Differential involvement of cAMP/PKA-, PLC/PKC- and Ca2+/calmodulin-dependent pathways in GnRH-induced prolactin secretion and gene expression in grass carp pituitary cells.

Gonadotropin-releasing hormone (GnRH) is a key stimulator for gonadotropin secretion in the pituitary and its pivotal role in reproduction is well conserved in vertebrates. In fish models, GnRH can also induce prolactin (PRL) release, but little is known for the corresponding effect on PRL gene expression as well as the post-receptor signalling involved. Using grass carp as a model, the functional role of GnRH and its underlying signal transduction for PRL regulation were examined at the pituitary level. Using laser capture microdissection coupled with RT-PCR, GnRH receptor expression could be located in carp lactotrophs. In primary cell culture prepared from grass carp pituitaries, the native forms of GnRH, GnRH2 and GnRH3, as well as the GnRH agonist [D-Arg6, Pro9, NEt]-sGnRH were all effective in elevating PRL secretion, PRL mRNA level, PRL cell content and total production. In pituitary cells prepared from the rostral pars distalis, the region in the carp pituitary enriched with lactotrophs, GnRH not only increased cAMP synthesis with parallel CREB phosphorylation and nuclear translocation but also induced a rapid rise in cytosolic Ca2+ by Ca2+ influx via L-type voltage-sensitive Ca2+ channel (VSCC) with subsequent CaM expression and NFAT2 dephosphorylation. In carp pituitary cells prepared from whole pituitaries, GnRH-induced PRL secretion was reduced/negated by inhibiting cAMP/PKA, PLC/PKC and Ca2+/CaM/CaMK-II pathways but not the signalling events via IP3 and CaN/NFAT. The corresponding effect on PRL mRNA expression, however, was blocked by inhibiting cAMP/PKA/CREB/CBP and Ca2+/CaM/CaN/NFAT2 signalling but not PLC/IP3/PKC pathway. At the pituitary cell level, activation of cAMP/PKA pathway could also induce CaM expression and Ca2+ influx via VSCC with parallel rises in PRL release and gene expression in a Ca2+/CaM-dependent manner. These findings, as a whole, suggest that the cAMP/PKA-, PLC/PKC- and Ca2+/CaM-dependent cascades are differentially involved in GnRH-induced PRL secretion and PRL transcript expression in carp lactotrophs. During the process, a functional crosstalk between the cAMP/PKA- and Ca2+/CaM-dependent pathways may occur with PRL release linked with CaMK-II and PKC activation and PRL gene transcription caused by nuclear action of CREB/CBP and CaN/NFAT2 signalling.

Neuregulin 4 (Nrg4) cooperates with melatonin to regulate the PRL expression via ErbB4/Erk signaling pathway as a potential prolactin (PRL) regulator.

Neuregulin-4 (Nrg4) and melatonin play vital roles in endocrine diseases. However, there is little discussion about the function and potential mechanism of Nrg4 and melatonin in prolactin (PRL) regulation. The human normal pituitary data from Gene Expression Profiling Interactive Analysis (GEPIA) database was used to explore the correlation between NRG4 and PRL. The expression and correlation of NRG4 and PRL were determined by Immunofluorescence staining (IF) and human normal pituitary tissue microarray. Western Blot (WB) was used to detect the expression of PRL, p-ErbB2/3/4, ErbB2/3/4, p-Erk1/2, Erk1/2, p-Akt and Akt in PRL-secreting pituitary GH3 and RC-4B/C cells treated by Nrg4, Nrg4-small interfering RNA, Erk1/2 inhibitor FR180204 and melatonin. The expression of NRG4 was significantly positively correlated with that of PRL in the GEPIA database and normal human pituitary tissues. Nrg4 significantly increased the expression and secretion of PRL and p-Erk1/2 expression in GH3 cells and RC-4B/C cells. Inhibition of Nrg4 significantly inhibited PRL expression. The increased levels of p-Erk1/2 and PRL induced by Nrg4 were abolished significantly in response to FR180204 in GH3 and RC-4B/C cells. Additionally, Melatonin promotes the expression of Nrg4, p-ErbB4, p-Erk1/2, and PRL and can further promote the expression of p-Erk1/2 and PRL in combination with Nrg4. Further investigation into the function of Nrg4 and melatonin on PRL expression and secretion may provide new clues to advance the clinical control of prolactinomas and hyperprolactinemia.

