Pituitary Folliculostellate Cells Research Articles

Renewing an old interest: Pituitary folliculostellate cells.

Anterior pituitary folliculostellate (FS) cells, first described almost 50years 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.

Distinctive actions of connexin 46 and connexin 50 in anterior pituitary folliculostellate cells.

Folliculostellate cell gap junctions establish a network for the transmission of information within the anterior pituitary. Connexins make up gap junction channels. Changes in connexin (Cx) turnover modify gap junction-mediated intercellular communication. We have reported that cytokines and hormones influence Cx43 turnover and coupling in folliculostellate cells and in the folliculostellate cell line TtT/GF. In addition, the expression of different connexins alters intercellular communication and connexins may have functions besides cell coupling. Here we assessed the expression, turnover and subcellular localization of Cx46 and Cx50 in the anterior pituitary and TtT/GF cells. Then, we assessed the impact of various natural (lactation, annual reproductive cycle, bFGF) and pathological (autoimmune orchitis, diabetes/obesity) conditions associated with altered anterior pituitary hormone secretion on Cx46 and Cx50. Anterior pituitary Cx46 and Cx50 expression and subcellular distribution were cell-dependent. Cx46 was expressed by folliculostellate, TtT/GF and endocrine cells. In the cytoplasm, Cx46 was chiefly associated with lysosomes. Variously sized Cx46 molecules were recovered exclusively in the TtT/GF cell nuclear fraction. In the nucleus, Cx46 co-localized with Nopp-140, a nucleolar factor involved in rRNA processing. Neither cytoplasmic nor nuclear Cx46 and Cx43 co-localized. Cx50 localized to folliculostellate and TtT/GF cells, and to the walls of blood capillaries, not to endocrine cells. Cx50 was cytoplasmic and associated with the cell membrane, not nuclear. Cx50 did not co-localize with Cx46 but it co-localized in the cytoplasm and co-immunoprecipitated with Cx43. Cx46 and Cx50 responses to various physiological and pathological challenges were different, often opposite. Cx46 and Cx43 expression and phosphorylation profiles differed in the anterior pituitary, whereas Cx50 and Cx43 were similar. The data suggest that Cx46 participates to cellular growth and proliferation and that Cx50, together with Cx43, contributes to folliculostellate cell coupling.

Update on Pituitary Folliculo-Stellate Cells

Folliculo-stellate cells (FSCs) are a non-endocrine population of sustentacular-like, star-shaped and follicle-forming cells, which contribute about 5-10% of elements from the anterior pituitary lobe. First identified with electron microscopy as non-hormone secreting accessory cells, light microscopy has revealed many of their cytophysiological features, and is known as positive for S-100 protein, a marker for FSCs. They are currently considered as functionally and phenotypically heterogeneous.

Macrophage colony-stimulating factor induces prolactin expression in rat pituitary gland.

We investigated the role of macrophage colony-stimulating factor (M-CSF) in the pituitary gland to understand the effect of M-CSF on pituitary hormones and the relationship between the endocrine and immune systems. When we attempted to establish pituitary cell lines from a thyrotropic pituitary tumor (TtT), a macrophage cell line, TtT/M-87, was established. We evaluated M-CSF-like activity in conditioned media (CM) from seven pituitary cell lines using TtT/M-87 cells. TtT/M-87 proliferation significantly increased in the presence of CM from TtT/GF cells, a pituitary folliculostellate (FS) cell line. M-CSF mRNA was detected in TtT/GF and MtT/E cells by reverse transcriptase-polymerase chain reaction (RT-PCR), and its expression in TtT/GF cells was increased in a lipopolysaccharide (LPS) dose-dependent manner. M-CSF mRNA expression was also increased in rat anterior pituitary glands by LPS. M-CSF receptor (M-CSFR) mRNA was only detected in TtT/ M-87 cells and increased in the LPS-stimulated rat pituitary glands. In rat pituitary glands, M-CSF and M-CSFR were found to be localized in FS cells and prolactin (PRL)-secreting cells, respectively, by immunohistochemistry. The PRL concentration in rat sera was significantly increased at 24 h after M-CSF administration, and mRNA levels significantly increased in primary culture cells of rat anterior pituitary glands. In addition, TNF-α mRNA was increased in the primary culture cells by M-CSF. These results revealed that M-CSF was secreted from FS cells and M-CSF regulated PRL expression in rat pituitary glands.

