Endocrine Abstracts

Using qRT-PCR, we found that normal human thyroid follicular cells in culture express the mRNAs for the receptors for vascular endothelial growth factors (VEGFRs). qRT-PCR revealed that the relative production of mRNAs for VEGFRs was neuropilin1=neuropilin2=VEGFR2>VEGFR1 >VEGFR3. Western blotting for phosphoVEGFR2 showed labelling of a 235 kDa protein with few degradation products. VEGFR2 is likely in its active, phosphorylated form, because thyroid cells secrete large amounts of VEGF-1 (5–10 ng VEGFA per day per million cells). We examined VEGFR expression and subcellular distribution in these cultures using specific antisera with confocal microscopy. VEGFR1 staining was predominantly nuclear, shown by coincidence of staining with the DNA stain, DAPI. VEGFR2 staining was also nuclear although in many cells the nuclei were ‘capped’ at opposite poles with an area of intense staining in the cytoplasm. TSH reorganises the cells into 3D follicular structures. VEGFR2 was found on the apical side of the cell, bordering the follicles. TSH decreased VEGFR2 mRNA levels dose dependently but this was not evident by immunostaining, consistent with a protein of long half life. VEGFR3 was also localised to the nucleus. Neuropilin1 was cytoplasmic whereas neuropilin2 was found on the nuclear membrane and, like VEGFR2, showed capping of the nucleus. We conclude that thyroid cells express VEGFRs. VEGFR2 is phosphorylated suggesting an autocrine role for the VEGFs secreted. Consistent with several other receptor tyrosine kinases, we found that VEGFRs (with the exception of neuropilin1) were predominantly located in the nucleus. Its function there (transcription factor? transporter of VEGFs? et al.?) is unclear We suggest that the association of VEGFR2 with the apical, follicular side of the cell is important for the structural reorganisation required for follicle formation, analogous to its role in tube formation in the endothelium.