Pituitary Stem Cell Regulation by Zeb2 and BMP Signaling.

Epithelial to mesenchymal transition (EMT) is important for many developing organs, and for wound healing, fibrosis, and cancer. Pituitary stem cells undergo an EMT-like process as they migrate and initiate differentiation, but little is known about the input of signaling pathways or the genetic hierarchy of the transcriptional cascade. Prop1 mutant stem cells fail to undergo changes in cellular morphology, migration, and transition to the Pou1f1 lineage. We used Prop1 mutant mice to identify the changes in gene expression that are affiliated with EMT-like processes. BMP and TGF-ß family gene expression was reduced in Prop1 mutants and Elf5, a transcription factor that characteristically suppresses EMT, had elevated expression. Genes involved in cell-cell contact such as cadherins and claudins were elevated in Prop1 mutants. To establish the genetic hierarchy of control, we manipulated gene expression in pituitary stem cell colonies. We determined that the EMT inducer, Zeb2, is necessary for robust BMP signaling and repression of Elf5. We demonstrated that inhibition of BMP signaling affects expression of target genes in the Id family, but it does not affect expression of other EMT genes. Zeb2 is necessary for expression of the SHH effector gene Gli2. However, knock down of Gli2 has little effect on the EMT-related genes, suggesting that it acts through a separate pathway. Thus, we have established the genetic hierarchy involved in the transition of pituitary stem cells to differentiation.

Mutual Effects of Orexin and Bone Morphogenetic Proteins on Gonadotropin Expression by Mouse Gonadotrope Cells.

Orexin plays a key role in the regulation of sleep and wakefulness and in feeding behavior in the central nervous system, but its receptors are expressed in various peripheral tissues including endocrine tissues. In the present study, we elucidated the effects of orexin on pituitary gonadotropin regulation by focusing on the functional involvement of bone morphogenetic proteins (BMPs) and clock genes using mouse gonadotrope LßT2 cells that express orexin type 1 (OX1R) and type 2 (OX2R) receptors. Treatments with orexin A enhanced LHß and FSHß mRNA expression in a dose-dependent manner in the absence of GnRH, whereas orexin A in turn suppressed GnRH-induced gonadotropin expression in LßT2 cells. Orexin A downregulated GnRH receptor expression, while GnRH enhanced OX1R and OX2R mRNA expression. Treatments with orexin A as well as GnRH increased the mRNA levels of Bmal1 and Clock, which are oscillational regulators for gonadotropin expression. Of note, treatments with BMP-6 and -15 enhanced OX1R and OX2R mRNA expression with upregulation of clock gene expression. On the other hand, orexin A enhanced BMP receptor signaling of Smad1/5/9 phosphorylation through upregulation of ALK-2/BMPRII among the BMP receptors expressed in LßT2 cells. Collectively, the results indicate that orexin regulates gonadotropin expression via clock gene expression by mutually interacting with GnRH action and the pituitary BMP system in gonadotrope cells.

MicroRNA-7a2 is Required for the Development of Pituitary Stem Cells.