Vascular Endothelial Growth Factor Secretion from Pituitary Folliculostellate Cells: Role of KATPChannels

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.

Transport features of pituitary folliculostellate cells increase in pregnancy

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Immunohistochemical and morphometric study of pituitary pars distalis folliculostellate cells of nonpregnant and pregnant viscachas

Pituitary folliculostellate cells (FSC) have several functions. Morphological, immunohistochemical and physiological characteristics of these cells have been reported for many mammalian species. There is little information, however, about the effect of pregnancy on FSC, particularly in wild animals. We conducted an immunohistochemical and morphometric study of the effect of pregnancy on FSC in viscacha, a seasonal breeder with a long gestation period. Immunohistochemistry and image analysis were used to measure the percentages of S-100-positive total, cellular, and colloidal areas and the number of FSC. In all groups studied, FSC were either isolated or arranged in small groups. These cells exhibited irregular nuclei, stellate shape, and cytoplasmic processes that delimited the follicles or contacted blood vessels. They expressed S-100 protein, which was localized both intracellularly (FSC) and extracellularly (follicular colloid). Some FSC were immunostained by anti-GFAP or anti-vimentin in the cytoplasm surrounding the nucleus and in the cytoplasmic processes, respectively. Morphometric parameters showed variation between nonpregnant and pregnant females. These variations were greatest during mid-pregnancy. We demonstrated that distribution of the FSC in pituitary pars distalis and expression of S-100 protein differ in nonpregnant and pregnant viscachas. Our findings suggest that FSC are involved in intrapituitary regulation of the hormone-secreting cells during pregnancy, probably by a paracrine mechanism.

Morphometric analysis of the human anterior pituitary's folliculostellate cells during the aging process

Folliculostellate cells represent non-endocrine cells of the anterior pituitary which influence the function of the endocrine cells via paracrine action. Though there is a lack of literature data on their presence during human aging, the aim of this research was to perform the quantification of anterior pituitary folliculostellate cells by the application of immunohistochemical and morphometric methods. The material for the study consisted of 15 anterior pituitaries taken from cadavers at routine autopsy. Their tissue was processed by standard histological procedure and the obtained histological slices were stained by S100 polyclonal antibody. Digital images of stained histological sections were analyzed by morphometric method with ImageJ system. The volume density of S100 positive cells was measured for each case. Results of morphometric and statistical analysis showed a significantly positive correlation between folliculostellate cell volume density and the age of the evaluated cases. Linear regression additionally showed that the age significantly predicts folliculostellate cells volume density in our sample. Further, all cases were classified into three age groups and One Way ANOVA showed that the volume density of folliculostellate cells was significantly higher only in the third age group in relation to the first and the second group, respectively. Volume densities of the first and the second age groups were not significantly different. So, the results of our study pointed to the conclusion that folliculostellate cells presence generally increases with age, but this increase is significant only in the oldest cases and might represent the modality of successful anterior pituitary aging.

Evidence From In Vitro and In Vivo Studies Showing that Nuclear Factor‐Kappa B Within the Pituitary Folliculostellate Cells and Corticotrophs Regulates Adrenocorticotrophic Hormone Secretion in Experimental Endotoxaemia

The hypothalamic-pituitary-adrenocortical (HPA) responses to bacterial infection are mediated, in part, by the actions of lipopolysaccharide (LPS) on pituitary folliculostellate (FS) cells that release pro-inflammatory cytokines [e.g. interleukin (IL)-6] and thereby facilitate adrenocorticotrophic hormone (ACTH) release from neighbouring corticotrophs. In the present study, two murine pituitary cell lines [TtT/GF (FS cells) and AtT20 D16:16 (corticotrophs)], alone and in co-culture, and an in vivo model of endotoxaemia were used to examine the potential role of nuclear factor-kappa B (NF-κB) in mediating LPS-induced ACTH secretion. Both cell lines expressed mRNAs for the key components of the LPS signalling system. LPS stimulated IL-6 release from TtT/GF cells via a glucocorticoid-sensitive, NF-κB-dependent mechanism; it also activated NF-κB in AtT20 cells, as did corticotrophin-releasing hormone (CRH). IL-6 potentiated (but LPS reduced) the stimulatory effects of CRH on ACTH release from AtT20 cells, whereas blockade of NF-κB (SC-514) increased the ACTH release induced by CRH in the presence or absence of LPS. In co-cultures, CRH and LPS acted synergistically to induce release of both IL-6 and ACTH. However, although SC-514 suppressed the release of IL-6 evoked by CRH and LPS, it potentiated the concomitant increase in ACTH release. In vivo both immunological (LPS) and psychological (restraint) stress increased intrapituitary NF-κB, whereas an NF-κB inhibitor (PHA781535E) attenuated the LPS-induced release of ACTH and abolished the HPA response to restraint stress. The results obtained in the present study support the premise that NF-κB plays an important role in mediating LPS signalling in the anterior pituitary gland, particularly in relation to IL-6 and ACTH secretion, and provide novel evidence that NF-κB blockade in vivo compromises stress-induced ACTH release.

