Anterior pituitary pyroglutamyl peptidase II activity controls TRH-induced prolactin release

doi:10.1016/j.peptides.2008.07.011

Peptides

Volume 29, Issue 11, November 2008, Pages 1953-1964

https://doi.org/10.1016/j.peptides.2008.07.011 Get rights and content

Abstract

Ecto-peptidases modulate the action of peptides in the extracellular space. The relationship between peptide receptor and ecto-peptidase localization, and the physiological role of peptidases is poorly understood. Current evidence suggests that pyroglutamyl peptidase II (PPII) inactivates neuronally released thyrotropin-releasing hormone (TRH). The impact of PPII localization in the anterior pituitary on the endocrine activities of TRH is unknown. We have studied whether PPII influences TRH signaling in anterior pituitary cells in primary culture. In situ hybridization (ISH) experiments showed that PPII mRNA was expressed only in 5–6% of cells. ISH for PPII mRNA combined with immunocytochemistry for prolactin, β-thyrotropin, or growth hormone, showed that 66% of PPII mRNA expressing cells are lactotrophs, 34% somatotrophs while none are thyrotrophs. PPII activity was reduced using a specific phosphorothioate antisense oligodeoxynucleotide or inhibitors. Compared with mock or scrambled oligodeoxynucleotide-treated controls, knock-down of PPII expression by antisense targeting increased TRH-induced release of prolactin, but not of thyrotropin. Similar data were obtained with either a transition-state or a tight binding inhibitor. These results demonstrate that PPII expression in lactotrophs coincides with its ability to control prolactin release. It may play a specialized role in TRH signaling in the anterior pituitary. Anterior pituitary ecto-peptidases may fulfill unique functions associated with their restricted cell-specific expression.

Introduction

Ecto-peptidases modify or limit the effects of peptides in the extracellular space. The physiological influence of some of these peptidases has been clearly demonstrated through the use of enzyme inhibitors or knock-out mice [41]. Peptide sensitivity to hydrolysis by an ecto-peptidase in vitro is not always coupled to in vivo susceptibility, since peptidases and their in vitro substrates do not co-localize consistently in vivo [26]. The anterior pituitary has a complex cyto-architecture with no clear-cut anatomical boundaries between endocrine cells of distinct phenotypes. However, cells are not uniformly distributed but mixed in distributions peculiar to each cell type, with some partially segregated from each other as multiple clusters, each consisting almost exclusively of the same phenotype [27], [31], [34]. Information regarding the role of ecto-peptidases in anterior pituitary is very limited, or difficult to interpret, in spite of the critical role of various peptides [11], [40]. Many ecto-peptidases are present in the gland with a limited distribution [11]. For example, within any one cluster of secretory cells in the pig anterior pituitary, only some cells are positive for neutral endopeptidase-24.11 while other clusters are wholly devoid of this enzyme [30]. The relevance of these peculiar distributions is unknown.

In mammals, thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH₂), secreted from parvocellular neurons of the paraventricular nucleus of the hypothalamus, is transported within the hypothalamic-hypophyseal portal system and acts in the anterior pituitary through a specific receptor, TRH-R1. TRH modulates the pituitary–thyroid axis [58], regulates prolactin (PRL) release during lactation [59] and, under specific conditions, growth hormone (GH) secretion [22].

Various lines of evidence suggest that TRH is hydrolyzed in vivo at the pyroglutamyl-histidyl bond, by the ectoenzyme pyroglutamyl peptidase II (PPII, EC 3.4.19.6). PPII is a cell surface peptidase located on neuronal and anterior pituitary cells where it seems strategically localized to play a significant role in the extracellular inactivation of TRH [5], [10]. PPII is a type II integral membrane protein containing a large extracellular region with a consensus sequence characteristic of the M1 family of gluzincins. The aminoacid sequence of the rat PPII has significant homology with human aminopeptidase N (APN; EC 3.4.11.2; 34%) and mouse aminopeptidase A (APA; EC 3.4.11.7; 32%) [43]. In contrast to the majority of ecto-peptidases, PPII is a narrow specificity peptidase hydrolyzing substrates with the general structure pGlu-X-Y, X being a moderately bulky and uncharged residue and Y, either Pro, Ala, Trp, Pro-Gly, ProNH₂, Pro-β-naphtylamine, or Pro-7-amino-4-methyl coumarin [3], [25], [33], [57]. Some structural determinants of this restricted specificity have been identified [14].