The pituitary gland is inhabited by a subpopulation of SOX2+ stem cells. However, the regulatory mechanisms underlying pituitary stem cell development remain poorly understood. In this study, we demonstrate that microRNA-7a (miR-7a) is enriched in the developing pituitary and is spatiotemporally expressed in the pituitary stem cells. Constitutive deletion of mir-7a2 in mice results in pituitary dysplasia emerging during birth, which is primarily manifested as malformed anterior lobes. Using immunofluorescence, immunohistochemistry, or in situ hybridization, we observe that the specification of hormone-expressing cells is not impeded post mir-7a2 deletion at birth, although the terminal differentiation of gonadotropes is inhibited. Further investigation of neonatal and adult pituitaries in mir-7a2 knockout mice reveals an expansion of the SOX2+ pituitary stem cell compartment. The inhibition of epithelial-mesenchymal like transition seems to be responsible for this phenotype, rather than abnormal proliferation or apoptosis. Furthermore, our data suggest that Gli3 and Ckap4 are potential targets of miR-7a in pituitary stem cells. In summary, our results identify miR-7a2 as a crucial factor involved in pituitary stem cell development.

Pituitary Somatotroph Adenoma-derived Exosomes: Characterization of Nonhormonal Actions.

CONTEXT: The identification and biological actions of pituitary-derived exosomes remain elusive. OBJECTIVE: This work aimed to validate production of exosomes derived from human and rat pituitary and elucidate their actions. METHODS: Isolated extracellular vesicles (EVs) were analyzed by Nanoparticle Tracking Analysis (NTA) and expressed exosomal markers detected by Western blot, using nonpituitary fibroblast FR and myoblast H9C2 cells as controls. Exosome inhibitor GW4869 was employed to detect attenuated EV release. Exosomal RNA contents were characterized by RNA sequencing. In vitro and in vivo hepatocyte signaling alterations responding to GH1-derived exosomes (GH1-exo) were delineated by mRNA sequencing. GH1-exo actions on protein synthesis, cAMP (3',5'-cyclic adenosine 5'-monophosphate) response, cell motility, and metastases were assessed. RESULTS: NTA, exosomal marker detection, and GW4869 attenuated EV release, confirming the exosomal identity of pituitary EVs. Hydrocortisone increased exosome secretion in GH1 and GH3 cells, suggesting a stress-associated response. Exosomal RNA contents showed profiles distinct for pituitary cells, and rat primary hepatocytes exposed to GH1-exo exhibited transcriptomic alterations distinct from those elicited by growth hormone or prolactin. Intravenous GH1-exo injection into rats attenuated hepatic Eif2ak2 and Atf4 mRNA expression, both involved in cAMP responses and amino acid biosynthesis. GH1-exo suppressed protein synthesis and forskolin-induced cAMP levels in hepatocytes. GH1-exo-treated HCT116 cells showed dysregulated p53 and mitogen-activated protein kinase (MAPK) pathways and attenuated motility of malignant HCT116 cells, and decreased tumor metastases in nude mice harboring splenic HCT116 implants. CONCLUSION: Our findings elucidate biological actions of somatotroph-derived exosomes and implicate exosomes as nonhormonal pituitary-derived messengers.

Perinatal DEHP exposure modulates pituitary estrogen receptor α and ß expression altering lactotroph and somatotroph cell growth in prepuberal and adult male rats.

Phthalates are synthetic chemicals widely used to make polyvinylchloride (PVC) soft and flexible. Of these, Di-(2-ethylhexyl) phthalate (DEHP) is the most commonly used, with high human exposure occurring as early as the fetal developmental stage and affecting the endocrine system. We focused on the perinatal DEHP effects on pituitary estrogen receptor (ER) expression in male rats, explored their impact on lactotroph and somatotroph cell growth, and evaluated the direct effects of this phthalate on pituitary cell cultures. Our results showed that DEHP perinatal exposure was unable to modify the ERα+ pituitary cell number from prepuberal rats, but increased ERß+ cells. In adulthood, the pituitary ERα+ cells underwent a slight decrease with ERß showing the greatest changes, and with a significant increase observed in somatotroph cells. Also, in vitro, DEHP reduced the ERα+ cells, increased the percentage of ERß+ pituitary cells and modified the Ki67 index, as well as decreasing the lactotrophs and increasing the somatotroph cells. In conclusion, the present study showed that DEHP induced ER expression changes in normal pituitary glands from male rats in in vivo and in vitro conditions, suggesting that DEHP could differentially modulate lactotroph and somatotroph cell growth, possibly as a consequence of ER imbalance.