Differentiation capacity of native pituitary folliculostellate cells and brain astrocytes

Pituitary folliculostellate (FS) cells are characterized by producing S100B protein, as do brain astrocytes. FS cells have some functions in the pituitary gland, i.e. scavenger functions, sustentacular cell activity through cytokines, and intercellular communication through gap junctions. However, the biological significances of FS cells, especially their differentiation capacities in the anterior pituitary gland, are still under discussion. To understand FS cells with new approaches, we generated a transgenic rat expressing GFP under S100b gene promoter, which regulates tissue-specific expression of S100b gene. Using the transgenic rat, we succeeded in inducing skeletal muscle cells from FS cells by culturing it in serum-free medium containing B-27 supplement, thyroid hormone (tri-iodothyronine), epidermal growth factor, and basic fibroblast growth factor. In this study, we also succeeded in inducing skeletal muscle cells from primary cultured astrocytes and astrocyte cell line, C6 cells. Hence, we concluded that pituitary FS cells have wide differentiation potential and have similar characteristics to astrocytes, which not only support cell activity but also support differentiation capacity.

Curcumin Inhibits the Growth, Induces Apoptosis and Modulates the Function of Pituitary Folliculostellate Cells

The polyphenol curcumin (diferuloylmethane) is the active componenet of the spice plant Curcuma longa and has been shown to exert multiple actions on mammalian cells. We have studied its effect on folliculostellate (FS) TtT/GF mouse pituitary cells, representative of a multifunctional, endocrine inactive cell type of the anterior pituitary. Proliferation of TtT/GF cells was inhibited by curcumin in a monolayer cell culture and in the colony formation assay in soft agar. Fluorescence-activated cell-sorting (FACS) analysis demonstrated curcumin-induced cell cycle arrest at G₂/M accompanied by inhibition of cyclin D₁ protein expression. Curcumin had a small effect on necrosis of TtT/GF cells, but it mainly stimulated apoptosis as demonstrated by FACS analysis (Annexin V-fluorescein isothiocyannate/7-aminoactinomycin D staining). Curcumin-induced apoptosis involved suppression of Bcl-2, stimulation of cleaved caspase-3 and induction of DNA fragmentation. Functional studies on FS cell-derived compounds showed that curcumin inhibited mRNA synthesis and release of angiogenic vascular endothelial growth factor-A (VEGF-A). Immune-like functions of FS cells were impaired since curcumin downregulated Toll-like receptor 4, reduced nuclear factor-ĸB expression and suppressed bacterial endotoxin-induced interleukin-6 (IL-6) secretion. The inhibitory action of curcumin on VEGF-A and IL-6 production was also found in primary rat pituitary cell cultures, in which FS cells are the only source of these proteins. The observed effects of curcumin on FS cell growth, apoptosis and functions may have therapeutic consequences for the intrapituitary regulation of hormone production and release as well as for pituitary tumor pathogenesis.

Medullary Thyroid Carcinoma Cell Lines Contain a Self-Renewing CD133+Population that Is Dependent onRETActivity

