Pituitary stem cells: past, present and future perspectives.

Pituitary cells that express the transcription factor SOX2 are stem cells because they can self-renew and differentiate into multiple pituitary hormone-producing cell types as organoids. Wounding and physiological challenges can activate pituitary stem cells, but cell numbers are not fully restored, and the ability to mobilize stem cells decreases with increasing age. The basis of these limitations is still unknown. The regulation of stem cell quiescence and activation involves many different signalling pathways, including those mediated by WNT, Hippo and several cytokines; more research is needed to understand the interactions between these pathways. Pituitary organoids can be formed from human or mouse embryonic stem cells, or from human induced pluripotent stem cells. Human pituitary organoid transplantation is sufficient to induce corticosterone release in hypophysectomized mice, raising the possibility of therapeutic applications. Today, pituitary organoids have the potential to assess the role of individual genes and genetic variants on hormone production ex vivo, providing an important tool for the advancement of exciting frontiers in pituitary stem cell biology and pituitary organogenesis. In this article, we provide an overview of notable discoveries in pituitary stem cell function and highlight important areas for future research.

Differential alternative splicing analysis links variation in ZRSR2 to a novel type of oral-facial-digital syndrome.

PURPOSE: Oral-facial-digital (OFD) syndromes are genetically heterogeneous developmental disorders, caused by pathogenic variants in genes involved in primary cilia formation and function. We identified a previously undescribed type of OFD with brain anomalies, ranging from alobar holoprosencephaly to pituitary anomalies, in 6 unrelated families. METHODS: Exome sequencing of affected probands was supplemented with alternative splicing analysis in patient and control lymphoblastoid and fibroblast cell lines, and primary cilia structure analysis in patient fibroblasts. RESULTS: In 1 family with 2 affected males, we identified a germline variant in the last exon of ZRSR2, NM_005089.4:c.1211_1212del NP_005080.1:p.(Gly404GlufsTer23), whereas 7 affected males from 5 unrelated families were hemizygous for the ZRSR2 variant NM_005089.4:c.1207_1208del NP_005080.1:p.(Arg403GlyfsTer24), either occurring de novo or inherited in an X-linked recessive pattern. ZRSR2, located on chromosome Xp22.2, encodes a splicing factor of the minor spliceosome complex, which recognizes minor introns, representing 0.35% of human introns. Patient samples showed significant enrichment of minor intron retention. Among differentially spliced targets are ciliopathy-related genes, such as TMEM107 and CIBAR1. Primary fibroblasts containing the NM_005089.4:c.1207_1208del ZRSR2 variant had abnormally elongated cilia, confirming an association between defective U12-type intron splicing, OFD and abnormal primary cilia formation. CONCLUSION: We introduce a novel type of OFD associated with elongated cilia and differential splicing of minor intron-containing genes due to germline variation in ZRSR2.

Phenotypic and genotypic landscape of PROKR2 in neuroendocrine disorders.

Prokineticin receptor 2 (PROKR2) encodes for a G-protein-coupled receptor that can bind PROK1 and PROK2. Mice lacking Prokr2 have been shown to present abnormal olfactory bulb formation as well as defects in GnRH neuron migration. Patients carrying mutations in PROKR2 typically present hypogonadotropic hypogonadism, anosmia/hyposmia or Kallmann Syndrome. More recently variants in PROKR2 have been linked to several other endocrine disorders. In particular, several patients with pituitary disorders have been reported, ranging from mild phenotypes, such as isolated growth hormone deficiency, to more severe ones, such as septo-optic dysplasia. Here we summarize the changing landscape of PROKR2-related disease, the variants reported to date, and discuss their origin, classification and functional assessment.

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency.

Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated individuals with hypopituitarism that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant-negative loss of function. Using a high-throughput splicing reporter assay, we tested 1,070 single-nucleotide variants in POU1F1. We identified 96 splice-disruptive variants, including 14 synonymous variants. In separate cohorts, we found two additional synonymous variants nominated by this screen that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in POU1F1.

Comprehensive Identification of Pathogenic Gene Variants in Patients With Neuroendocrine Disorders.