New insights into the role and origin of pituitary S100ß-positive cells.

In the anterior pituitary, S100ß protein (S100ß) has been assumed to be a marker of folliculo-stellate cells, which are one of the non-hormone-producing cells existing in the parenchyma of the adult anterior lobe and are composed of subpopulations with various functions. However, recent accumulating studies on S100ß-positive cells, including non-folliculo-stellate cells lining the marginal cell layer (MCL), have shown the novel aspect that most S100ß-positive cells in the MCL and parenchyma of the adult anterior lobe are positive for sex determining region Y-box 2 (SOX2), a marker of pituitary stem/progenitor cells. From the viewpoint of SOX2-positive cells, the majority of these cells in the MCL and in the parenchyma are positive for S100ß, suggesting that S100ß plays a role in the large population of stem/progenitor cells in the anterior lobe of the adult pituitary. Reportedly, S100ß/SOX2-double positive cells are able to differentiate into hormone-producing cells and various types of non-hormone-producing cells. Intriguingly, it has been demonstrated that extra-pituitary lineage cells invade the pituitary gland during prenatal pituitary organogenesis. Among them, two S100ß-positive populations have been identified: one is SOX2-positive population which invades at the late embryonic period through the pituitary stalk and another is a SOX2-negative population that invades at the middle embryonic period through Atwell's recess. These two populations are likely the substantive origin of S100ß-positive cells in the postnatal anterior pituitary, while S100ß-positive cells emerging from oral ectoderm-derived cells remain unclear.

Proliferating primary pituitary cells as a model for studying regulation of gonadotrope chromatin and gene expression.

The chromatin organization of the gonadotropin gene promoters in the pituitary gonadotropes plays a major role in determining how these gene are activated, but is difficult to study because of the low numbers of these cells in the pituitary gland. Here, we set out to create a cell model to study gonadotropin chromatin, and found that by optimizing cell culture conditions, we can maintain stable proliferating cultures of primary non-transformed gonadotrope cells over weeks to months. Although expression of the gonadotropin genes drops very low, these cells are enriched in gonadotrope markers and respond to GnRH. Furthermore, >85% of the cells contained Lhb and/or Fshb mature transcripts; though these were virtually restricted to the nuclei. The gonadotropes were harvested initially due to expression of dTOMATO, following activation of Cre recombinase by the Gnrhr promoter. Over 6 mo in culture, a similar proportion of the recombined DNA was maintained (i.e. cells derived from the original gonadotropes or having acquired Gnrhr-promoter activity), together with cells of a distinct origin. The cells are enriched with markers of proliferating pituitary and stem cells, including Sox2, suggesting that multipotent precursor cells might have proliferated and differentiated into gonadotrope-like cells. These cell cultures offer a new and versatile methodology for research in gonadotrope differentiation and function, and can provide enough primary cells for chromatin immunoprecipitation and epigenetic analysis, while our initial studies also indicate a possible regulatory mechanism that might be involved in the nuclear export of gonadotropin gene mRNAs.

Hypothalamic-pituitary organoid generation through the recapitulation of organogenesis.

This paper overviews the development and differentiation of the hypothalamus and pituitary gland from embryonic stem (ES) and induced pluripotent stem (iPS) cells. It is important to replicate the developmental process in vivo to create specific cells/organoids from ES/iPS cells. We also introduce the latest findings and discuss future issues for clinical application. Neuroectodermal progenitors are induced from pluripotent stem cells by strictly removing exogenous patterning factors during the early differentiation period. The induced progenitors differentiate into rostral hypothalamic neurons, in particular magnocellular vasopressin+  neurons. In three-dimensional cultures, ES/iPS cells differentiate into hypothalamic neuroectoderm and nonneural head ectoderm adjacently. Rathke's pouch-like structures self-organize at the interface between the two layers and generate various endocrine cells, including corticotrophs and somatotrophs. Our next objective is to sophisticate our stepwise methodology to establish a novel transplantation treatment for hypopituitarism and apply it to developmental disease models.

