The biological relevance of thyroid hormone receptors in immortalized human umbilical vein endothelial cells.
The gene expression of thyroid hormone receptors (TR) in ECRF24 immortalized human umbilical vein endothelial cells (HUVECs) was investigated at both the mRNA and the protein level. Endothelin-1 (ET-1) and von Willebrand factor (vWF) production were measured in response to triiodothyronine (T(3)) administration. A real-time PCR technique was used to quantify the presence of mRNAs encoding for the different isoforms of the TR. The binding of T(3) to nuclear TRs was studied in isolated endothelial cell nuclei by Scatchard analysis. Expression of TR at the protein level was investigated by immunocytochemistry and Western blotting using TR-isoform-specific polyclonal rabbit antisera. ET-1 and vWF were measured in cell supernatants with a two-site immunoenzymatic assay. Scatchard analysis yielded a maximum binding capacity of 55 fmol T(3)/mg DNA (+/-200 sites/cell) with a K(d) of 125 pmol/l. Messenger RNAs encoding for the TRalpha1 and the TRalpha2 and the TRbeta1 were observed. The approximate number of mRNA molecules per cell was at least 50 molecules per cell for TRalpha1, five for TRalpha2 and two for TRbeta1. Immunocytochemistry revealed (peri)nuclear staining for TRbeta1, TRalpha1 and TRalpha2. ET-1 and vWF secretion did not increase upon addition of T(3) (10(-10)-10(-6) M). Immortalized ECRF24 HUVECs express TR, but at low levels. The number of TRs per endothelial cell is probably too low to be functional and no change in ET-1 or vWF production was found after addition of T(3). Therefore we conclude that the genomic effects of T(3) are unlikely to occur in these immortalized HUVECs.
Department of Endocrinology, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
M J
MJ
Diekman
B
B
Zandieh Doulabi
M
M
Platvoet-Ter Schiphorst
E
E
Fliers
O
O
Bakker
W M
WM
Wiersinga
D015153:Blotting, Western; D018929:Cell Culture Techniques; D002460:Cell Line; D019332:Endothelin-1 / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007124:Immunoenzyme Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism*; D014841:von Willebrand Factor / Q000096:biosynthesis
D019332:Endothelin-1; D012333:RNA, Messenger; D011988:Receptors, Thyroid Hormone; D014841:von Willebrand Factor