PURPOSE: Congenital hypopituitarism (CH) can present in isolation or with other birth defects. Mutations in multiple genes can cause CH, and the use of a genetic screening panel could establish the prevalence of mutations in known and candidate genes for this disorder. It could also increase the proportion of patients that receive a genetic diagnosis. METHODS: We conducted target panel genetic screening using single-molecule molecular inversion probes sequencing to assess the frequency of mutations in known hypopituitarism genes and new candidates in Argentina. We captured genomic deoxyribonucleic acid from 170 pediatric patients with CH, either alone or with other abnormalities. We performed promoter activation assays to test the functional effects of patient variants in LHX3 and LHX4. RESULTS: We found variants classified as pathogenic, likely pathogenic, or with uncertain significance in 15.3% of cases. These variants were identified in known CH causative genes (LHX3, LHX4, GLI2, OTX2, HESX1), in less frequently reported genes (FOXA2, BMP4, FGFR1, PROKR2, PNPLA6) and in new candidate genes (BMP2, HMGA2, HNF1A, NKX2-1). CONCLUSION: In this work, we report the prevalence of mutations in known CH genes in Argentina and provide evidence for new candidate genes. We show that CH is a genetically heterogeneous disease with high phenotypic variation and incomplete penetrance, and our results support the need for further gene discovery for CH. Identifying population-specific pathogenic variants will improve the capacity of genetic data to predict eventual clinical outcomes.

Corrigendum to "Angiogenesis in Pituitary Adenomas: Human Studies and New Mutant Mouse Models".

[This corrects the article DOI: 10.1155/2014/608497.].

From Pituitary Stem Cell Differentiation to Regenerative Medicine.

The anterior pituitary gland is comprised of specialized cell-types that produce and secrete polypeptide hormones in response to hypothalamic input and feedback from target organs. These specialized cells arise during embryonic development, from stem cells that express SOX2 and the pituitary transcription factor PROP1, which is necessary to establish the stem cell pool and promote an epithelial to mesenchymal-like transition, releasing progenitors from the niche. Human and mouse embryonic stem cells can differentiate into all major hormone-producing cell types of the anterior lobe in a highly plastic and dynamic manner. More recently human induced pluripotent stem cells (iPSCs) emerged as a viable alternative due to their plasticity and high proliferative capacity. This mini-review gives an overview of the major advances that have been achieved to develop protocols to generate pituitary hormone-producing cell types from stem cells and how these mechanisms are regulated. We also discuss their application in pituitary diseases, such as pituitary hormone deficiencies.

Regulation of pituitary stem cells by epithelial to mesenchymal transition events and signaling pathways.

The anterior pituitary gland is comprised of specialized cell-types that produce and secrete polypeptide hormones in response to hypothalamic input and feedback from target organs. These specialized cells arise from stem cells that express SOX2 and the pituitary transcription factor PROP1, which is necessary to establish the stem cell pool and promote an epithelial to mesenchymal-like transition, releasing progenitors from the niche. The adult anterior pituitary responds to physiological challenge by mobilizing the SOX2-expressing progenitor pool and producing additional hormone-producing cells. Knowledge of the role of signaling pathways and extracellular matrix components in these processes may lead to improvements in the efficiency of differentiation of embryonic stem cells or induced pluripotent stem cells into hormone producing cells in vitro. Advances in our basic understanding of pituitary stem cell regulation and differentiation may lead to improved diagnosis and treatment for patients with hypopituitarism.

Chronic hyperprolactinemia evoked by disruption of lactotrope dopamine D2 receptors impacts on liver and adipocyte genes related to glucose and insulin balance.

We studied the impact of high prolactin titers on liver and adipocyte gene expression related to glucose and insulin homeostasis in correlation with obesity onset. To that end we used mutant female mice that selectively lack dopamine type 2 receptors (D2Rs) from pituitary lactotropes (lacDrd2KO), which have chronic high prolactin levels associated with increased body weight, marked increments in fat depots, adipocyte size, and serum lipids, and a metabolic phenotype that intensifies with age. LacDrd2KO mice of two developmental ages, 5 and 10 mo, were used. In the first time point, obesity and increased body weight are marginal, although mice are hyperprolactinemic, whereas at 10 mo there is marked adiposity with a 136% increase in gonadal fat and a 36% increase in liver weight due to lipid accumulation. LacDrd2KO mice had glucose intolerance, hyperinsulinemia, and impaired insulin response to glucose already in the early stages of obesity, but changes in liver and adipose tissue transcription factors were time and tissue dependent. In chronic hyperprolactinemic mice liver Prlr were upregulated, there was liver steatosis, altered expression of the lipogenic transcription factor Chrebp, and blunted response of Srebp-1c to refeeding at 5 mo of age, whereas no effect was observed in the glycogenesis pathway. On the other hand, in adipose tissue a marked decrease in lipogenic transcription factor expression was observed when morbid obesity was already settled. These adaptive changes underscore the role of prolactin signaling in different tissues to promote energy storage.

