Mouse Pituitary AtT-20 Cells Research Articles

Metformin inhibits cell proliferation and ACTH secretion in AtT20 cells via regulating the MAPK pathway

We investigated the impact of metformin on ACTH secretion and tumorigenesis in pituitary corticotroph tumors. The mouse pituitary tumor AtT20 cell line was treated with varying concentrations of metformin. Cell viability was assessed using the CCK-8 assay, ACTH secretion was measured using an ELISA kit, changes in the cell cycle were analyzed using flow cytometry, and the expression of related proteins was evaluated using western blotting. RNA sequencing was performed on metformin-treated cells. Additionally, an in vivo BALB/c nude xenograft tumor model was established in nude mice, and immunohistochemical staining was conducted for further verification. Following metformin treatment, cell proliferation was inhibited, ACTH secretion decreased, and G1/S phase arrest occurred. Analysis of differentially expressed genes revealed cancer-related pathways, including the MAPK pathway. Western blotting confirmed a decrease in phosphorylated ERK1/2 and phosphorylated JNK. Combining metformin with the ERK1/2 inhibitor Ulixertinib resulted in a stronger inhibitory effect on cell proliferation and POMC (Precursors of ACTH) expression. In vivo studies confirmed that metformin inhibited tumor growth and reduced ACTH secretion. In conclusion, metformin inhibits tumor progression and ACTH secretion, potentially through suppression of the MAPK pathway in AtT20 cell lines. These findings suggest metformin as a potential drug for the treatment of Cushing's disease.

EGFL7-blocking antibody inhibit pituitary adenoma proliferation and invasion

Objective: To study the effect of EGFL7-blocking monoclonal antibody inhibiting the proliferation and invasion of mouse adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (ACTHomas) AtT-20 cells. Methods: After treated with different concentration of EGFL7-blocking monoclonal antibody for 24, 48, 72 h, the proliferation of AtT-20 cells was determined by MTS. Simultaneously, ACTH secretion of AtT-20 cells by ELISA and the mRNA expression of invasion related genes such as E-cadherin, snail, MMP-2 and MMP-9 by Real-time PCR were detected. Results: EGFL7-blocking monoclonal antibody could effectively inhibit the proliferation and ACTH secretion of AtT-20 cells in a dose-dependent and time-dependent manner. At the same time, EGFL7-blocking monoclonal antibody significant down-regulated the mRNA level of snail, MMP-2 and MMP-9 in AtT-20 cells. Conclusion: EGFL7-blocking monoclonal antibody could inhibit mouse pituitary adenoma AtT-20 cells proliferation and invasion.

Fascin promotes the invasion of pituitary adenoma through partial dependence on epithelial-mesenchymal transition.

The aim of the present study was to investigate the role and potential regulatory mechanisms of fascin in the invasion and epithelial-to-mesenchymal transition of pituitary adenoma cells. A total of 30 specimens were assessed in the present study. The expression levels of fascin in the invasive pituitary adenoma group and non-invasive pituitary adenoma group were determined by immunochemistry. Fascin was downregulated via small interfering RNA in mouse pituitary AtT-20 cells. The proliferation, cell cycle and apoptosis of AtT-20 cells were assessed using Cell Counting Kit‑8 and flow cytometry. The invasion of AtT-20 cells was detected using a Transwell assay. Transmission electron microscopy was utilized to observe the ultrastructure of AtT-20 cells. Real-time quantitative PCR, Western blotting and immunofluorescence staining were utilized to detect the expression levels of fascin and EMT markers. In the present study, fascin expression and clinical characteristics were not significantly correlated in pituitary adenoma. The protein expression level of fascin in invasive pituitary adenoma was higher than that in non-invasive pituitary adenoma, as assessed by immunochemistry. Downregulation of fascin resulted in significant decreases in cell viability, proliferation and invasion, arrested the cell cycle at the G1 phase and increased apoptosis. In addition, downregulation of fascin significantly decreased the expression levels of N-cadherin, the mesenchymal cell marker vimentin and the transcription factor Twist but significantly increased the expression levels of the epithelial cell marker E-cadherin. Further experiments revealed that overexpression of E-cadherin resulted in significant decreases in cell viability, proliferation, invasion, and the expression of fascin and transcription factor Twist and also arrested the cell cycle at the G2 phase. The results of the present study suggest that suppressing the expression level of fascin could regulate the invasion, proliferation and apoptosis of pituitary tumour cells and alter the expression level of various EMT markers. The present study identified that fascin effectively promotes the invasion, proliferation and apoptosis of pituitary tumour cells partially via the EMT pathway.

