Montserrat Garcia-Lavandeira

Craniopharyngiomas Express Embryonic Stem Cell Markers (SOX2, OCT4, KLF4, and SOX9) as Pituitary Stem Cells but Do Not Coexpress RET/GFRA3 Receptors

CONTEXT Adult stem cells maintain some markers expressed by embryonic stem cells and express other specific markers depending on the organ where they reside. Recently, stem/progenitor cells in the rodent and human pituitary have been characterized as expressing GFRA2/RET, PROP1, and stem cell markers such as SOX2 and OCT4 (GPS cells). OBJECTIVE Our objective was to detect other specific markers of the pituitary stem cells and to investigate whether craniopharyngiomas (CRF), a tumor potentially derived from Rathkes pouch remnants, express similar markers as normal pituitary stem cells. DESIGN We conducted mRNA and Western blot studies in pituitary extracts, and immunohistochemistry and immunofluorescence on sections from normal rat and human pituitaries and 20 CRF (18 adamantinomatous and two papillary). RESULTS Normal pituitary GPS stem cells localized in the marginal zone (MZ) express three key embryonic stem cell markers, SOX2, OCT4, and KLF4, in addition to SOX9 and PROP1 and β-catenin overexpression. They express the RET receptor and its GFRA2 coreceptor but also express the coreceptor GFRA3 that could be detected in the MZ of paraffin pituitary sections. CRF maintain the expression of SOX2, OCT4, KLF4, SOX9, and β-catenin. However, RET and GFRA3 expression was altered in CRF. In 25% (five of 20), both RET and GFRA3 were detected but not colocalized in the same cells. The other 75% (15 of 20) lose the expression of RET, GFRA3, or both proteins simultaneously. CONCLUSIONS Human pituitary adult stem/progenitor cells (GPS) located in the MZ are characterized by expression of embryonic stem cell markers SOX2, OCT4, and KLF4 plus the specific pituitary embryonic factor PROP1 and the RET system. Redundancy in RET coreceptor expression (GFRA2 and GFRA3) suggest an important systematic function in their physiological behavior. CRF share the stem cell markers suggesting a common origin with GPS. However, the lack of expression of the RET/GFRA system could be related to the cell mislocation and deregulated growth of CRF.

Deletion of miRNA processing enzyme Dicer in POMC-expressing cells leads to pituitary dysfunction, neurodegeneration and development of obesity.

MicroRNAs (miRNAs) have recently emerged as key regulators of metabolism. However, their potential role in the central regulation of whole-body energy homeostasis is still unknown. In this study we show that the expression of Dicer, an essential endoribonuclease for miRNA maturation, is modulated by nutrient availability and excess in the hypothalamus. Conditional deletion of Dicer in POMC-expressing cells resulted in obesity, characterized by hyperphagia, increased adiposity, hyperleptinemia, defective glucose metabolism and alterations in the pituitary-adrenal axis. The development of the obese phenotype was paralleled by a POMC neuron degenerative process that started around 3 weeks of age. Hypothalamic transcriptomic analysis in presymptomatic POMCDicerKO mice revealed the downregulation of genes implicated in biological pathways associated with classical neurodegenerative disorders, such as MAPK signaling, ubiquitin-proteosome system, autophagy and ribosome biosynthesis. Collectively, our results highlight a key role for miRNAs in POMC neuron survival and the consequent development of neurodegenerative obesity.

Direct promoter induction of p19Arf by Pit-1 explains the dependence receptor RET/Pit-1/p53-induced apoptosis in the pituitary somatotroph cells

Somatotrophs produce growth hormone (GH) and are the most abundant secretory cells of the pituitary. Somatotrophs express the transcription factor Pit-1 and the dependence receptor RET, its co-receptor GFRa1 and ligand GDNF. Pit-1 is a transcription factor essential for somatotroph proliferation and differentiation and for GH expression. GDNF represses excess Pit-1 expression preventing excess GH. In the absence of GDNF, RET behaves as a dependence receptor, becomes intracellularly processed and induces strong Pit-1 expression leading to p53 accumulation and apoptosis. How accumulation of Pit-1 leads to p53 expression is unknown. We have unveiled the relationship of Pit-1 with the p19Arf gene. There is a parallel correlation of RET processing, Pit-1 increase and ARF protein and mRNA expression. Interfering the pathway with RET, Pit-1 or p19Arf siRNA blocked apoptosis. We have found a Pit-1 DNA-binding element within the ARF promoter. Pit-1 directly regulates the CDKN2A locus and binds to the p19Arft promoter inducing p19Arf gene expression. The Pit-1-binding element is conserved in rodents and humans. RET/Pit-1 induces p19Arf/p53 and apoptosis not only in a somatotroph cell line but also in primary cultures of pituitary somatotrophs, where ARF siRNA interference also blocks p53 and apoptosis. Analyses of the somatotrophs in whole pituitaries supported the above findings. Thus Pit-1, a differentiation factor, activates the oncogene-induced apoptosis (OIA) pathway as oncogenes exerting a tight control in somatotrophs to prevent the disease due to excess of GH (insulin-resistance, metabolic disease, acromegaly).