Context: Medullary thyroid carcinoma (MTC) is a cancer of the parafollicular C cells commonly caused by an inherited or acquired RET protooncogene mutation. Therapeutic resistance and recurrence of the disease imply the presence of cancer stem cells in MTC. Objective: In this study, we sought to identify and characterize cancer stem cell-like cells in MTC. Main Outcome Measures: The characterization of stem cell properties was performed using immunostaining, flow cytometry, sphere formation assay, rederivation assay, Western blotting, and quantitative RT-PCR of defined markers of neural stem and progenitor cells. The role of RET activation was assessed through RNA interference knockdown. Results: CD133 positivity was identified by immunostaining patient MTC. Flow cytometry confirmed a subpopulation of CD133 cells in two MTC cell lines. The CD133 cells could be expanded by sphere formation assay, passaged multiple times, and expressed neural progenitor markers -tubulin 3 and glial fibrillary acidic protein. The MZ-CRC-1 cell line, which harbors a M918T RET mutation, had greater CD133 cell numbers and sphere-forming ability than the TT cell line, which harbors the less active C634W mutation. Sphere formation was more dependent on RET activity than epidermal growth factor or fibroblast growth factor. Conclusion: Our data support the existence of cancer stem-like cells in MTC, which exhibit the features of self-renewal and of multiple lineage differentiation that is dependent on RET receptor activity. These findings may provide new insights to develop more promising therapy for MTC. Differential Ligand-Mediated Pituitary Somatostatin Receptor Subtype Signaling: Implications for Corticotroph Tumor Therapy Anat Ben-Shlomo, Herbert Schmid, Kolja Wawrowsky, Oxana Pichurin, Erika Hubina, Vera Chesnokova, Ning-Ai Liu, Michael Culler, and Shlomo Melmed (J Clin Endocrinol Metab, published October 9, 2009, 10.1210/jc.2009-1311) ABSTRACT Objective: Pituitary targeted pharmacotherapy for Cushing’s disease is challenging and ineffective. Unlike octreotide and lanreotide, the multisomatostatin receptor (SST) analog pasireotide that exhibits SST5 greater than SST2 binding affinity offers potential for treating Cushing’s disease. Because corticotroph cells express SST5 more abundantly than SST2, pasireotide likely exerts superior corticotroph action mainly through SST5. However, there is no direct evidence for this assumption, and moreover, the ligand effect on corticotroph SST2 is not known. Results: We used AtT20 mouse pituitary corticotroph tumor cells stably overexpressing SST2 or SST5 and TtT/GF mouse pituitary folliculostellate cells stably or transiently expressing SST receptors to examine ligand-receptor activation by SST2and SST5selective agonists. We show that pasireotide was more potent than either octreotide or somatostatin-14 in mouse corticotroph cells. Pasireotide potency is not affected by SST2 abundance, SST2 antagonist treatment, or octreotide cotreatment in SST2overexpressing cells. Pasireotide also does not induce SST2 internalization and attenuates octreotide or SRIF14-induced SST2 internalization only at superphysiological dose ranges. In contrast, octreotide attenuates pasireotide potency in SST5-overexpressing cells. Moreover, short exposure to pasireotide causes prolonged inhibition of forskolin or CRH-induced cAMP accumulation, in contrast to somatostatin-14and SST2-selective agonists that induced postwithdrawal cAMP rebound. Long-term pasireotide signaling effects are enhanced by SST5 overexpression. Conclusion: The results indicate that SST5 determines shortand long-term enhanced pasireotide action in corticotroph cells, whereas the ligand action on SST2 is negligible. Endocrine Reviews, December 2009, 30(7):926–934 edrv.endojournals.org 933Objective: Pituitary targeted pharmacotherapy for Cushing’s disease is challenging and ineffective. Unlike octreotide and lanreotide, the multisomatostatin receptor (SST) analog pasireotide that exhibits SST5 greater than SST2 binding affinity offers potential for treating Cushing’s disease. Because corticotroph cells express SST5 more abundantly than SST2, pasireotide likely exerts superior corticotroph action mainly through SST5. However, there is no direct evidence for this assumption, and moreover, the ligand effect on corticotroph SST2 is not known. Results: We used AtT20 mouse pituitary corticotroph tumor cells stably overexpressing SST2 or SST5 and TtT/GF mouse pituitary folliculostellate cells stably or transiently expressing SST receptors to examine ligand-receptor activation by SST2and SST5selective agonists. We show that pasireotide was more potent than either octreotide or somatostatin-14 in mouse corticotroph cells. Pasireotide potency is not affected by SST2 abundance, SST2 antagonist treatment, or octreotide cotreatment in SST2overexpressing cells. Pasireotide also does not induce SST2 internalization and attenuates octreotide or SRIF14-induced SST2 internalization only at superphysiological dose ranges. In contrast, octreotide attenuates pasireotide potency in SST5-overexpressing cells. Moreover, short exposure to pasireotide causes prolonged inhibition of forskolin or CRH-induced cAMP accumulation, in contrast to somatostatin-14and SST2-selective agonists that induced postwithdrawal cAMP rebound. Long-term pasireotide signaling effects are enhanced by SST5 overexpression. Conclusion: The results indicate that SST5 determines shortand long-term enhanced pasireotide action in corticotroph cells, whereas the ligand action on SST2 is negligible. Endocrine Reviews, December 2009, 30(7):926–934 edrv.endojournals.org 933