MIF Inhibition Suppresses Cell Viability and Induces Apoptosis via the ATF4-CHOP Pathway in Mouse Pituitary AtT-20 Cells

Cushing’s disease (CD) is characterized by cortisol overproduction due to ACTH hypersecretion from a pituitary tumour (PT). With an incidence of approximately 1.2 to 2.4 cases per million per year, CD patients have higher rates of morbidity and mortality than the general population. Surgical management is currently the first therapeutic option. However, remission rates vary between studies, and patients may suffer from complications caused by hormonal abnormalities from remnant PT tissues, the surgery itself, as medical treatment options are limited. Macrophage migratory inhibitory factor (MIF) is a cytokine expressed in various tumors, including ACTH-producing PTs, and has been found to play a crucial role in tumorigenesis. Previous studies demonstrate that MIF regulates cell growth via the signal transducer and activator of transcription 3 (STAT3) pathway, the mammalian target of rapamycin (mTOR) pathway, and autophagy. Together, these indicate MIF as a potential therapeutic target for PTs. However, the role of MIF in ACTH-producing PTs remains unknown. Using mouse ACTH-producing PT cells, AtT-20 cells as a model, we established that MIF overexpression led to increased cell growth. In contrast, pharmacological MIF inhibition by 4-iodo-6-phenylpyrimidine (4-IPP) and (S,R)-3-(4-Hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (ISO-1) and genetic MIF downregulation by siRNA both suppressed cell viability and induced apoptosis, suggesting an anti-apoptotic role of MIF. Genetic MIF downregulation also increased the expression of apoptosis-inducible genes such as activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and reduced ACTH production. However, pharmacological MIF inhibition had no effect on ACTH production, which suggests that the mechanism of pharmacological MIF inhibition may be different from MIF downregulation. Neither MIF upregulation nor downregulation affected cell signalling pathways such as the STAT3 pathway, the mTOR pathway, or autophagy. Our findings suggest that MIF inhibition can be a viable therapeutic approach for ACTH-producing PTs.

The activation of G protein-coupled receptor 30 increases pro-opiomelanocortin gene expression through cAMP/PKA/NR4A pathway in mouse pituitary corticotroph AtT-20 cells

G protein-coupled receptor 30 (GPR30) signaling plays an important role in many regulatory pathways, such as gene expression, cell proliferation and migration. However, whether GPR30 is involved in transcription of the pro-opiomelanocortin (Pomc) gene in pituitary corticotroph cells is currently unknown. Here, we report that GPR30 signaling, activated by the GPR30 specific agonist G-1, increases Pomc expression in the mouse corticotroph cell line AtT-20. G-1 also increased nuclear receptor subfamily 4 group A member 1- and 2-dependent transcription activity and phosphorylation of cyclic adenosine monophosphate response element binding protein. Furthermore, protein kinase A inhibitors strongly attenuated G-1-mediated transactivation. The findings suggest that G-1 stimulates GPR30-mediated mechanisms via cyclic adenosine monophosphate/protein kinase A/nuclear receptor subfamily 4 group A members activity in the regulation of Pomc in corticotroph cells.

