The astroglial and stem cell functions of adult rat folliculostellate cells.

The mammalian pituitary gland is a complex organ consisting of hormone-producing cells, anterior lobe folliculostellate cells (FSCs), posterior lobe pituicytes, vascular pericytes and endothelial cells, and Sox2-expressing stem cells. We present single-cell RNA sequencing and immunohistofluorescence analyses of pituitary cells of adult female rats with a focus on the transcriptomic profiles of nonhormonal cell types. Samples obtained from whole pituitaries and separated anterior and posterior lobe cells contained all expected pituitary resident cell types and lobe-specific vascular cell subpopulations. FSCs and pituicytes expressed S100B, ALDOC, EAAT1, ALDH1A1, and VIM genes and proteins, as well as other astroglial marker genes, some common and some cell type-specific. We also found that the SOX2 gene and protein were expressed in ~15% of pituitary cells, including FSCs, pituicytes, and a fraction of hormone-producing cells, arguing against its stem cell specificity. FSCs comprised two Sox2-expressing subclusters; FS1 contained more cells but lower genetic diversity, while FS2 contained proliferative cells, shared genes with hormone-producing cells, and expressed genes consistent with stem cell niche formation, regulation of cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. FS1 cells were randomly distributed in the anterior and intermediate lobes, while FS2 cells were localized exclusively in the marginal zone between the anterior and intermediate lobes. These data indicate the identity of the FSCs as anterior pituitary-specific astroglia, with FS1 cells representing differentiated cells equipped for classical FSC roles and FS2 cells exhibiting additional stem cell-like features.

Co-expression of intermediate filaments glial fibrillary acidic protein and cytokeratin in pituitary adenoma.

PURPOSE: To analyze the co-expression of the intermediate filaments GFAP and cytokeratin in 326 pituitary adenomas with regard to the distribution pattern, the subtype of the adenoma and clinical prognostic data. METHODS: Tissue from 326 pituitary adenomas and 13 normal anterior pituitaries collected in the Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, between 2006 and 2009 was investigated by immunohistochemistry, immunofluorescence and electron microscopy. RESULTS: Co-expression of intermediate filaments GFAP and cytokeratin was associated with hormone expression in 62/278 cases (22%), but only found in 2/48 (4%) of null cell adenomas (p < 0.01). Simultaneous co-expression of GFAP and cytokeratin in the same cells was demonstrated in 26 out of 326 pituitary adenomas and in all 13 pituitaries. In pituitary intermediate filaments were demonstrated in a larger area of the cytoplasm than in adenoma (p < 0.01), however, overlapping expression was seen in 2.6% of the total area in both, pituitary and adenoma. Congenially, cells with overlapping expression were found near vessels and in follicles. Furthermore, adenomas with cellular co-expression of GFAP and cytokeratin were associated with a lower recurrence rate (7.7%) compared to adenomas without co-expression of intermediate filaments (17.8%). CONCLUSIONS: Cellular co-expression of the intermediate filaments GFAP and cytokeratin in pituitary adenomas and the pituitary was demonstrated and shown to be associated with hormone expression and low recurrence rate. The results are discussed with regard to the biology of folliculostellate cells, neural transformation and tumor stem cells. This study may complement the understanding of pituitary adenoma biology.

Biological and Therapeutic Implications of the Tumor Microenvironment in Pituitary Adenomas.

Pituitary adenomas (PAs) are neoplasms derived from the endocrine cells of the anterior pituitary gland. Most frequently, they are benign tumors, but may sometimes display an aggressive course, and in some cases metastasize. Their biology, including their wide range of behavior, is only partly understood. In terms of therapeutic targeting, most PAs are easily treated with available medical treatments, surgery, and sometimes radiotherapy. Nevertheless, gonadotroph adenomas lack medical therapeutic options, and treatment of aggressive PAs and pituitary carcinomas remains challenging. Here, we present an overview of the implications of the tumor microenvironment in PAs, reviewing its composition and function, as well as published cases that have been treated thus far using tumor microenvironment-targeting therapies. Additionally, we discuss emerging views, such as the concept of nonangiogenic tumors, and present perspectives regarding treatments that may represent future potential therapeutic options. Tumor-infiltrating lymphocytes, tumor-associated macrophages, folliculostellate cells, tumor-associated fibroblasts, angiogenesis, as well as the extracellular matrix and its remodeling, all have complex roles in the biology of PAs. They have been linked to hormone production/secretion, size, invasion, proliferation, progression/recurrence, and treatment response in PAs. From a therapeutic perspective, immune-checkpoint inhibitors and bevacizumab have already shown a degree of efficacy in aggressive PAs and pituitary carcinomas, and the use of numerous other tumor microenvironment-targeting therapies can be foreseen. In conclusion, similar to other cancers, understanding the tumor microenvironment improves our understanding of PA biology beyond genetics and epigenetics, and constitutes an important tool for developing future therapies.