Genetics of Combined Pituitary Hormone Deficiency: Roadmap into the Genome Era.

The genetic basis for combined pituitary hormone deficiency (CPHD) is complex, involving 30 genes in a variety of syndromic and nonsyndromic presentations. Molecular diagnosis of this disorder is valuable for predicting disease progression, avoiding unnecessary surgery, and family planning. We expect that the application of high throughput sequencing will uncover additional contributing genes and eventually become a valuable tool for molecular diagnosis. For example, in the last 3 years, six new genes have been implicated in CPHD using whole-exome sequencing. In this review, we present a historical perspective on gene discovery for CPHD and predict approaches that may facilitate future gene identification projects conducted by clinicians and basic scientists. Guidelines for systematic reporting of genetic variants and assigning causality are emerging. We apply these guidelines retrospectively to reports of the genetic basis of CPHD and summarize modes of inheritance and penetrance for each of the known genes. In recent years, there have been great improvements in databases of genetic information for diverse populations. Some issues remain that make molecular diagnosis challenging in some cases. These include the inherent genetic complexity of this disorder, technical challenges like uneven coverage, differing results from variant calling and interpretation pipelines, the number of tolerated genetic alterations, and imperfect methods for predicting pathogenicity. We discuss approaches for future research in the genetics of CPHD.

Pituitary and brain dopamine D2 receptors regulate liver gene sexual dimorphism.

Liver sexual gene dimorphism, which depends mainly on specific patterns of GH secretion, may underlie differential susceptibility to some liver diseases. Because GH and prolactin secretion are regulated by dopaminergic pathways, we studied the participation of brain and lactotrope dopamine 2 receptors (D2Rs) on liver gene sexual dimorphism, to explore a link between the brain and liver gene expression. We used global D2R knockout mice (Drd2(-/-)) and conducted a functional dissection strategy based on cell-specific Drd2 inactivation in neurons (neuroDrd2KO) or pituitary lactotropes. Disruption of neuronal D2Rs (which impaired the GH axis) decreased most of male or female-predominant class I liver genes and increased female-predominant class II genes in males, consistent with the positive (class I) or negative (class II) regulation of these genes by GH. Notably, sexual dimorphism was lost for class I and II genes in neuroDrd2KO mice. Disruption of lactotrope D2Rs did not modify class I or II genes in either sex, because GH axis was preserved. But surprisingly, 1 class II gene (Prlr) and female-predominant class I genes were markedly up-regulated in lacDrd2KO females, pointing to direct or indirect effects of prolactin in the regulation of selected female-predominant liver genes. This suggestion was strengthened in the hyperprolactinemic Drd2(-/-) female mouse, in which increased expression of the same 4 liver genes was observed, despite a decreased GH axis. We hereby demonstrate endocrine-mediated D2R actions on sexual dimorphic liver gene expression, which may be relevant during chronic dopaminergic medications in psychiatric disease.

Angiogenesis in pituitary adenomas: human studies and new mutant mouse models.

The role of angiogenesis in pituitary tumor development has been questioned, as pituitary tumors have been usually found to be less vascularized than the normal pituitary tissue. Nevertheless, a significantly higher degree of vasculature has been shown in invasive or macropituitary prolactinomas when compared to noninvasive and microprolactinomas. Many growth factors and their receptors are involved in pituitary tumor development. For example, VEGF, FGF-2, FGFR1, and PTTG, which give a particular vascular phenotype, are modified in human and experimental pituitary adenomas of different histotypes. In particular, vascular endothelial growth factor, VEGF, the central mediator of angiogenesis in endocrine glands, was encountered in experimental and human pituitary tumors at different levels of expression and, in particular, was higher in dopamine agonist resistant prolactinomas. Furthermore, several anti-VEGF techniques lowered tumor burden in human and experimental pituitary adenomas. Therefore, even though the role of angiogenesis in pituitary adenomas is contentious, VEGF, making permeable pituitary endothelia, might contribute to adequate temporal vascular supply and mechanisms other than endothelial cell proliferation. The study of angiogenic factor expression in aggressive prolactinomas with resistance to dopamine agonists will yield important data in the search of therapeutical alternatives.