BP-1-102 and silencing of Fascin-1 by RNA interference inhibits the proliferation of mouse pituitary adenoma AtT20 cells via the signal transducer and activator of transcription 3/fascin-1 pathway

Introduction The expression levels of signal transducer and activator of transcription 3 (STAT3) protein and Fascin-1 were inhibited using the STAT3 inhibitor BP-1-102 and RNA interference, respectively, to investigate the expression of AtT20 in mouse pituitary cells. The proliferative capacity and related molecular mechanisms of pituitary tumor cells were then analyzed. Methods Mouse AtT20 pituitary adenoma cells were divided into a control group (Pa group), a STAT3 inhibitor vehicle group (PA + DMSO group), a STAT3 inhibitor group (PA + BP-1-102 group), a Fascin-1 negative control group (PA + neg-siRNA group) and a Fascin-1 silenced group (PA + Fascin-siRNA group). The related protein expression and cell proliferation of the five groups were measured using immunofluorescence, Western blot and real-time RT-PCR, whereas their apoptosis and cell cycle were evaluated using CCK-8 and flow cytometry. Results Proliferation of AtT20 cells is inhibited with BP-1-102 enhanced apoptosis, at the same time reduced the expression of Fascin-1 and N-cadherin, and increased the expression of E-cadherin. After inhibiting Fascin-1, the expression of STAT3 decreased, the expression of N-cadherin decreased and the expression of E-cadherin increased. Conclusions BP-1-102 is a novel drug with a great potential in pituitary tumors. Given their important roles in the growth of pituitary adenomas, STAT3 and Fascin-1 can be used as new treatment targets.

O-linked β-N-acetylglucosamine transferase is involved in pro-opiomelanocortin gene expression in mouse pituitary corticotroph AtT-20 cells

Glucocorticoids and glucocorticoid receptors (GRs) suppress pituitary pro-opiomelanocortin (Pomc) gene expression. O-linked β-N-acetylglucosamine (O-GlcNAc) modification, mediated by O-GlcNAc transferase (OGT), plays an important role during gene transcription. However, whether OGT is involved in the GR-mediated transrepression that occurs in pituitary corticotroph cells is currently unknown. Here, we report that OGT regulates Pomc expression in the mouse corticotroph cell line AtT-20. The overexpression of OGT has an additive effect on the GR-mediated negative transcription pathway. Both the knockdown of OGT by RNA interference and the use of a chemical OGT inhibitor abolished the repressive effects of Pomc expression induced by GRs. OGT inhibition leads to both the decreased recruitment of GRs and the increased recruitment of RNA polymerase II to the Pomc locus. O-GlcNAc modification is involved in the negative regulation of Pomc transcription in corticotroph cells. OGT may be a promising therapeutic target for the treatment of Cushing’s disease.

MiR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic islet tumours, and is due to mutations of the MEN1 gene, which encodes the tumour suppressor protein menin. Menin has multiple roles in genome stability, transcription, cell division and proliferation, but its mechanistic roles in tumourigenesis remain to be fully elucidated. miRNAs are non-coding single-stranded RNAs that post-transcriptionally regulate gene expression and have been associated with tumour development, although the contribution of miRNAs to MEN1-associated tumourigenesis and their relationship with menin expression are not fully understood. Alterations in miRNA expression, including downregulation of three putative ‘tumour suppressor’ miRNAs, miR-15a, miR-16-1 and let-7a, have been reported in several tumour types including non-MEN1 pituitary adenomas. We have therefore investigated the expression of miR-15a, miR-16-1 and let-7a in pituitary tumours that developed after 12 months of age in female mice with heterozygous knockout of the Men1 gene (Men1+/− mice). The miRNAs miR-15a, miR-16-1 and let-7a were significantly downregulated in pituitary tumours (by 2.3-fold, P < 0.05; 2.1-fold P < 0.01 and 1.6-fold P < 0.05, respectively) of Men1+/− mice, compared to normal WT pituitaries. miR-15a and miR-16-1 expression inversely correlated with expression of cyclin D1, a known pro-tumourigenic target of these miRNAs, and knockdown of menin in a human cancer cell line (HeLa), and AtT20 mouse pituitary cell line resulted in significantly decreased expression of miR-15a (P < 0.05), indicating that the decrease in miR-15a may be a direct result of lost menin expression.