Intratumoural spatial distribution of S100B + folliculostellate cells is associated with proliferation and expression of FSH and ERα in gonadotroph tumours.

Folliculostellate cells are S100B-expressing cells with numerous functions in the normal anterior pituitary. These cells have also been identified in pituitary neuroendocrine tumours (PitNETs), where their precise role remains elusive. Here, we aimed to build a refined cartography of S100B-expressing cells to characterise their interpatient and intratumoural spatial distribution, and to start identifying their potential functions in PitNETs. High-throughput histological analysis of S100B-stained tumour sections of 54 PitNETs revealed a significant decrease in S100B + cells in PitNETs compared to the normal anterior pituitary. A Ki67 index ≥ 3, a mitosis count > 2/10 per high power fields, and a proliferative status, were all associated with fewer S100B + cells in gonadotroph tumours. Gonadotroph tumours also showed interpatient and intratumoural heterogeneity in the spatial distribution of S100B + cells. The existence of an intratumoural heterogeneity was further confirmed by the incorporation to our spatial analysis of additional markers: Ki67, FSH, LH, ERα and SSTR2. The tumour areas with fewer S100B + cells displayed a higher percentage of Ki67 + cells, whereas strong positive correlations were observed between S100B + , FSH + , and ERα + cells. Such spatial associations suggest that S100B + folliculostellate cells could play a role in gonadotroph tumorigenesis, and may contribute to the maintenance of tumour cells in a low proliferating, FSH + /ERα + differentiated state. Albeit, further in-depth functional studies are required to decipher the mechanisms underlying these spatial associations and to potentially identify a therapeutic use.

Renewing an old interest: Pituitary folliculostellate cells.

Anterior pituitary folliculostellate (FS) cells, first described almost 50 years ago, have a wide range of functions with respect to supporting and coordinating endocrine cell function, in particular through paracrine and gap junction-mediated signalling. Our previous studies identified the morphological organisation of FS cells, which mediates coordinated calcium activity throughout the homotypic FS network and allows signalling across the whole pituitary gland. It is also clear that FS cells can modify endocrine output and feedback on pituitary axes over a range of timescales. Recently, several studies have defined FS cells as a source of anterior pituitary endocrine cell renewal, which has resulted in a renaming of FS cells as "Sox2+ve stem cells". Here, we highlight the broader potential of the FS cell population in fine-tuning and coordinating pituitary axes function. In addition, we identify a need for: the definition of the possible subtypes of FS cell and their relationship with the stem cell population; the potential role of FS cells in pulsatile hormone secretion and coordination of heterotypic cell networks; and the roles that FS cells may play in both early-life programming of pituitary axes and in memory, or anticipation, of demand. Further studies of FS cells may demonstrate the fundamental importance of this cell type and its potential as a therapeutic target to correct pituitary gland dysfunction, one of which is stem cell therapy. Clearly, a thorough understanding of all of these interactions and relationships of FS and endocrine cells is required whatever therapeutic use is suggested by their various roles.

Follicular cells in pituitary neuroendocrine tumors.

