P078
T3-DEPENDENT RABBIT SERCA2a PROMOTER ACTIVITY IS MODIFIED BY A MEMBER OF THE ZN FINGER/HOMEODOMAIN FAMILY, ZFHEP, IN JEG-3 CELLS.
Cabanillas AM§, Romero MR§,
§Departamento Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba,
The thyroid hormone (T3) affects the function of the cardiomyocyte, largely due to a direct effect on cardiac genes such as sarcoplasmic reticulum calcium ATPase (SERCA2a), a and b myosin heavy chain and others, through its receptor (TR), which is a DNA-binding protein. We found that the zinc finger homeodomain transcription factor Zfhep can bind similar DNA sequences as TR and inhibited T3-mediated activation of the rat growth hormone (GH) gene. Zfhep is highly expressed in rat heart, but its role in cardiocyte is still unknown. Our goal was to examine the ability of Zfhep to regulate transcription of a T3-responsive cardiac gene, SERCA2a. Transfection assays were performed in JEG-3 cells, using the rabbit SERCA2a promoter (562 base pairs of 5´-flanking sequence) driving the luciferase gene as reporter. Where appropiate, expression vector for Zfhep (pCDNAI/Zfhep), and T3 at 100 nM were present. An expression vector for TRa (pSG5-TRa) was always included. CMVb (expressing b-galactosidase) was used to correct for transfection efficiency. As an additional control, we performed transfections in parallel with GH TRE and DR4 TRE to check T3- and Zfhep- responses of the cells. As expected, GH TRE was responsive to T3 and to Zfhep and DR4 TRE was not responsive to Zfhep. Prior to transfection studies, we examined the endogenous expression of Zfhep in JEG-3 and other cell lines by Western analysis. We found no expression of Zfhep in nuclear extracts from JEG-3, and positive expression in COS-1 and GH4C1 cells. According to literature, T3 stimulated SERCA promoter activity 3.2 fold. Zfhep repressed T3-induced luciferase activity from 16.55 ± 0.50 to 9.29 ± 2.14 (arbitrary light units, n=3, P<0.05). T3-response (T3-r) was diminished by Zfhep to 30% of the control in JEG-3 cells. Seemingly, T3-r was decreased to 33 % in a rat heart-derived cell line H9C2. Our results suggest that: 1- JEG-3 cells represent a good model to study the effect of Zfhep on T3 action; 2- Zfhep could modulate TR-dependent transactivation of heart specific genes such as SERCA2a; 3- Zfhep effect might not be dependent on cell specific factors since it is unrelated to cardiocyte specific cell lines.