Metformin suppresses growth and adrenocorticotrophic hormone secretion in mouse pituitary corticotroph tumor AtT20 cells.

Pituitary corticotroph tumors lead to excess adrenocorticotrophic hormone (ACTH) secretion, resulting in Cushing's disease (CD), which is associated with significant mortality. Standard treatments include neurosurgery, radiotherapy and medical therapy. Both surgery and radiotherapy have undesirable complications and high recurrence rates. At present, there is only one medical option available that targets pituitary adenoma and ACTH secretion, the drug pasireotide. However, hyperglycemia is common during pasireotide treatment. In addition, some patients have discontinued pasireotide treatment because of hyperglycemia-related adverse events or uncontrolled diabetes. New medical treatments directly targeting the corticotroph cells and suppressing ACTH secretion are urgently required. Metformin is a commonly used antidiabetic drug that has been widely used to control the hyperglycemia that occurs in patients with CD, which is secondary to both cortisol excess and pasireotide treatment. Recent studies suggest that metformin has direct anticancer activities against many tumor cell lines. In the present study, we investigated whether metformin exerts an anti-tumor effect by directly targeting pituitary corticotroph tumors and exploring the underlying mechanisms. Using the mouse corticotroph tumor cells, AtT20 cells, we report that metformin inhibited cell proliferation, promoted cell apoptosis and decreased ACTH secretion but did not block the cell cycle in cells. The apoptosis induced by metformin was accompanied by increased caspase-3 activity. Meanwhile, metformin down-regulated the anti-apoptotic protein B cell lymphoma 2 (Bcl-2) but up-regulated the pro-apoptotic protein Bcl2-associated X (BAX), which suggests the involvement of the mitochondrial-mediated apoptosis pathway. Furthermore, metformin promoted AMP-activated protein kinase (AMPK) phosphorylation but inhibited insulin-like growth factor-1 receptor (IGF-1R) expression, protein kinase B (PKB/AKT) phosphorylation and mammalian target of rapamycin (mTOR) phosphorylation. Finally, the IGF-1R inhibitor picropodophyllin (PPP) significantly inhibited the cell proliferation of AtT20 cells. We conclude that metformin inhibits cell proliferation and induces apoptosis in AtT20 cells by activating AMPK/mTOR and inhibiting IGF-1R/AKT/mTOR signaling pathways. Metformin may have direct antitumor activity against pituitary corticotroph tumors.

Regulation of cAMP Responsive Element Binding Protein 3-Like 1 (Creb3l1) Expression by Orphan Nuclear Receptor Nr4a1.

Cyclic AMP (cAMP) inducible transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1) is strongly activated in the hypothalamus in response to hyperosmotic cues such as dehydration (DH). We have recently shown that Creb3l1 expression is upregulated by cAMP pathways in vitro, however the exact mechanisms are not known. Here we show that increasing Creb3l1 transcription by raising cAMP levels in mouse pituitary AtT20 cells automatically initiates cleavage of Creb3l1, leading to a greater abundance of the transcriptionally active N-terminal portion. Inhibiting protein synthesis indicated that de novo protein synthesis of an intermediary transcription factor was required for Creb3l1 induction. Strategic mining of our microarray data from dehydrated rodent hypothalamus revealed four candidates, reduced to two by analysis of acute hyperosmotic-induced transcriptional activation profiles in the hypothalamus, and one, orphan nuclear receptor Nr4a1, by direct shRNA mediated silencing in AtT20 cells. We show that activation of Creb3l1 transcription by Nr4a1 involves interaction with a single NBRE site in the promoter region. The ability to activate Creb3l1 transcription by this pathway in vitro is dictated by the level of methylation of a CpG island within the proximal promoter/5′UTR of this gene. We thus identify a novel cAMP-Nr4a1-Creb3l1 transcriptional pathway in AtT20 cells and also, our evidence would suggest, in the hypothalamus.