Follicular cells (FCs) are thought to be agranular, non-hormone-producing stellate cells distributed throughout the adenohypophysis, occasionally arranged around colloid-filled follicles, and thought to be more prominent in the vicinity of necrosis and apoptotic cells. A distinct but similar cell type, the folliculostellate cell (FSC), is a sustentacular cell that is negative for keratins and stains for S100, GFAP, and SOX10. While several studies have examined FSCs in pituitary neuroendocrine tumors (PitNETs), the distribution and derivation of FCs in these lesions is unclear. We examined the presence and distribution of FCs in 104 PitNETs obtained by trans-sphenoidal surgery, using immunohistochemistry for keratins as well as the full complement of immunohistochemical stains for tumor characterization. The tumors included 9 somatotroph, 5 mammosomatotroph, 7 lactotroph, 7 immature PIT1-lineage, 2 acidophil stem cell, 17 corticotroph, 53 gonadotroph, 2 null cell, and 2 unusual plurihormonal tumors. CK-positive FCs were only identified in gonadotroph PitNETs and were found in 12 (23%) of those tumors; all other tumor types were negative for FCs. FCs express keratins identified by CAM5.2, AE1/AE3, CK18, and CK19 antibodies. FCs were identified scattered singly among hormone-producing neuroendocrine cells, in small clusters of 3-5 cells and surrounding colloid-filled follicles, as well as linearly along intratumoral blood vessels. Sequential stains showed that FCs express nuclear SF1 and GATA3, transcription factors of gonadotrophs, and multiplex immunohistochemistry confirmed colocalization of SF1 in the nucleus of keratin-positive FCs. In this series, FCs were exclusively found in gonadotroph PitNETs and occurred in 23% of those tumors. Co-expression of gonadotroph transcription factors in FCs supports the concept of cellular plasticity and transformation of neoplastic hormone-producing neuroendocrine cells to FCs. Further studies are required to determine if and why gonadotrophs alone undergo this transformation, the function of these cells and whether they have prognostic value.

Macrophages are activated in the rat anterior pituitary under chronic inflammatory conditions.

In the anterior lobe of the pituitary gland (AP), non-endocrine cells regulate hormone secretion by endocrine cells. However, the functions of non-endocrine cells in the AP during chronic pain are largely unclear. Here, we show that macrophages, but not folliculostellate (FS) cells, were selectively increased in the AP in the complete Freund's adjuvant (CFA)-induced chronic inflammatory pain model in rats. In addition, IL-1ß expression was increased in the AP, and the IL-1ß-immunopositive cells were identified as macrophages. On the other hand, increased macrophage density and IL-1ß expression were not detected in a neuropathic pain model induced by partial sciatic nerve ligation (PSL). Furthermore, we found c-Fos expression specifically in the somatotrophs under the chronic inflammatory pain condition. Because IL-1ß promotes growth hormone (GH) synthesis and release, our results suggest that AP macrophage contributes to GH release through IL-1ßduring chronic inflammatory pain.　.

Adenosine regulates thrombomodulin and endothelial protein C receptor expression in folliculostellate cells of the pituitary gland.

Adenosine stimulates the release of interleukin 6 (IL-6) and vascular endothelial growth factor from folliculostellate cells of the anterior pituitary gland indicating that such cells are also involved in the communication between the immune and endocrine systems during stress and inflammation. In order to understand the precise actions of adenosine on folliculostellate cells, DNA microarray analysis was used to determine global changes in gene expression. Hierarchical clusters revealed, of the genes that had altered expression, the majority were suppressed and many, such as B cell translocation gene 2 and cyclin-dependent kinase inhibitor 2b were related to cell cycle arrest or inhibition of proliferation. Several of the up-regulated genes were associated with cytokine signalling or membrane receptor activity. The most notable of these being IL-6, sulfiredoxin 1, endothelial protein C receptor (EPCR) and thrombomodulin (THBD) which can all play a role in controlling inflammation. The EPCR and THBD pathway is well known in anti-coagulation but also has anti-inflammatory and anti-apoptotic properties. Up-regulation of EPCR and THBD in folliculostellate cells was confirmed by qRT-PCR and western blotting analysis and their expression were also demonstrated in many of the hormone-secreting cells of the anterior pituitary gland. Our findings suggest that adenosine can stimulate expression of stress and inflammation related genes from folliculostellate cells of the anterior pituitary gland. These genes include EPCR and THBD, neither of which has been previously identified in the pituitary gland.

Cell Type- and Sex-Dependent Transcriptome Profiles of Rat Anterior Pituitary Cells.

