Thyrotropin chronically regulates the pool of thyroperoxidase and its intracellular distribution: A quantitative confocal microscopic study

Abstract

The regulation of thyroperoxidase (TPO) expression and of its intracellular distribution was studied in porcine thyroid cells cultured on porous bottom filters. Cells were cultured for 18 days in the absence or in the presence of thyrotropin (TSH) and with or without iodide. Microsomes were purified and analyzed by electrophoresis. TPO was detected by immunoblotting with polyclonal anti-porcine TPO antibodies and quantified by scanning the bands. The amount of TPO was increased 2-fold by TSH. High concentrations of iodide (1-50 microM, added daily) decreased the level of TPO. Confocal microscopy served to determine the intracellular localization of TPO and its quantitative distribution. Intracellular and surface-located TPO was detected by fluorescein-labeled antibodies on saponin-treated cells. Quantitative confocal microscopy showed that TSH increased the total amount of TPO 2-fold as for immunoblotting. The highest amount of TPO was found in the perinuclear area and between the nucleus and the Golgi apparatus. Only 4% of TPO was present on the apical surface and about 1% on the basolateral membrane; the remainder (about 95%) was inside the cells. TSH did not change these relative contents. TSH modified the intracellular distribution of the enzyme, increasing the TPO pool from the perinuclear area to apical membrane. This domain could be a site of storage of TPO. Adding a physiological concentration of iodide (0.5 microM, daily) did not influence the intracellular distribution of TPO. We concluded that chronic TSH stimulation 1) increased 2-fold the pool of TPO but did not change the relative proportion of TPO inside the cells and on the apical surface, and 2) modified the intracellular distribution of vesicular TPO, the major part of which was accumulated in the perinuclear and cytoplasmic area under the subapical domain of the polarized cells.

Cell Type-dependent Differences in Thyroid Peroxidase Cell Surface Expression

“…10). In this context, it is interesting to note a recent report that by immunofluorescence in thyrocytes after 18 days in primary culture, Յ5% of TPO is detected at the plasma membrane (24). Although such prolonged primary thyrocyte culture has not been extensively characterized, almost certainly these cells have an extremely low mitotic index.…”

“…Despite this, lingering questions persist about the trafficking of TPO to the cell surface in the thyroid and in various cell culture systems (24). In primary porcine thyrocytes after 7 days in culture, only ϳ30% of endogenously expressed TPO could be chemically modified at the cell surface with a non-permeant biotinylation reagent (25).…”

The effect of oxythioquinox exposure on normal human mammary epithelial cell gene expression: A microarray analysis study

“…Slides were washed in triplicate with phosphate buffered saline (PBS) and cover slips were added. Slides were dried for one hour at room temperature before viewing, using the laser scanning confocal microscope BX50 (Olympus), and quantitative analysis was performed [22]. Relative p53 expression was quantified between different time points and strains by determination of the area under the integrated intensity curve (Fluoview, Olympus, B & B Microscopes, Pittsburgh, PA).…”

Thyroid Hormone Synthesis

“…[207][208][209] The first 735 amino acids of TPO display a 42% sequence identity with MPO 210 and the polypeptide region interacting with the heme group is 74% homologous between TPO and MPO. [218][219][220][221] In contrast, TPO reaches the plasma membrane efficiently in transiently transfected Chinese hamster ovary (CHO) cells and stably transfected rat PCCl3 thyroid cells. 212,213 Based on this structure, theoretical three-dimensional structures have been derived for other mammalian peroxidases, 201 but the threedimensional structure of TPO has not been modeled.…”