AT-101 acts as anti-proliferative and hormone suppressive agent in mouse pituitary corticotroph tumor cells

Gossypol, a naturally occurring compound in cottonseeds, has anticancer effects against several tumor cell lines. It has been extensively studied in clinical trials and is well tolerated with a favorable safety profile. AT-101, a derivative of R (-)-gossypol, binds to Bcl-2 family proteins and induces apoptosis in vitro. Although transsphenoidal surgical excision of the pituitary corticotroph adenoma is the gold standard of care, it is not successful all the time. Medical therapy for Cushing's disease still remains a challenge for the clinicians. We aimed to investigate the cytotoxic and apoptotic effects of AT-101 in mouse pituitary corticotroph tumor AtT20 cells. Cytotoxic effect of AT-101 was assessed by XTT cell viability assay. Apoptosis was shown by measuring DNA fragmentation and Caspase-3/7 activity. Changes in mRNA expressions of apoptosis-related genes were investigated by qPCR array after treatment with AT-101. ACTH was measured by ACTH-EIA Kit. AT-101 induced cytotoxicity and apoptosis in AtT20 cells. mRNA levels of pro-apoptotic genes such as TNFR-SF-10B, Bid, PYCARD, Caspase-8, Caspase-3, and Caspase-7 were induced by 2.0-, 1.5-, 1.7-, 1.5-, 1.6-, and 2-fold, respectively, in AtT20 cells by AT-101 treatment. Moreover, some of the anti-apoptotic genes such as BCL2L10, NAIP1, and PAK-7 were reduced by 2.1-, 2.3-, 4.0-fold, respectively, in AtT20 cells. AT-101 also decreased ACTH secretion significantly. AT-101 induces apoptosis in mouse pituitary corticotroph tumor cells.

RETRACTED ARTICLE: Silencing of the Smad nuclear interacting protein 1 (SNIP1) by siRNA inhibits proliferation and induces apoptosis in pituitary adenoma cells

Smad nuclear interacting protein 1 (SNIP1) gene encodes a protein that contains a conservative C-terminal forkhead-associated domain and functions as a transcriptional coactivator to regulate cell proliferation and cancer progression. This study aimed to investigate the clinical and biological significance of SNIP1 expression in pituitary adenoma. We analyzed SNIP1 expressions in mouse fibroblast L929 cells and mouse pituitary adenoma AtT-20 cells by Western blotting. SNIP1 gene knockdown by small interfering RNA (siRNA) transfection was performed to evaluate SNIP1 function in pituitary adenoma cell lines. As expected, SNIP1 was found to be upregulated in pituitary adenoma cells. The mRNA and protein levels of SNIP1 were inhibited in AtT-20 cells transfected with siRNAs, which led to decreased proliferation, increased apoptosis, and cycle arrest of pituitary adenoma cells. Concomitantly, c-Myc and cyclin D1 protein levels were reduced. These findings may provide novel targets for the treatment of pituitary adenoma.

Correction: Growth Suppression of Mouse Pituitary Corticotroph Tumor AtT20 Cells by Curcumin: A Model for Treating Cushing's Disease

Correction: Growth Suppression of Mouse Pituitary Corticotroph Tumor AtT20 Cells by Curcumin: A Model for Treating Cushing's Disease

Growth suppression of mouse pituitary corticotroph tumor AtT20 cells by curcumin: a model for treating Cushing's disease.