Effectiveness of Columbianadin, a Bioactive Coumarin Derivative, in Perturbing Transient and Persistent INa.

Columbianadin (CBN) is a bioactive coumarin-type compound with various biological activities. However, the action of CBN on the ionic mechanism remains largely uncertain, albeit it was reported to inhibit voltage-gated Ca2+ current or to modulate TRP-channel activity. In this study, whole-cell patch-clamp current recordings were undertaken to explore the modifications of CBN or other related compounds on ionic currents in excitable cells (e.g., pituitary GH3 cells and HL-1 atrial cardiomyocytes). GH3-cell exposure to CBN differentially decreased peak or late component of voltage-gated Na+ current (INa) with effective IC50 of 14.7 or 2.8 µM, respectively. The inactivation time course of INa activated by short depolarization became fastened in the presence of CBN with estimated KD value of 3.15 µM. The peak INa diminished by 10 µM CBN was further suppressed by subsequent addition of either sesamin (10 µM), ranolazine (10 µM), or tetrodotoxin (1 µM), but it was reversed by 10 µM tefluthrin (Tef); however, further application of 10 µM nimodipine failed to alter CBN-mediated inhibition of INa. CBN (10 µM) shifted the midpoint of inactivation curve of INa to the leftward direction. The CBN-mediated inhibition of peak INa exhibited tonic and use-dependent characteristics. Using triangular ramp pulse, the hysteresis of persistent INa enhanced by Tef was noticed, and the behavior was attenuated by subsequent addition of CBN. The delayed-rectifier or erg-mediated K+ current was mildly inhibited by 10 µM CBN, while it also slightly inhibited the amplitude of hyperpolarization-activated cation current. In HL-1 atrial cardiomyocytes, CBN inhibited peak INa and raised the inactivation rate of the current; moreover, further application of 10 µM Tef attenuated CBN-mediated decrease in INa. Collectively, this study provides an important yet unidentified finding revealing that CBN modifies INa in electrically excitable cells.

Alcohol metabolite acetic acid activates BK channels in a pH-dependent manner and decreases calcium oscillations and exocytosis of secretory granules in rat pituitary GH3 cells.

Acetaldehyde and acetic acid/acetate, the active metabolites of alcohol (ethanol, EtOH), generate actions of their own ranging from behavioral, physiological, to pathological/cancerogenic effects. EtOH and acetaldehyde have been studied to some depth, whereas the effects of acetic acid have been less well explored. In this study, we investigated the effect of acetic acid on big conductance calcium-activated potassium (BK) channels present in GH3 rat pituitary tumor cells in more detail. In whole cell voltage clamp recordings, extracellular application of acetic acid increased total outward currents in a dose-dependent manner. This effect was prevented after the application of the specific BK channel blocker paxilline. Acetic acid action was pH-dependent-in whole cell current and single BK channel recordings, open probability (Po) was significantly increased by extracellular pH reduction and decreased by neutral or base pH. Acetic acid hyperpolarized the membrane potential, whereas acidic physiological solution had a depolarizing effect. Moreover, acetic acid reduced calcium (Ca2+) oscillations and exocytosis of growth hormone contained secretory granules from GH3 cells. These effects were partially prevented by BK inhibitors-tetraethylammonium or paxillin. In conclusion, our experiments indicate that acetic acid activates BK channels in GH3 cells which eventually contribute to acetic acid-induced membrane hyperpolarization, cessation of Ca2+ oscillations, and decrease of growth hormone release.

Cluster of differentiation (CD) 9-positive mouse pituitary cells are adult stem/progenitor cells.