Differential Ligand-Mediated Pituitary Somatostatin Receptor Subtype Signaling: Implications for Corticotroph Tumor Therapy

Pituitary targeted pharmacotherapy for Cushing's disease is challenging and ineffective. Unlike octreotide and lanreotide, the multisomatostatin receptor (SST) analog pasireotide that exhibits SST5 greater than SST2 binding affinity offers potential for treating Cushing's disease. Because corticotroph cells express SST5 more abundantly than SST2, pasireotide likely exerts superior corticotroph action mainly through SST5. However, there is no direct evidence for this assumption, and moreover, the ligand effect on corticotroph SST2 is not known. We used AtT20 mouse pituitary corticotroph tumor cells stably overexpressing SST2 or SST5 and TtT/GF mouse pituitary folliculostellate cells stably or transiently expressing SST receptors to examine ligand-receptor activation by SST2- and SST5-selective agonists. We show that pasireotide was more potent than either octreotide or somatostatin-14 in mouse corticotroph cells. Pasireotide potency is not affected by SST2 abundance, SST2 antagonist treatment, or octreotide cotreatment in SST2-overexpressing cells. Pasireotide also does not induce SST2 internalization and attenuates octreotide or SRIF14-induced SST2 internalization only at superphysiological dose ranges. In contrast, octreotide attenuates pasireotide potency in SST5-overexpressing cells. Moreover, short exposure to pasireotide causes prolonged inhibition of forskolin or CRH-induced cAMP accumulation, in contrast to somatostatin-14- and SST2-selective agonists that induced postwithdrawal cAMP rebound. Long-term pasireotide signaling effects are enhanced by SST5 overexpression. The results indicate that SST5 determines short- and long-term enhanced pasireotide action in corticotroph cells, whereas the ligand action on SST2 is negligible.

Fibroblast Growth Factor-2 Autofeedback Regulation in Pituitary Folliculostellate TtT/GF Cells

To investigate paracrine regulation of pituitary cell growth, we tested fibroblast growth factor (FGF) regulation of TtT/GF folliculostellate (FS) cells. FGF-2, and FGF-4 markedly induced cell proliferation, evidenced by induction of pituitary tumor transforming gene-1 (Pttg1) mRNA expression and percentage of cells in S phase. Signaling for FGF-2-induced FS cell proliferation was explored by specific pharmacological inhibition. A potent inhibitory effect on FGF-2 action was observed by blocking of Src tyrosine kinase with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (>or=0.1 microm), followed by protein kinase C (PKC) inhibition with GF109203X. Treatment with FGF-2 (30 ng/ml; 10 min) activated phosphorylation of signal transducer and activator of transcription-3, ERK, stress-activated protein kinase/c-Jun N-terminal kinase, Akt, and focal adhesion kinase. Src inhibition with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine suppressed FGF-2-induced Akt and focal adhesion kinase, indicating effects downstream of FGF-2-induced Src activation. FGF-2 also markedly induced its own mRNA expression, peaking at 2-4 h, and this effect was suppressed by Src tyrosine kinase inhibition. The PKC inhibitor GF109203X abolished FGF-2 autoinduction, indicating PKC as the primary pathway involved in FGF-2 autoregulation in these cells. In addition to pituitary FGF-2 paracrine activity on hormonally active cells, these results show an autofeedback mechanism for FGF-2 in non-hormone-secreting pituitary FS cells, inducing cell growth and its own gene expression, and mediated by Src/PKC signaling.