Understanding the physiology and pathology of an organ composed of a variety of cell populations depends critically on genome-wide information on each cell type. Here, we report single-cell transcriptome profiling of over 6,800 freshly dispersed anterior pituitary cells from postpubertal male and female rats. Six pituitary-specific cell types were identified based on known marker genes and characterized: folliculostellate cells and hormone-producing corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. Also identified were endothelial and blood cells from the pituitary capillary network. The expression of numerous developmental and neuroendocrine marker genes in both folliculostellate and hormone-producing cells supports that they have a common origin. For several genes, the validity of transcriptome analysis was confirmed by qRT-PCR and single cell immunocytochemistry. Folliculostellate cells exhibit impressive transcriptome diversity, indicating their major roles in production of endogenous ligands and detoxification enzymes, and organization of extracellular matrix. Transcriptome profiles of hormone-producing cells also indicate contributions toward those functions, while also clearly demonstrating their endocrine function. This survey highlights many novel genetic markers contributing to pituitary cell type identity, sexual dimorphism, and function, and points to relationships between hormone-producing and folliculostellate cells.

The Microenvironment of Pituitary Tumors-Biological and Therapeutic Implications.

The tumor microenvironment (TME) includes resident and infiltrative non-tumor cells, as well as blood and lymph vessels, extracellular matrix molecules, and numerous soluble factors, such as cytokines and chemokines. While the TME is now considered to be a prognostic tool and a therapeutic target for many cancers, little is known about its composition in pituitary tumors. This review summarizes our current knowledge of the TME within pituitary tumors and the strong interest in TME as a therapeutic target. While we cover the importance of angiogenesis and immune infiltrating cells, we also address the role of the elusive folliculostellate cells, the emerging literature on pituitary tumor-associated fibroblasts, and the contribution of extracellular matrix components in these tumors. The cases of human pituitary tumors treated with TME-targeting therapies are reviewed and emerging concepts of vascular normalization and combined therapies are presented. Together, this snapshot overview of the current literature pinpoints not only the underestimated role of TME components in pituitary tumor biology, but also the major promise it may offer for both prognosis and targeted therapeutics.

The anterior pituitary gap junctions: potential targets for toxicants.

The anterior pituitary regulates endocrine organs and physiological activities in the body. Environmental pollutants and drugs deleterious to the endocrine system may affect anterior pituitary activity through direct action on anterior pituitary cells. Within the gland, endocrine and folliculostellate cells are organized into and function as individual tridimensional networks, each network regulating its activity by coordinating the connected cells' responses to physiological or pathological cues. The gap junctions connecting endocrine cells and/or folliculostellate cells allow transmission of information among cells that is necessary for adequate network function. Toxicants may affect gap junctions as well as the physiology of the anterior pituitary. However, whether toxicants effects on anterior pituitary hormone secretion involve gap junctions is unknown. The folliculostellate cell gap junctions are sensitive to hormones, cytokines and growth factors. These cells may be an interesting experimental model for evaluating whether toxicants target anterior pituitary gap junctions.

Morphometric analysis of the folliculostellate cells and luteinizing hormone gonadotropic cells of the anterior pituitary of the men during the aging process.

The aim of this research was to quantify the changes in the morphology and density of the anterior pituitary folliculostellate (FS) and luteinizing hormone (LH) cells. Material was tissue of the pituitary gland of the 14 male cadavers. Tissue slices were immunohistochemically stained with monoclonal anti-LH antibody and polyclonal anti-S100 antibody for the detection of LH and FS cells, respectively. Digital images of the stained slices were afterwards morphometrically analyzed by ImageJ. Results of the morphometric analysis showed significant increase of the FS cells volume density in cases older than 70 years. Volume density of the LH cells did not significantly change, whereas their area significantly increased with age. Nucleocytoplasmic ratio of the LH cells gradually decreased and became significant after the age of 70. Finally, volume density of the FS cell significantly correlated with LH cells area and nucleocytoplasmic ratio. From all above cited, we concluded that in men, density and size of the FS cells increase with age. Long-term hypertrophy of the LH cells results in their functional decline after the age of 70. Strong correlation between FS cells and LH cells morphometric parameters might point to age-related interaction between these two cell groups.

The "game" of glial fibrillary acidic and S100 proteins in pituitary adenomas: two players or several?