BackgroundPituitary corticotroph tumors secrete excess adrenocorticotrophic hormone (ACTH) resulting in Cushing's disease (CD). Standard treatment includes surgery and, if not successful, radiotherapy, both of which have undesirable side effects and frequent recurrence of the tumor. Pharmacotherapy using PPARγ agonists, dopamine receptor agonists, retinoic acid or somatostatin analogs is still experimental. Curcumin, a commonly used food additive in South Asian cooking, has potent growth inhibitory effects on cell proliferation. Our laboratory recently demonstrated that curcumin inhibited growth and induced apoptosis in prolactin- and growth hormone-producing tumor cells [1]. Subsequently, Schaaf et.al. confirmed our findings and also showed the in vivo effectiveness of curcumin to suppress pituitary tumorigenesis. However the molecular mechanism that mediate this effect of curcumin are still unknown.Principal FindingsUsing the mouse corticotroph tumor cells, AtT20 cells, we report that curcumin had a robust, irreversible inhibitory effect on cell proliferation and clonogenic property. The curcumin-induced growth inhibition was accompanied by decreased NFκB activity. Further, curcumin down-regulated the pro-survival protein Bcl-xL, depolarized the mitochondrial membrane, increased PARP cleavage, which led to apoptotic cell death. Finally, curcumin had a concentration-dependent suppressive effect on ACTH secretion from AtT20 cells.ConclusionThe ability of curcumin to inhibit NFκB and induce apoptosis in pituitary corticotroph tumor cells leads us to propose developing it as a novel therapeutic agent for the treatment of CD.

Curcumin acts as anti-tumorigenic and hormone-suppressive agent in murine and human pituitary tumour cells in vitro and in vivo

Curcumin (diferuloylmethane) is the active ingredient of the spice plant Curcuma longa and has been shown to act anti-tumorigenic in different types of tumours. Therefore, we have studied its effect in pituitary tumour cell lines and adenomas. Proliferation of lactosomatotroph GH3 and somatotroph MtT/S rat pituitary cells as well as of corticotroph AtT20 mouse pituitary cells was inhibited by curcumin in monolayer cell culture and in colony formation assay in soft agar. Fluorescence-activated cell sorting (FACS) analysis demonstrated curcumin-induced cell cycle arrest at G2/M. Analysis of cell cycle proteins by immunoblotting showed reduction in cyclin D(1), cyclin-dependent kinase 4 and no change in p27(kip). FACS analysis with Annexin V-FITC/7-aminoactinomycin D staining demonstrated curcumin-induced early apoptosis after 3, 6, 12 and 24 h treatment and nearly no necrosis. Induction of DNA fragmentation, reduction of Bcl-2 and enhancement of cleaved caspase-3 further confirmed induction of apoptosis by curcumin. Growth of GH3 tumours in athymic nude mice was suppressed by curcumin in vivo. In endocrine pituitary tumour cell lines, GH, ACTH and prolactin production were inhibited by curcumin. Studies in 25 human pituitary adenoma cell cultures have confirmed the anti-tumorigenic and hormone-suppressive effects of curcumin. Altogether, the results described in this report suggest this natural compound as a good candidate for therapeutic use on pituitary tumours.

Impact of curcumin on growth and apoptosis of pituitary tumor cells

Curcumin (Diferuloylmethan) is the active ingredient in the traditional herbal remedy and dietary spice turmeric (Curcuma Longa). Curcumin has a wide range of beneficial properties, including anti-inflammatory, antioxidant, chemopreventive and chemotherapeutic activity. The pleiotropic activities of curcumin derive from its ability to influence multiple signalling pathways. We have investigated in vitro effects of curcumin in lactosomatotroph GH3 and somatotroph MtT/S rat pituitary cells, in corticotroph AtT20 mouse pituitary cells and in folliculostellate TtT/GF mouse pituitary cells. Curcumin inhibited growth of all cell lines in monolayer cell culture and suppressed colony formation in soft agar (IC50: 19µM). At least for GH3 cells it could be shown, that GH3 cell tumor formation was reduced by curcumin in nude mice. FACS analysis with Annexin-V-FITC/7AAD-staining demonstrated curcumin-induced early apoptosis after 12 and 24h treatment and nearly no necrosis. In endocrine cell types, hormone production was inhibited by curcumin. Angiogenic components (VEGF, VEGF receptor type 2, HIF-1alpha) and immunomodulatory factors (IL-6, TLr4) were all suppressed by curcumin in pituitary cells. Regarding the mechanism of action, results indicate that curcumin suppresses the PI3 kinase/Akt pathway and inhibits CDK4. Reduction of Bcl-2 and enhancement of cleaved caspase 9 suggest induction of apoptosis by curcumin, which was confirmed by cell death ELISA. In summary, curcumin shows clear anti-tumorigenic properties and its effects on human pituitary tumors will be tested in future studies.

