription factors. In addition, the presence of different T3 receptor isoforms (T3Rs) may contribute to another level of complexity in providing specificity for T3 action. In this study, we investigated T3Ra1vs. T3Rb1-specific interactions with the myocyte enhancer-specific factor-2 (MEF-2) on the expression of the SERCA 2 gene in transient transfection assays in embryonal heart-derived H9c2 cells. MEF-2a in combination with either T3Ra1 or T3Rb1 isoforms resulted in a 2.5-fold increase in SERCA 2 transgene expression in the absence of T3. Addition of T3 did not induce any further increase in SERCA 2 expressionwhenT3Ra1 andMEF-2a expression vectorswere cotransfected. In contrast, in the presence of T3Rb1 and MEF-2, the addition of T3 increased chlorampenicol acetyltransferase activity by an additional 2.2-fold to a total 5.5-fold increase. The interaction between MEF-2a and T3R is transcription factor specific because another factor that binds to MEF-2 consensus sites (heart factor 1b) was not able to interact with T3R. In addition, MEF-2a failed to interact with other nuclear factors (cAMP response element-binding protein and Egr-1) that stimulate SERCA 2 gene transcription. In addition, we found that a single homologous thyroid hormone response element is not able to mediate the interactions between MEF-2a and T3Rs to increase SERCA 2 gene transcription. Our findings point to T3R isoform-specific interactions with a cell type-specific transcription factor (MEF-2) in the regulation of SERCA 2 gene expression. (Endocrinology 138: 26–32, 1997) T HEART is an important target organ for thyroid hormone action, and T3-induced increases in the transcription of specific cardiac genes such as those coding for the Ca adenosine triphosphatase (ATPase) of the sarcoplasmic reticulum (SERCA 2) (1–4) or myosin heavy chain a (MHCa) have been reported (3, 5). Details of the transcriptional regulation of cardiac genes by T3 remain unexplored. It is, for example, unclearwhether interactions between transcription factors that are important in regulating the myocyte-specific gene program, such as myocyte enhancer-specific factor-2a (MEF-2a) (6) and T3 receptors (T3Rs) occur. In addition, it is currently unclear whether the simultaneous occurrence of different T3R isoforms, in particular T3Ra1 and T3Rb1, in cardiacmyocytes serve T3R isoform-specific regulatory functions. Previous studies, focusing on the central nervous system, showed that messenger RNAs coding for different T3R isoforms are differentially expressed in a developmental and spacial fashion, suggesting that T3R isoforms probably exhibit distinct functions (7). Furthermore, there is evidence, from T3Rb knock-out mice, that the T3Rb isoform is necessary for inner ear development. This is a function that cannot be assumed by T3Ra1 (8). Functional analyses of T3Rs have shown that they contain two types of transcription activation functions (AF). The AF2, mediated by the carboxyl-terminal D/E/F domain has been strictly associated with hormonedependent transactivation (9). The domain D/E/F is well conserved among the T3R isoforms, and the ligand-dependent transactivation function mediated by this domain is similar as well (10). On the other hand, the AF1 function, mediated by the N-terminus A/B domain, has been reported as having both ligand-dependent and independent transactivation properties and is, at the moment, the less well understood activation activity (9, 11). Furthermore, this domain presents no significant amino acid sequence similarity among the T3R isoforms able to bind ligand, suggesting that the N-terminus probably mediates T3R isoform-specific actions. We used the gene coding for the Ca ATPase of the sarcoplasmic reticulum as a model for studying interactions between T3Rs and the MEF-2a transcription factor. SERCA 2 plays an important role in heart function by lowering free cytosolic calcium levels during diastole, and T3-induced increases in the speed of diastolic relaxation in the heart are largely mediated through increased expression of this gene (12–14). In addition, we identified three thyroid hormone response elements (TRE) located in the regulatory region of Received May 21, 1997. Address requests for reprints to: Wolfgang H. Dillmann, M.D., Department of Medicine, University of California-San Diego, 9500 Gilman Drive (BSB/5063), La Jolla, California 92093-0618. * This work was supported by NIH Grant HL-25022. † Recipient of a fellowship from the Brazilian Conselho Nacional de Pesquisa. 0013-7227/97/$03.00/0 Vol. 138, No. 1 Endocrinology Printed in U.S.A. Copyright © 1997 by The Endocrine Society