INTRODUCTION: S100 protein and GFAP expression in pituitary adenomas tumour cells is not well known; few correlations with other prognostic or therapeutic factors have previously been reported in pituitary adenomas. We aim to elucidate their involvement in the pathogenesis of pituitary adenomas and to establish the correlation of their expression with different growth factors and growth factor receptors known to have a prognostic and/or therapeutic role. MATERIAL AND METHODS: Sixty-one cases of pituitary adenomas were immunohistochemically assessed for the expression of GFAP and S100 protein in both tumour cells and FS cells, in close relationship with hormone profile, and correlated with vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) expression, previously studied by our team. RESULTS: GFAP and S100 protein were expressed both in tumour cells and FS cells. Differences between morphology, distribution, and density of GFAP+ FS cells and S100+ FS cells were observed according to the hormone profile of pituitary adenomas. GFAP and S100 protein expression in tumour cells was significantly related to hormone profile of pituitary adenomas and also with VEGF and EGFR expression. CONCLUSIONS: GFAP and S100 protein expressions in tumour cells from pituitary adenomas are influenced by hormone profile. Our re-sults support the presence of two molecular subtypes of FS cells GFAP+/VEGF+/S100 respectively and another one that is GFAP-/S100+/EGFR+ simultaneously with the classical variant GFAP+/S100+. It is possible that S100+/EGFR+ pituitary adenomas represent a group of pituitary adenomas with an aggressive behaviour and a high ability of invasion and recurrence.

Intrapituitary mechanisms underlying the control of fertility: key players in seasonal breeding.

Recent studies have shown that, in conjunction with dynamic changes in the secretion of GnRH from the hypothalamus, paracrine interactions within the pituitary gland play an important role in the regulation of fertility during the annual reproductive cycle. Morphological studies have provided evidence for close associations between gonadotropes and lactotropes and gap junction coupling between these cells in a variety of species. The physiological significance of this cellular interaction was supported by subsequent studies revealing the expression of prolactin receptors in both the pars distalis and pars tuberalis regions of the pituitary. This cellular interaction is critical for adequate gonadotropin output because, in the presence of dopamine, prolactin can negatively regulate the LH response to GnRH. Receptor signaling studies showed that signal convergence at the level of protein kinase C and phospholipase C within the gonadotrope underlies the resulting inhibition of LH secretion. Although this is a conserved mechanism present in all species studied so far, in seasonal breeders such as the sheep and the horse, this mechanism is regulated by photoperiod, as it is only apparent during the long days of spring and summer. At this time of year, the nonbreeding season of the sheep coincides with the breeding season of the horse, indicating that this inhibitory system plays different roles in short- and long-day breeders. Although in the sheep, it contributes to the complete suppression of the reproductive axis, in the horse, it is likely to participate in the fine-tuning of gonadotropin output by preventing gonadotrope desensitization. The photoperiodic regulation of this inhibitory mechanism appears to rely on alterations in the folliculostellate cell population. Indeed, electron microscopic studies have recently shown increased folliculostellate cell area together with upregulation of their adherens junctions during the spring and summer. The association between gonadotropes and lactotropes could also underlie an interaction between the gonadotropic and prolactin axes in the opposite direction. In support of this alternative, a series of studies have demonstrated that GnRH stimulates prolactin secretion in sheep through a mechanism that does not involve the mediatory actions of LH or FSH and that this stimulatory effect of GnRH on the prolactin axis is seasonally regulated. Collectively, these findings highlight the importance of intercellular communications within the pituitary in the control of gonadotropin and prolactin secretion during the annual reproductive cycle in seasonal breeders.

Biphasic Effect of Basic Fibroblast Growth Factor on Anterior Pituitary Folliculostellate TtT/GF Cell Coupling, and Connexin 43 Expression and Phosphorylation.