Once hypophysiotropic TRH is released, it may be hydrolyzed by PPII in the perivascular space of the median eminence, or by thyroliberinase, an isoform of PPII originating in the liver [44], in the portal vessels or by PPII in the anterior pituitary. In the anterior pituitary, the activity of PPII is lower than in the brain [53]. The separation of anterior pituitary cells by sedimentation at unit gravity shows that PPII activity is expressed in lactotrophs, and not in another cell type [5]. Anterior pituitary PPII activity is tightly regulated by estrogens and thyroid hormones [1], [2], [28], [37], as well as by hypothalamic/paracrine factors [50], [52], [54]. It might control TRH-stimulated hormone release [12]. However, this hypothesis has not been tested experimentally.

To understand the specific role of PPII in the anterior pituitary, we performed an in vitro study as we cannot yet manipulate specifically anterior pituitary PPII without affecting median eminence PPII or portal vessel thyroliberinase in vivo. We used primary cultures of dispersed anterior pituitary cells, instead of tissue slices or aggregate cell cultures, due to the uncertain efficiency of the knock-down technique in these settings. We analyzed the cellular distribution of PPII mRNA and used PPII antisense oligodeoxynucleotides (ASO), or PPII inhibitors, to study the effect of selective reduction of the expression or activity of the enzyme on thyrotropin (TSH) or PRL release in response to TRH. We demonstrate that PPII mRNA is not expressed in most of anterior pituitary TRH target cells and that PPII controls TRH-induced PRL release.

Section snippets

Animals

Random cycle female Wistar rats weighing 250–300 g were housed on a 12 h light–12 h dark schedule with free access to food and water. Rats were killed by decapitation between 10:00 and 11:00 A.M. The guidelines for the use of animals in neuroscience research of the Society for Neuroscience (USA) were followed. Procedures were approved by the Bioethics Committee of our Institute.

Cell culture

Cultures of anterior pituitary cells were performed as previously described [50], with minor modifications. For in situ

PPII mRNA is expressed in a small proportion of anterior pituitary cells in culture

PPII activity and mRNA levels are relatively low in anterior pituitaries of adult rats [21], [23], [28], [53] but are markedly up regulated by T₃ [1], [2], [28], [37]. If T₃ is not added, PPII activity and mRNA levels are barely detectable in primary cultures [5], [51], probably because thyroid hormone levels are very low in the culture medium. To identify the cells expressing PPII mRNA, we carried out radioactive ISH studies on pituitary cells pre-treated for 120 h with 10 nM T₃. This dose of T₃

Discussion

The anterior pituitary gland is one of the most anatomically complex endocrine glands in vertebrates and serves critical homeostatic functions by regulating key organs in response to signals from brain and periphery. Some of these signals are peptides, and the role of ecto-peptidases in this organ is poorly understood. In the present study, we show that in cultured anterior pituitary cells, PPII mRNA is expressed in small sub-populations of lactotrophs and somatotrophs but not in thyrotrophs.

Conclusions

Anterior pituitary PPII activity controls TRH signaling in a sub-population of lactotrophs since PPII activity is markedly regulated by various of the effectors that regulate PRL release, it is localized on some lactotrophs and the reduction of its activity enhances TRH-induced PRL release. As we could not observe a similar situation for TSH release, we suggest that the anterior pituitary role of PPII is cell-type specific. We propose that the specific localization and level of expression of

Acknowledgments

The authors thank the technical help of M. Cisneros, as well as the animal care by S. Gonzalez. This work was supported by grants 39931 from CONACYT to J.L.C., 24975 from CONACYT to M.A.V., IN226406-3 from DGAPA-UNAM to J.L.C., 3276/2 from International Foundation for Sciences and the Wood Wheland Program (IUBMB) to I.P.