Pituitary gland development and disease: from stem cell to hormone production.

Many aspects of pituitary development have become better understood in the past two decades. The signaling pathways regulating pituitary growth and shape have emerged, and the balancing interactions between the pathways are now appreciated. Markers for multipotent progenitor cells are being identified, and signature transcription factors have been discovered for most hormone-producing cell types. We now realize that pulsatile hormone secretion involves a 3D integration of cellular networks. About a dozen genes are known to cause pituitary hypoplasia when mutated due to their essential roles in pituitary development. Similarly, a few genes are known that predispose to familial endocrine neoplasia, and several genes mutated in sporadic pituitary adenomas are documented. In the next decade, we anticipate gleaning a deeper appreciation of these processes at the molecular level, insight into the development of the hypophyseal portal blood system, and evolution of better therapeutics for congenital and acquired hormone deficiencies and for common craniopharyngiomas and pituitary adenomas.

Efficient, specific, developmentally appropriate cre-mediated recombination in anterior pituitary gonadotropes and thyrotropes.

Tissue-specific expression of cre recombinase is a well-established genetic tool to analyze gene function, and it is limited only by the efficiency and specificity of available cre mouse strains. Here, we report the generation of a transgenic line containing a cre cassette with codon usage optimized for mammalian cells (iCre) under the control of a mouse glycoprotein hormone α-subunit (αGSU) regulatory sequences in a bacterial artificial chromosome genomic clone. Initial analysis of this transgenic line, Tg(αGSU-iCre), with cre reporter strains reveals onset of cre activity in the differentiating cells of the developing anterior pituitary gland at embryonic day 12.5, with a pattern characteristic of endogenous αGSU. In adult mice, αGSU-iCre was active in the anterior lobe of the pituitary gland and in the cells that produce αGSU (gonadotropes and thyrotropes) with high penetrance. Little or no activity was observed in other tissues, including skeletal and cardiac muscle, brain, kidney, lungs, testis, ovary, and liver. This αGSU-iCre line is suitable for efficient, specific, and developmentally regulated deletion of floxed alleles in anterior pituitary gonadotropes and thyrotropes.

Enhanced nestin expression and small blood vessels in human pituitary adenomas.

The role of angiogenesis in human pituitary tumor progression is questioned. Our aim was to characterize the morphologic changes that occur in the vasculature of pituitary adenomas, in correlation with the expression of nestin, a protein found in endothelial cells of newly formed vessels of developing organs. We also evaluated the relation of angiogenic markers and nestin with Ki-67 index. Immunohistochemical studies were performed on paraffin embedded samples of 47 pituitary adenomas and six normal pituitaries. We determined microvessel density (number of CD31+ or CD34+ vessels per square millimetre), vascular area (cumulative area occupied by vessels), average vessel size, and further classified vessels as small (< 100 µm2) or large (> 100 µm2). We correlated the above parameters with nestin expression and Ki-67 index. Lower vascular area compared to normal tissue was found in adenomas (p < 0.05). Interestingly, pituitary adenomas had significantly more small vessels than control pituitaries (p < 0.04 for CD31 and CD34). In tumors many capillaries were positive for nestin, while scarce staining was detected in controls, so that nestin positive area was significantly higher in tumors. Furthermore, nestin area correlated positively with the % of small vessels. Ki-67 correlated neither with vascular area nor with nestin expression. In human pituitary tumors there was a predominance of small capillaries in correlation with increased expression of the progenitor marker nestin. We suggest that angiogenesis is an active process in these tumors, in spite of their low total vascular area when compared to normal pituitaries.

Inhibitory effects of antivascular endothelial growth factor strategies in experimental dopamine-resistant prolactinomas.