Histamine H 4 receptors regulate ACTH release in AtT-20 cells

The early research described that adrenocorticotropic hormone (ACTH) release from mouse pituitary tumor AtT-20 cells was regulated by histamine H 3 receptors. Here, we provide the evidence that histamine H 4 receptor was expressed in AtT-20 cell, and that the accelerated ACTH secretions from the cells by histamine and R-α-methylhistamine were blocked by JNJ7777120, a specific H 4 receptor antagonist, in concentration dependent manner, but not by the H 1 and H 2 receptor antagonists. The results indicate, for the first time, that histamine H 4 receptor, rather than histamine H 3 receptor, played a role in regulation of ACTH release from mouse pituitary AtT-20 cells.

Tyrosine Phosphorylation of Kir3.1 in Spinal Cord Is Induced by Acute Inflammation, Chronic Neuropathic Pain, and Behavioral Stress

Tyrosine phosphorylation is an important means of regulating ion channel function. Our previous gene expression studies using the Xenopus laevis oocyte system suggested that tyrosine phosphorylation of G-protein-gated inwardly rectifying potassium channels (K(ir)3 or GIRK) suppressed basal channel conductance and accelerated channel deactivation. To assess whether similar mechanisms regulate K(ir)3 function in mammalian cells, we developed and characterized a phosphoselective antibody recognizing K(ir)3.1 phosphorylated at tyrosine 12 in the N-terminal domain and then probed for evidence of K(ir)3.1 phosphorylation in cultured mammalian cells and spinal cord. The antibody was found to discriminate between the phospho-Tyr(12) of K(ir)3.1 and the native state in transfected cell lines and in primary cultures of mouse atria. Following either mouse hindpaw formalin injection or sciatic nerve ligation, pY12-K(ir)3.1 immunoreactivity was enhanced unilaterally in the superficial layers of the spinal cord dorsal horn, regions previously described as expressing K(ir)3.1 channels. Mice lacking K 3.1 following targeted gene disruption did not show specific pY12-K(ir)3.1 immunoreactivity after sciatic nerve ligation. Further, mice exposed to repeatedly forced swim stress showed bilateral enhancement in pY12-K(ir)3.1 in the dorsal horn. This study provides evidence that K(ir)3 tyrosine phosphorylation occurred during acute and chronic inflammatory pain and under behavioral stress. The reduction in K(ir)3 channel activity is predicted to enhance neuronal excitability under physiologically relevant conditions and may mediate a component of the adaptive physiological response.

Distinct Conformations of the Corticotropin Releasing Factor Type 1 Receptor Adopted following Agonist and Antagonist Binding Are Differentially Regulated

The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit beta-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or beta-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.

Basal adrenocorticotropin (ACTH) secretion is negatively modulated by protein phosphatase 5 in mouse pituitary corticotropin AtT20 cells

siRNA oligonucleotides for protein phosphatase 5 (PP5) were designed and transfected into mouse corticotroph AtT20 cells to induce lower PP5 expression levels. PP5-siRNA transfections (at 3 days) produced a ∼50% down-regulation in targeted protein levels. PP5-underexpressing cells released significantly more ir-ACTH (10–12-fold) relative to baseline levels and promoted POMC release into the media. Neither CRF-mediated ACTH release nor dexamethasone-induced ACTH repression were affected in PP5-siRNA transfected cells. In summary, our observations suggest that endogenous PP5 can exert a negative modulatory effect on basal ACTH release in neurosecretion-competent AtT20 cells through a mechanism as yet unknown but which does not directly involve regulated CRF or glucocorticoid receptor-dependent pathways. However, PP5 may cause mis-sorting of POMC and POMC-derived peptides at the constitutive-like secretory pathway level in an unregulated manner. Such a missorting could lead to impaired processing of POMC.