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Thyroid hormones (THs) are ancient signaling molecules that contribute to the regulation of metabolism, energy homeostasis and growth. In vertebrates, the hypothalamus-pituitary-thyroid (HPT) axis links the corresponding organs through hormonal signals, including thyrotropin releasing factor (TRF), and thyroid stimulating hormone (TSH) that ultimately activates the synthesis and secretion of THs from the thyroid gland. Although this axis is conserved among most vertebrates, the identity of the hypothalamic TRF that positively regulates TSH synthesis and secretion varies. We review the evolution of the hypothalamic factors that induce TSH secretion, including thyrotropin-releasing hormone (TRH), corticotrophin-releasing hormone (CRH), urotensin-1–3, and sauvagine, and non-mammalian glucagon-like peptide in metazoans. Each of these peptides is part of an extracellular communication unit likely composed of at least 3 elements: the peptide, G-protein coupled receptor and bioavailability regulator, set up on the central neuroendocrine articulation. The bioavailability regulators include a TRH-specific ecto-peptidase, pyroglutamyl peptidase II, and a CRH-binding protein, that together with peptide secretion/transport rate and transduction coupling and efficiency at receptor level shape TRF signal intensity and duration. These vertebrate TRF communication units were coopted from bilaterian ancestors. The bona fide elements appeared early in chordates, and are either used alternatively, in parallel, or sequentially, in different vertebrate classes to control centrally the activity of the HPT axis. Available data also suggest coincidence between apparition of ligand and bioavailability regulator.
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PPII is present in neuronal cells and cells of the anterior pituitary gland (Charli et al., 1998). TRH specifically inhibits PPII activity in TRH-responsive lactotrophic cells (Cruz et al., 2008; Vargas et al., 1994). Studies on circadian levels of TSH indicate lower TRH levels during the day and higher levels at night.
Thyrotropin-releasing hormone (TRH) causes a variety of thyroidal and non-thyroidal effects, the best known being the feedback regulation of thyroid hormone levels. This was employed in the TRH stimulation test, which is currently little used. The role of TRH as a cancer biomarker is minor, but exaggerated responses to TSH and prolactin levels in breast cancer led to the hypothesis of a potential role for TRH in the pathogenesis of this disease. TRH is a rapidly degraded peptide with multiple targets, limiting its suitability as a biomarker and drug candidate. Although some studies reported efficacy in neural diseases (depression, spinal cord injury, amyotrophic lateral sclerosis, etc.), therapeutic use of TRH is presently restricted to spinocerebellar degenerative disease. Regulation of TRH production in the hypothalamus, patterns of expression of TRH and its receptor in the body, its role in energy metabolism and in prolactin secretion are addressed in this review.
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The more accepted role of AH PPII is to regulate TRH-induced PRL release. In vitro studies have shown that by inhibiting PPII activity, the addition of TRH into cultured adenohypophyseal cells is able to increase PRL release but not that of TSH (Cruz et al., 2008). In vivo and in vitro studies show that TRH is able to down-regulate PPII mRNA expression and activity in the AH (Vargas et al., 1994, 1998, 2002).
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Anterior pituitary pyroglutamyl peptidase II activity controls TRH-induced prolactin release

Abstract

Introduction

Section snippets

Animals

Cell culture

PPII mRNA is expressed in a small proportion of anterior pituitary cells in culture

Discussion

Conclusions

Acknowledgments

Biochimie

TEM

Neuropeptides

Neurochem Int

J Biol Chem

Biochem Biophys Res Commun

Anal Biochem

Neurochem Int

J Biol Chem

Neuroscience

Int J Biochem Cell Biol

TPS

Neuropharmacology

Biochim Biophys Acta

Biochem Pharmacol

Neurosci Let

Neurochem Int

Adenohypophyseal degradation of thyrotropin releasing hormone regulated by thyroid hormones

Nature

Purification and characterization of the thyrotropin-releasing-hormone-degrading ectoenzyme

Eur J Biochem

Regulation and cellular localization of the membrane bound thyrotropin-releasing hormone-degrading enzyme in primary cultures of neuronal, glial and adenohypophyseal cells

Endocrinology

Analysis by plaque assay of GH and prolactin release from individual cells in culture of male pituitaries: evidence for functional heterogeneity within rat mammotrope and somatotrope

Neuroendocrinology

Thyrotropin-releasing hormone (TRH) gene expression in the anterior pituitary. I. Presence of pro-TRH messenger ribonucleic acid and pro-TRH-derived peptide in a subpopulation of somatotrophs

Endocrinology

Thyrotropin-releasing hormone gene expression in the anterior pituitary. II. Stimulation by glucocorticoids

Endocrinology

Post-secretory inactivation of peptides in the hypothalamic-adenohypophyseal axis: focus on pyroglutamyl peptidase II, the thyrotropin releasing hormone inactivating ectoenzyme

TRH inactivation in the extracellular compartment: role of pyroglutamyl peptidase II

Neurobiology

A truncated isoform of pyroglutamyl aminopeptidase II produced by exon extension has dominant negative activity

J Neurochem

3,3′,5′-Triiodo-l-thyronine reduces efficiency of mRNA knockdown by antisense oligodeoxynucleotides: a study with pyroglutamyl aminopeptidase II in adenohypophysis

Oligonucleotides

Stage-specific modulation of neprilysin and aminopeptidase N in the limbic system during kindling progression

J Mol Neurosci

Prolactin: structure, function, and regulation of secretion

Physiol Rev

Delineation of a particulate thyrotropin-releasing-hormone-degrading enzyme in rat brain by the use of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase

J Neurochem