Basic fibroblast growth factor (bFGF) is a mitogenic and differentiating cytokine. In the anterior pituitary, folliculostellate (FS) cells constitute the major source of bFGF. bFGF affects endocrine cell proliferation and secretion in the anterior pituitary. In addition, bFGF increases its own expression by acting directly on FS cells. FS cell Cx43-mediated gap junction intercellular communication allows the establishment of an intrapituitary network for the transmission of information. In the present study, we assessed how bFGF regulates FS cell coupling. Time course studies were carried out on the FS cell line TtT/GF. Short-term bFGF treatment induced a transient cell uncoupling and the phosphorylation in Ser368 of membrane-bound Cx43 without modifying Cx43 levels. We demonstrated the involvement of the protein kinase C (PKC) isoform α in the phosphorylation of Cx43 in S368. Moreover, we showed that bFGF induced PKCα activation by stimulating its expression, phosphorylation and association with the plasma membrane. The long-term incubation with bFGF increased TtT/GF cell coupling, total Cx43 levels and Cx43 accumulation at the cell membrane of cytoplasmic projections. The Cx43 level increase was a result of the stimulation of Cx43 gene transcription as mediated by the extracellular-regulated kinase 1/2 signalling pathway. Taken together, the data show that bFGF modulates TtT/GF cell coupling by activating different pathways that lead to opposite effects on Cx43 phosphorylation and expression depending on the duration of the exposure of the cells to bFGF. A short-term bFGF exposure reduces cell-to-cell communication as a mean of desynchronising FS cells. By contrast, long-term exposure to bFGF enhances cell-to-cell communication and facilitates coordination among FS cells.

Vascular endothelial growth factor secretion from pituitary folliculostellate cells: role of KATP channels.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen responsible for physiological and pathological angiogenesis. Abnormal regulation of VEGF expression in anterior pituitary folliculostellate (FS) cells has been implicated in pituitary tumour progression. FS and endocrine cells express VEGF, which is considered to be secreted by the constitutive pathway. The present study investigated the mechanism of VEGF secretion in TtT/GF cells, a mouse FS cell line. TtT/GF cells were shown to express VEGF(164), the most potent and bioavailable isoform of VEGF. Immunofluorescence and immunogold electron microscopy localised VEGF to the cytoplasm and small electron-lucent vesicles. Pituitary adenylate cyclase-activating polypeptide (PACAP), a well-documented stimulant of VEGF secretion, caused a robust increase in VEGF secretion over 24 h. Glyburide, an ABCA1 and K(ATP) channel blocker, also caused an increase in VEGF secretion when applied alone, and amplified the response to PACAP. Other ABCA1 transport blockers did not affect VEGF secretion. Exposure of TtT/GF cells to cycloheximide with PACAP or glyburide inhibited the increased secretion of VEGF, consistent with control of secretion at the transcription level. The SUR2B/Kir6.1 form of K(ATP) channels was shown to be expressed by TtT/GF cells. Diazoxide, a K(ATP) activator, inhibited PACAP- and PACAP + glyburide-stimulated VEGF secretion but not that of glyburide alone. These data suggest that K(ATP) channels are expressed by FS cells and play a significant role in the control of VEGF secretion, and also that activation of K(ATP) channels inhibits the secretion of VEGF at the level of transcription.

Reassembly of anterior pituitary organization by hanging drop three-dimensional cell culture.

The anterior pituitary gland comprises 5 types of hormone-producing cells and non-endocrine cells, such as folliculostellate (FS) cells. The cells form a lobular structure surrounded by extracellular matrix (ECM) but are not randomly distributed in each lobule; hormone-producing cells have affinities for specific cell types (topographic affinity), and FS cells form a homotypic meshwork. To determine whether this cell and ECM organization can be reproduced in vitro, we developed a 3-dimensional (3D) model that utilizes hanging drop cell culture. We found that the topographic affinities of hormone-producing cells were indeed maintained (ie, GH to ACTH cells, GH to TSH cells, PRL to LH/FSH cells). Fine structures in hormone-producing cells retained their normal appearance. In addition, FS cells displayed well-developed cytoplasmic protrusions, which interconnected with adjacent FS cells to form a 3D meshwork. In addition, reassembly of gap junctions and pseudofollicles among FS cells was observed in cell aggregates. Major ECM components-collagens and laminin-were deposited and distributed around the cells. In sum, the dissociated anterior pituitary cells largely maintained their in vivo anterior pituitary architectures. This culture system appears to be a powerful experimental tool for detailed analysis of anterior pituitary cell organization.