Prolactin-secreting adenomas are the most frequent type among pituitary tumors, and pharmacological therapy with dopamine agonists remains the mainstay of treatment. But some adenomas are resistant, and a decrease in the number or function of dopamine D2 receptors (D2Rs) has been described in these cases. D2R knockout [Drd2(-/-)] mice have chronic hyperprolactinemia and pituitary hyperplasia and provide an experimental model for dopamine agonist-resistant prolactinomas. We described previously that disruption of D2Rs increases vascular endothelial growth factor (VEGF) expression. We therefore designed two strategies of antiangiogenesis using prolactinomas generated in Drd2(-/-) female mice: direct intra-adenoma mVEGF R1 (Flt-1)/Fc chimera (VEGF-TRAP) injection for 3 weeks [into subcutaneously transplanted pituitaries from Drd2(-/-) mice] and systemic VEGF neutralization with the specific monoclonal antibody G6-31. Both strategies resulted in substantial decrease of prolactin content and lactotrope area, and a reduction in tumor size was observed in in situ prolactinomas. There were significant decreases in vascularity, evaluated by cluster of differentiation molecule 31 vessel staining, and proliferation (proliferating cell nuclear antigen staining) in response to both anti-VEGF treatments. These data demonstrate that the antiangiogenic approach was effective in inhibiting the growth of in situ dopamine-resistant prolactinomas as well as in the transplanted adenomas. No differences in VEGF protein expression were observed after either anti-VEGF treatment, and, although serum VEGF was increased in G6-31-treated mice, pituitary activation of the VEGF receptor 2 signaling pathway was reduced. Our results indicate that, even though the role of angiogenesis in pituitary adenomas is contentious, VEGF might contribute to adequate vascular supply and represent a supplementary therapeutic target in dopamine agonist-resistant prolactinomas.

New insights into the endocrine and metabolic roles of dopamine D2 receptors gained from the Drd2 mouse.

Dopamine D2 receptor (D2R) participation in prolactin regulation is well documented, but the role of D2Rs in the control of other hormones involved in growth, food intake and glucose metabolism has not been extensively studied. The study of D2R knockout mice (Drd2(-/-)) puts forward new insights into the role of the D2R in growth hormone (GH)-releasing hormone-GH regulation, peptides involved in food intake, glucose homeostasis, as well as in prolactinoma development. The expected phenotype of chronic hyperprolactinemia and prolactinoma development was found in the Drd2(-/-) mouse, and this model constitutes a valuable tool in the study of dopamine-resistant prolactinomas. Unexpectedly, these mice were growth retarded, and the importance of functional hypothalamic D2Rs in the neonatal period was revealed. In the Drd2(-/-) mouse there was a failure of high neonatal GH levels and therefore the expansion of pituitary somatotropes was permanently altered. These mice also had increased food intake, and a sexually dimorphic participation of the D2R in food intake regulation is suggested. The effect described is probably secondary to D2R regulation of prolactin secretion. Furthermore, the negative modulation of D2Rs on α-melanocyte-stimulating hormone release and positive action on the hypothalamic expression of orexins reveals the complex D2R regulation of food intake. Finally, pancreatic D2Rs inhibit glucose-stimulated insulin release. Lack of dopaminergic inhibition throughout development in the Drd2(-/-) mouse may exert a gradual deteriorating effect on insulin homeostasis, so that eventually glucose intolerance develops. These results highlight the complex endocrine actions of the D2Rs at different levels, hypothalamus, pituitary or pancreas, which function to improve fitness, reproductive success and survival.

VEGF and CD31 association in pituitary adenomas.

Pituitary tumors are usually less vascularized than the normal pituitary, and the role of angiogenesis in these adenomas is contentious. Appraisal of microvascular density and expression of the potent angiogenic vascular endothelial growth factor (VEGF) by immunohistochemistry has yielded controversial results, as a broad spectrum of immunostaining can be found. We determined the protein expression of VEGF and CD31, an endothelial marker, in a series of 56 surgically removed pituitary adenomas using Western blot assay. Prolactinomas had higher VEGF protein expression compared to nonfunctioning or ACTH- and GH-secreting adenomas, while CD31 was similar in the different adenoma histotypes. VEGF and CD31 were not affected by sex, age, years of adenoma evolution, or proliferation rate (Ki67 and PCNA) for all adenoma types. Only in nonfunctioning adenomas CD31 concentration increased significantly with age. There was a positive correlation between CD31 and VEGF expression when all adenoma histotypes were considered, or when prolactinomas and nonfunctioning adenomas were evaluated separately. The positive association of VEGF and CD31 expression suggests the participation of angiogenesis in adenoma development, while epithelial cell proliferation in pituitary tumors is not directly related to VEGF or CD31 expression, and other factors, such as primary genetic alterations may be involved.