SOX2-positive cells are stem/progenitor cells that supply hormone-producing cells; they are found in the anterior lobe of the rodent pituitary gland. However, they are likely composed of several subpopulations. In rats, a SOX2-positive cell populations can be distinguished by the presence of S100ß. We identified the novel markers cluster of differentiation (CD) CD9 and CD81, members of the tetraspanin superfamily, for the identification of S100ß/SOX2-positive cells. Recently, CD9/CD81 double-knockout mice were generated. Although they grew normally until 3 weeks after birth, they exhibited atrophy of the pituitary gland. These findings suggested that CD9/CD81/S100ß/SOX2-positive cells in the mouse pituitary are adult stem/progenitor cells. To substantiate this hypothesis, we examined CD9 and CD81 expression in the adult and developing anterior lobe. Immunohistochemistry showed that CD9/CD81-positive cells began appearing from postnatal day 0 and settled in the stem cell niches (marginal cell layer and parenchyma) of the adult anterior lobe while expressing S100ß. We next isolated CD9 -positive cells from the adult anterior lobe, using the anti-CD9 antibody for cell characterisation. The cells in culture formed free-floating three-dimensional clusters (pituispheres); moreover, induction into all types of hormone-producing cells was successful. Furthermore, reduction of CD9 and CD81 mRNAs by siRNAs inhibited cell proliferation. These findings indicate that CD9/CD81/S100ß/SOX2-positive cells may play a role as adult stem/progenitor cells in SOX2-positive subpopulations, thus supplying hormone-producing cells in the postnatal anterior lobe. Furthermore, CD9 and CD81 are implicated in cell proliferation. The current findings provide novel insights into adult pituitary stem/progenitor cells.

Human pituitary development and application of iPSCs for pituitary disease.

The pituitary plays a pivotal role in maintaining systemic homeostasis by secreting several hormones. During fetal development, the pituitary develops from the oral ectoderm in contact with the adjacent hypothalamus. This process is regulated by the fine-tuned expression of transcription and growth factors. Impairments of this process result in congenital pituitary hypoplasia leading to dysfunction of the pituitary. Although animal models such as knockout mice have helped to clarify these underlying mechanisms, the developmental processes of the human pituitary gland and the mechanisms of human pituitary disorders have not been fully understood. This is because, at least in part, of the lack of a human pituitary developmental model. Recently, methods for in vitro induction of the pituitary gland from human pluripotent stem cells were developed. These models can be utilized not only for regenerative medicine but also for human pituitary studies on developmental biology and for modeling of pituitary disorders, such as hypopituitarism and pituitary tumors. In this review, we provide an overview of recent progress in the applications of pluripotent stem cells for pituitary research and discuss further perspectives for pituitary studies.

HSP70 inhibits pig pituitary gonadotrophin synthesis and secretion by regulating the corticotropin-releasing hormone signaling pathway and targeting SMAD3.

High levels or long periods of stress have been shown to negatively impact cell homeostasis, including with respect to abnormalities in domestic animal reproduction, which are typically activated through the hypothalamus-pituitary-adrenal axis, in which corticotropin-releasing hormone (CRH) and heat shock protein 70 (HSP70) are involved. In addition, CRH has been reported to inhibit pituitary gonadotrophin synthesis, and HSP70 is expressed in the pituitary gland. The aim of this study was to determine whether HSP70 was involved in regulating gonadotrophin synthesis and secretion by mediating the CRH pathway in the porcine pituitary gland. Our results showed that HSP70 was highly expressed in the porcine pituitary gland, with over 90% of gonadotrophic cells testing HSP70 positive. The results of functional studies demonstrated that the HSP70 inducer decreased FSH and LH levels in cultured porcine primary pituitary cells, whereas an HSP70 inhibitor blocked the negative effect of CRH on gonadotrophin synthesis and secretion. Furthermore, our results demonstrated that HSP70 inhibited gonadotrophin synthesis and secretion by blocking GnRH-induced SMAD3 phosphorylation, which acts as the targeting molecule of HSP70, while CRH upregulated HSP70 expression through the PKC and ERK pathways. Collectively, these data demonstrate that HSP70 inhibits pituitary gonadotrophin synthesis and secretion by regulating the CRH signaling pathway and inhibiting SMAD3 phosphorylation, which are important for our understanding the mechanisms of the stress affects domestic animal reproductive functions.