Spindle cell oncocytoma of the adenohypophysis: report of a case with marked cellular atypia and recurrence despite adjuvant treatment

Spindle cell oncocytoma (SCO) of the adenohypophysis is a recently defined pituitary tumor mimicking a non-functioning macroadenoma and composed of mitochondrion rich tumor cells, positive for S-100, vimentin, epithelial membrane antigen and galectin-3 but lacking cytokeratins, pituitary hormones, and neuroendocrine markers. Derivation from pituitary folliculostellate cells (FSCs) has been suggested based upon immunohistochemical and ultrastructural characteristics shared by SCO and FSCs. 10 cases of SCO have been reported to date; of these, 8 underwent a benign clinical course and 2 recurred. We report a case of SCO with typical histologic and immunohistochemical features in addition to marked cellular pleomorphism and nuclear atypia. It showed slow regrowth over a 30-month period of follow-up despite combined surgical and radiotherapy. Despite the benign course of most reported cases, additional experience with longer follow-up are needed to assess clinical, histopathologic, and proliferative indices and their relevance to optimal therapy for this rare pituitary tumor.

NOD proteins expression and function in the pituitary folliculostellate cells

Folliculostellate (FS) cells were found to regulate hormonal secretion in pituitary endocrine cells. This cell type share properties with components of the innate immune response such as dendritic cells and macrophages, and it's suggested that they are able to initiate immune responses and act as part of an immunoendocrine regulation system. Members of toll-like receptor (TLR) family recognize bacterial cell wall components and initiate an immune response. Recently, we detected by RT-PCR the TLR4 mRNA expression in FS cell line, TtT/GF. In the present study, we detected the constitutive expression of two members of a novel nucleotide-binding oligomerization domain (NOD) family of proteins that act as cytosolic receptors. We show that NOD1 and NOD2 are expressed in the human and mouse pituitary glands, and in TtT/GF cells. Furthermore, NOD1-agonistic diaminopimelic acid (DAP) and NOD2-agonistic muramyl dipeptide (MDP) induced (NF)-κB activation in TtT/GF cell cultures. Both NOD agonists in combination with TLR4 agonistic, LPS enhanced NF-κB transcriptional activity, and interleukin-6 (IL-6) production in these cells. Knocking-down NOD1 and NOD2 markedly inhibited LPS induced IL-6 production in TtT/GF cells. RNA interference for NOD2, but not NOD1, downregulated NF-κB activity. LPS, MDP and LPS plus MDP increased NF-κB expression and transcriptional activity, in a mechanism involving STAT3, since knocking-down this protein abolished NF-κB activation. All together, we revealed that NOD1 and NOD2 are expressed in the pituitary gland and in TtT/GF cells. In addition our findings suggest that FS cells play a predominant role in the context of imune-endocrine interactions during bacterial infection and inflammation and NOD proteins have a central participation in these processes.

Tumor Necrosis Factor-α-Induced Anterior Pituitary Folliculostellate TtT/GF Cell Uncoupling Is Mediated by Connexin 43 Dephosphorylation

The anterior pituitary folliculostellate (FS) cells are key elements of the paracrine control of the pituitary function. These cells are the source and the target of growth factors and cytokines, and are connected to other pituitary cells via Cx43-mediated gap junctions. Here, we show that acute treatment of the FS TtT/GF cell line with TNF-alpha caused a transient cell uncoupling that was accompanied by the dephosphorylation of Cx43 in Ser368. These TNF-alpha-evoked effects were dependent on protein phosphatase 2A (PP2A) and protein kinase C (PKC) activities. TNF-alpha did not affect total cell Cx43-PP2A catalytic subunit interaction, but it did induce PP2A catalytic subunit recruitment to the Triton X-100 insoluble subcellular fraction, in which Cx43-gap junction plaques are recovered. This recruitment temporally coincided with Cx43 phosphorylated in Ser368-Cx43 dephosphorylation. Cx43 did not interact with the conventional PKC-alpha, but it did interact with the atypical PKC-zeta. Moreover, this interaction was weakened by TNF-alpha. Cx43 dephosphorylation in Ser368 was followed by the tyrosine phosphorylation of the protein. The temporary closure of gap junctions during acute TNF-alpha challenge may constitute a protective mechanism to limit or confine the spread of inflammatory signals among the FS cells.