Pituitary Cell Turnover: From Adult Stem Cell Recruitment through Differentiation to Death

The recent demonstration using genetic tracing that in the adult pituitary stem cells are normally recruited from the niche in the marginal zone and differentiate into secretory cells in the adenopituitary has elegantly confirmed the proposal made when the pituitary stem cell niche was first discovered 5 years ago. Some of the early controversies have also been resolved. However, many questions remain, such as which are the markers that make a pituitary stem cell truly unique and the exact mechanisms that trigger recruitment from the niche. Little is known about the processes of commitment and differentiation once a stem cell has left the niche. Moreover, the acceptance that pituitary cells are renewed by stem cells implies the existence of regulated mechanisms of cell death in differentiated cells which must themselves be explained. The demonstration of an apoptotic pathway mediated by RET/caspase 3/Pit-1/Arf/p53 in normal somatotrophs is therefore an important step towards understanding how pituitary cell number is regulated. Further work will elucidate how the rates of the three processes of cell renewal, differentiation and apoptosis are balanced in tissue homeostasis after birth, but altered in pituitary hyperplasia in response to physiological stimuli such as puberty and lactation. Thus, we can aim to understand the mechanisms underlying human disease due to insufficient (hypopituitarism) or excess (pituitary tumor) cell numbers.

Functional Role of the RET Dependence Receptor, GFRa Co-Receptors and Ligands in the Pituitary

The RET receptor is a tyrosine kinase receptor implicated in kidney and neural development. In the adenopituitary RET and the co-receptor GFRa1 are expressed exclusively in the somatotrophs secreting GH. RET is implicated in a clever pathway to maintain at physiological levels the number of somatotrophs and the GH production. Thus, in absence of its ligand GDNF, RET induces apoptosis through massive expression of Pit-1 leading to p53 accumulation. In the presence of the ligand GDNF, RET activates its tyrosine kinase and promotes survival at the expense of reducing Pit-1 expression and downregulating GH. Recent data suggest that RET can also have a second role in pituitary plasticity through a second co-receptor GFRa2.

Somatotropinomas, But Not Nonfunctioning Pituitary Adenomas, Maintain a Functional Apoptotic RET/Pit1/ARF/p53 Pathway That Is Blocked by Excess GDNF

Acromegaly is caused by somatotroph cell adenomas (somatotropinomas [ACROs]), which secrete GH. Human and rodent somatotroph cells express the RET receptor. In rodents, when normal somatotrophs are deprived of the RET ligand, GDNF (Glial Cell Derived Neurotrophic Factor), RET is processed intracellularly to induce overexpression of Pit1 [Transcription factor (gene : POUF1) essential for transcription of Pituitary hormones GH, PRL and TSHb], which in turn leads to p19Arf/p53-dependent apoptosis. Our purpose was to ascertain whether human ACROs maintain the RET/Pit1/p14ARF/p53/apoptosis pathway, relative to nonfunctioning pituitary adenomas (NFPAs). Apoptosis in the absence and presence of GDNF was studied in primary cultures of 8 ACROs and 3 NFPAs. Parallel protein extracts were analyzed for expression of RET, Pit1, p19Arf, p53, and phospho-Akt. When GDNF deprived, ACRO cells, but not NFPAs, presented marked level of apoptosis that was prevented in the presence of GDNF. Apoptosis was accompanied by RET processing, Pit1 accumulation, and p14ARF and p53 induction. GDNF prevented all these effects via activation of phospho-AKT. Overexpression of human Pit1 (hPit1) directly induced p19Arf/p53 and apoptosis in a pituitary cell line. Using in silico studies, 2 CCAAT/enhancer binding protein alpha (cEBPα) consensus-binding sites were found to be 100% conserved in mouse, rat, and hPit1 promoters. Deletion of 1 cEBPα site prevented the RET-induced increase in hPit1 promoter expression. TaqMan qRT-PCR (real time RT-PCR) for RET, Pit1, Arf, TP53, GDNF, steroidogenic factor 1, and GH was performed in RNA from whole ACRO and NFPA tumors. ACRO but not NFPA adenomas express RET and Pit1. GDNF expression in the tumors was positively correlated with RET and negatively correlated with p53. In conclusion, ACROs maintain an active RET/Pit1/p14Arf/p53/apoptosis pathway that is inhibited by GDNF. Disruption of GDNFs survival function might constitute a new therapeutic route in acromegaly.