SDF-1α/MicroRNA-134 Axis Regulates Nonfunctioning Pituitary Neuroendocrine Tumor Growth via Targeting VEGFA.

Background: Nonfunctioning pituitary neuroendocrine tumor (NF-PitNET) is difficult to resect. Except for surgery, there is no effective treatment for NF-PitNET. MicroRNA-134 (miR-134) has been reported to inhibit proliferation and invasion ability of tumor cells. Herein, the mechanism underlying the effect of miR-134 on alleviating NF-PitNET tumor cells growth is explored. Methods: Mouse pituitary αT3-1 cells were transfected with miR-134 mimics and inhibitor, followed by treatment with stromal cell-derived factor-1α (SDF-1α) in vitro. MiR-134 expression level: we used quantitative real-time PCR (qRT-PCR) to detect the expression of miR-134. Cell behavior level: cell viability and invasion ability were assessed using a cell counting kit-8 (CCK8) assay and Transwell invasion assay respectively. Cytomolecular level: tumor cell proliferation was evaluated by Ki-67 staining; propidium iodide (PI) staining analyzed the effect of miR-134 on cell cycle arrest; western blot analysis and immunofluorescence staining evaluated tumor migration and invasive ability. Additionally, we collected 27 NF-PitNET tumor specimens and related clinical data. The specimens were subjected to qRT-PCR to obtain the relative miR-134 expression level of each specimen; linear regression analysis was used to analyze the miR-134 expression level in tumor specimens and the age of the NF-PitNET population, gender, tumor invasion, prognosis, and other indicators. Results: In vitro experiment, miR-134 was observed to significantly inhibit αT3-1 cells proliferation characterized by inhibited cell viability and expressions of vascular endothelial growth factor A (VEGFA) and cell cycle transition from G1 to S phase (P < 0.01). VEGFA was verified as a target of miR-134. Additionally, miR-134-induced inhibition of αT3-1 cell proliferation and invasion was attenuated by SDF-1α and VEGFA overexpression (P < 0.01). In primary NF-PitNET tumor analysis, miR-134 expression level was negatively correlated with tumor invasion (P = 0.003). Conclusion: The regulation of the SDF-1α/miR-134/VEGFA axis represents a novel mechanism in the pathogenesis of NF-PitNETs and may serve as a potential therapeutic target for the treatment of NF-PitNETs.

Characterisation of a mural cell network in the murine pituitary gland.

The anterior and intermediate lobes of the pituitary are composed of endocrine cells, as well as vasculature and supporting cells, such as folliculostellate cells. Folliculostellate cells form a network with several postulated roles in the pituitary, including production of paracrine signalling molecules and cytokines, coordination of endocrine cell hormone release, phagocytosis, and structural support. Folliculostellate cells in rats are characterised by expression of S100B protein, and in humans by glial fibrillary acid protein. However, there is evidence for another network of supporting cells in the anterior pituitary that has properties of mural cells, such as vascular smooth muscle cells and pericytes. The present study aims to characterise the distribution of cells that express the mural cell marker platelet derived growth factor receptor beta (PDGFRß) in the mouse pituitary and establish whether these cells are folliculostellate. By immunohistochemical localisation, we determine that approximately 80% of PDGFRß+ cells in the mouse pituitary have a non-perivascular location and 20% are pericytes. Investigation of gene expression in a magnetic cell sorted population of PDGFRß+ cells shows that, despite a mostly non-perivascular location, this population is enriched for mural cell markers but not enriched for rat or human folliculostellate cell markers. This is confirmed by immunohistochemistry. The present study concludes that a mural cell network is present throughout the anterior pituitary of the mouse and that this population does not express well-characterised human or rat folliculostellate cell markers.