Generation of Transgenic Rats Expressing Green Fluorescent Protein in S-100β-Producing Pituitary Folliculo-Stellate Cells and Brain Astrocytes

Folliculo-stellate (FS) cells are known to act as sustentacular cells or scavenger cells in the anterior lobe. However, the precise function and origin of FS cells are still under discussion. Like brain astrocytes, FS cells contain S-100beta protein, and FS cells can be detected immunocytochemically using antibodies for S-100beta protein after fixation; however, living FS cells can not be detected. The generation of transgenic rats expressing green fluorescent protein (GFP) under the control of S-100beta protein gene promoter may allow the detection of living FS cells, which may be an excellent tool for the study of FS cells. With the aim of generation of transgenic rats, we analyzed the promoter activity of the S-100beta gene and found that intron 1 is important for cell-specific expression of the S-100beta gene. Therefore, we obtained a DNA construct containing GFP gene under a part of the S-100 promoter with intron 1. We transfected the construct into rat embryos and succeeded in generating transgenic rats. The transgenic rats expressed GFP in FS cells specifically in the anterior lobe. GFP is also expressed in other known S-100beta-expressing cells, i.e. brain astrocytes, adipocytes, and chondrocytes. We believe that the newly generated transgenic rats will provide a new approach for the study of FS cells and other S-100beta protein-producing cells.

Adenosine stimulates connexin 43 expression and gap junctional communication in pituitary folliculostellate cells

Adenosine is known to stimulate interleukin (IL)-6 and vascular endothelial growth factor (VEGF) secretion from pituitary TtT/GF folliculostellate [corrected] (FS) cells indicating that it is an important paracrine regulator of anterior pituitary function. This study demonstrates that rodent anterior pituitary cell lines produce extracellular adenosine that is able to increase intercellular gap junction communication in FS cells. Ecto-5'-nucleotidase (CD73), the enzyme that generates adenosine from AMP, was demonstrated by immunocytochemistry in approximately 20% of anterior pituitary cells, and some of these cells colocalized with prolactin and growth hormone. CD73 mRNA and protein were detected in GH3 and MMQ (somatotroph-lactotroph lineages) and TtT/GF cells, and enzyme activity was demonstrated by the conversion of exogenously added fluorescent ethenoAMP to ethenoadenosine. Adenosine production, as measured by HPLC, was detected in GH3 (1 microM/h) and MMQ (3 microM/h) but not in TtT/GF cells. Adenosine (EC50: 0.5 microM) and NECA (universal adenosine receptor agonist; EC50 0.1 microM) stimulated connexin 43 (Cx43) mRNA and protein expression within 1-2 h in TtT/GF cells. Adenosine and NECA also stimulated gap junctional intercellular communication (as assessed by transmission of Alexa Fluor 488) by 6- to 8-fold in comparison with untreated TtT/GF cells. In cocultures of MMQ and TtT/GF cells, Cx43 expression in TtT/GF cells increased in proportion to the number of MMQ cells plated out. These data suggest that adenosine, formed locally in the anterior pituitary gland can stimulate gap junction communication in FS cells.

Mechanisms for Growth Factor-Induced Pituitary Tumor Transforming Gene-1 Expression in Pituitary Folliculostellate TtT/GF Cells

PTTG1, a securin protein, also behaves as a transforming gene and is overexpressed in pituitary tumors. Because pituitary folliculostellate (FS) cells regulate pituitary tumor growth factors by paracrine mechanisms, epidermal growth factor (EGF) receptor (EGFR)-mediated PTTG1 expression and cell proliferation was tested in pituitary FS TtT/GF cells. EGFR ligands caused up to 3-fold induction of Pttg1 mRNA expression, enhanced proliferating cell nuclear antigen, and increased entry of G0/1-arrested cells into S-phase. PTTG binding factor mRNA expression was not altered. EGF-induced Pttg1 expression and cell proliferation was abolished by preincubation of TtT/GF cells with EGFR inhibitors AG1478 and gefitinib. Phosphatidylinositol 3 kinase, protein kinase C, and MAPK, but not c-Jun N-terminal kinase and Janus activating kinase signaling regulated EGF-induced Pttg1, as well as proliferating cell nuclear antigen mRNA expression and entry into S-phase. EGF-induced EGFR and ERK1/2 phosphorylation was followed by rapid MAPK kinase/ERK kinase-dependent activation of Elk-1 and c-Fos. EGF-induced Pttg1 expression peaked at the S-G2 transition and declined thereafter. Pttg1 cell cycle dependency was confirmed by suppression of EGF-induced Pttg1 mRNA by blockade of cells in early S-phase. The results show that PTTG1 and its binding protein PTTG binding factor are expressed in pituitary FS TtT/GF cells. EGFR ligands induce PTTG1 and regulate S-phase, mediated by phosphatidylinositol 3 kinase, protein kinase C, and MAPK pathways. PTTG1 is therefore a target for EGFR-mediated paracrine regulation of pituitary cell growth.