Tanycyte pyroglutamyl peptidase II contributes to regulation of the hypothalamic-pituitary-thyroid axis through glial-axonal associations in the median eminence

Abstract

Pyroglutamyl peptidase II (PPII), a highly specific membrane-bound metallopeptidase that inactivates TRH in the extracellular space, is tightly regulated by thyroid hormone in cells of the anterior pituitary. Whether PPII has any role in the region where axons containing hypophysiotropic TRH terminate, the median eminence, is unknown. For this purpose, we analyzed the cellular localization and regulation of PPII mRNA in the mediobasal hypothalamus in adult, male rats. PPII mRNA was localized in cells lining the floor and infralateral walls of the third ventricle and coexpressed with vimentin, establishing these cells as tanycytes. PPII mRNA extended in a linear fashion from the tanycyte cell bodies in the base of the third ventricle to its cytoplasmic and end-feet processes in the external zone of the median eminence in close apposition to pro-TRH-containing axon terminals. Compared with vehicle-treated, euthyroid controls, animals made thyrotoxic by the i.p. administration of 10 microg L-T(4) daily for 1-3 d, showed dramatically increased accumulation of silver grains in the mediobasal hypothalamus and an approximately 80% increase in enzymatic activity. PPII inhibition in mediobasal hypothalamic explants increased TRH secretion, whereas i.p. injection of a specific PPII inhibitor increased cold stress- and TRH-induced TSH levels in plasma. We propose that an increase in circulating thyroid hormone up-regulates PPII activity in tanycytes and enhances degradation of extracellular TRH in the median eminence through glial-axonal associations, contributing to the feedback regulation of thyroid hormone on anterior pituitary TSH secretion.

Figure 1

Rostrocaudal distribution of PPII mRNA in the MBH of an adult male rat. PPII mRNA is present in the most rostral tip of the floor of third ventricle (III, arrow) (A) and extends into the ventrolateral walls of the third ventricle in more caudal sections (arrows in E) (B–F) and into the pituitary stalk (arrow in F) in a distribution typical for α- and β-tanycytes (depicted in E). Cytoplasmic processes containing PPII mRNA are seen to extend through the internal zone of the median eminence (arrow in D) to terminate in the external zone. ARC, Arcuate nucleus; DMN, dorsomedial nucleus; ME, median eminence; VMN, ventromedial nucleus. Scale bar, 100 μm.

Figure 2

Colocalization of PPII mRNA and vimentin immunoreactivity in tanycytes of the third ventricle. Bright-field microscopy showing the distribution and subtypes of tanycytes in the third ventricular wall (vimentin immunostaining with diaminobenzidine). Note the presence of PPII mRNA (silver grains) in α- and β-tanycyte cell bodies (A) and basal processes in the median eminence (arrow) (B). EZ, External zone of the median eminence; IZ, internal zone of the median eminence. Scale bar, 20 μm.

Figure 3

Association between tanycytes and pro-TRH-containing axon terminals in the median eminence. A, Low-powered magnification of a triple-labeled section showing vimentin immunofluorescence in green, pro-TRH immunofluorescence in red, and DAPI fluorescence in blue. Pro-TRH axon terminals are associated primarily with β2-tanycyte cytoplasmic processes. B, High magnification of inset shown in A. C–E, Confocal images of β2-tanycyte end-feet processes immunostained with vimentin (C) and pro-TRH-containing axon terminals (D). Note that pro-TRH-containing axon terminals establish close associations with tanycyte end-feet processes in the external zone of the median eminence (E), near portal capillaries (F, arrow). III, Third ventricle; Arc, arcuate nucleus; EPZ, ependymal zone of the median eminence; EZ, external zone of the median eminence; IZ, internal zone of the median eminence. PT, Pars tuberalis. Arrows in B and E point to portal capillaries. Scale bars, 200 μm (A) and 50 μm (C–E).

Figure 4

PPII response in the MBH to T₄. Distribution of PPII mRNA in tanycytes of vehicle-treated animals using an antisense probe (A) and absence of signal when using a sense probe (B). Note marked increase of PPII mRNA in both the cell bodies (arrows in C) and cytoplasmic processes (arrow in C and D) of tanycytes in the median eminence (C) 7 h after a single dose of T₄ and persistence of the response 2, 3, and 4 d after T₄ injections (D–F). Scale bar, 200 μm. G, Diagram representing computerized image analysis of the in situ hybridization autoradiograms (five animals per group). **, P < 0.01; *, P < 0.05. Regions of the third ventricle (III) included in the analysis are outlined with dashes in E. H, Diagram representing enzymatic activity of PPII in membranes of the MBH obtained from a separate group of male rats treated with vehicle or T₄. Data are the mean of PPII activities expressed as picomoles per minute per milligram protein and sem. ***, P < 0.001; **, P < 0.01.

Figure 5

A, Effect of the PPII inhibitor HcPI on PPII enzymatic activity in the MBH and anterior pituitary of adult male rats and serum PPII (thyroliberinase) in plasma. Values are percentage of control: serum thyroliberinase, 2.25 ± 0.05 pmol/min · ml; MBH, 40.9 ± 1.3 pmol/min · mg; anterior pituitary, 22.5 ± 0.9 pmol/min · mg protein. B, Effect of TRH and HcPI on serum levels of TSH. C, Effect of HcPI on serum levels of TSH in animals exposed to cold. **, P < 0.01; ***, P < 0.001.

Figure 6

Proposed mechanism for the role of β2-tanycyte PPII in the median eminence. Once released from axon terminals, TRH is degraded by tanycyte PPII in the periportal space in response to elevated circulating levels of thyroid hormone, reducing the amount of active TRH transported in the portal system and, consequently, the secretion of TSH. The increase in tanycyte PPII occurs after active transport of T₄ (or T₃) into tanycytes by thyroid hormone transporters and the conversion of T₄ to T₃ in the tanycyte cytoplasm by type 2 iodothyronine deiodinase.