Pharmacological and Genetic Manipulation of p53 in Brown Fat at Adult But Not Embryonic Stages Regulates Thermogenesis and Body Weight in Male Mice

p53 is a well-known tumor suppressor that plays multiple biological roles, including the capacity to modulate metabolism at different levels. However, its metabolic role in brown adipose tissue (BAT) remains largely unknown. Herein we sought to investigate the physiological role of endogenous p53 in BAT and its implication on BAT thermogenic activity and energy balance. To this end, we generated and characterized global p53-null mice and mice lacking p53 specifically in BAT. Additionally we performed gain-and-loss-of-function experiments in the BAT of adult mice using virogenetic and pharmacological approaches. BAT was collected and analyzed by immunohistochemistry, thermography, real-time PCR, and Western blot. p53-deficient mice were resistant to diet-induced obesity due to increased energy expenditure and BAT activity. However, the deletion of p53 in BAT using a Myf5-Cre driven p53 knockout did not show any changes in body weight or the expression of thermogenic markers. The acute inhibition of p53 in the BAT of adult mice slightly increased body weight and inhibited BAT thermogenesis, whereas its overexpression in the BAT of diet-induced obese mice reduced body weight and increased thermogenesis. On the other hand, pharmacological activation of p53 improves body weight gain due to increased BAT thermogenesis by sympathetic nervous system in obese adult wild-type mice but not in p53(-/-) animals. These results reveal that p53 regulates BAT metabolism by coordinating body weight and thermogenesis, but these metabolic actions are tissue specific and also dependent on the developmental stage.

The syndrome of central hypothyroidism and macroorchidism: IGSF1 controls TRHR and FSHB expression by differential modulation of pituitary TGFβ and Activin pathways

IGSF1 (Immunoglobulin Superfamily 1) gene defects cause central hypothyroidism and macroorchidism. However, the pathogenic mechanisms of the disease remain unclear. Based on a patient with a full deletion of IGSF1 clinically followed from neonate to adulthood, we investigated a common pituitary origin for hypothyroidism and macroorchidism, and the role of IGSF1 as regulator of pituitary hormone secretion. The patient showed congenital central hypothyroidism with reduced TSH biopotency, over-secretion of FSH at neonatal minipuberty and macroorchidism from 3 years of age. His markedly elevated inhibin B was unable to inhibit FSH secretion, indicating a status of pituitary inhibin B resistance. We show here that IGSF1 is expressed both in thyrotropes and gonadotropes of the pituitary and in Leydig and germ cells in the testes, but at very low levels in Sertoli cells. Furthermore, IGSF1 stimulates transcription of the thyrotropin-releasing hormone receptor (TRHR) by negative modulation of the TGFβ1-Smad signaling pathway, and enhances the synthesis and biopotency of TSH, the hormone secreted by thyrotropes. By contrast, IGSF1 strongly down-regulates the activin-Smad pathway, leading to reduced expression of FSHB, the hormone secreted by gonadotropes. In conclusion, two relevant molecular mechanisms linked to central hypothyroidism and macroorchidism in IGSF1 deficiency are identified, revealing IGSF1 as an important regulator of TGFβ/Activin pathways in the pituitary.

Humanized Medium (h7H) Allows Long-Term Primary Follicular Thyroid Cultures From Human Normal Thyroid, Benign Neoplasm, and Cancer

CONTEXT Mechanisms of thyroid physiology and cancer are principally studied in follicular cell lines. However, human thyroid cancer lines were found to be heavily contaminated by other sources, and only one supposedly normal-thyroid cell line, immortalized with SV40 antigen, is available. In primary culture, human follicular cultures lose their phenotype after passage. We hypothesized that the loss of the thyroid phenotype could be related to culture conditions in which human cells are grown in medium optimized for rodent culture, including hormones with marked differences in its affinity for the relevant rodent/human receptor. OBJECTIVE The objective of the study was to define conditions that allow the proliferation of primary human follicular thyrocytes for many passages without losing phenotype. METHODS Concentrations of hormones, transferrin, iodine, oligoelements, antioxidants, metabolites, and ethanol were adjusted within normal homeostatic human serum ranges. Single cultures were identified by short tandem repeats. Human-rodent interspecies contamination was assessed. RESULTS We defined an humanized 7 homeostatic additives medium enabling growth of human thyroid cultures for more than 20 passages maintaining thyrocyte phenotype. Thyrocytes proliferated and were grouped as follicle-like structures; expressed Na+/I- symporter, pendrin, cytokeratins, thyroglobulin, and thyroperoxidase showed iodine-uptake and secreted thyroglobulin and free T3. Using these conditions, we generated a bank of thyroid tumors in culture from normal thyroids, Graves hyperplasias, benign neoplasms (goiter, adenomas), and carcinomas. CONCLUSIONS Using appropriate culture conditions is essential for phenotype maintenance in human thyrocytes. The bank of thyroid tumors in culture generated under humanized humanized 7 homeostatic additives culture conditions will provide a much-needed tool to compare similarly growing cells from normal vs pathological origins and thus to elucidate the molecular basis of thyroid disease.