Growth differentiation factor-15 stimulates the synthesis of corticotropin-releasing factor in hypothalamic 4B cells.

Growth differentiation factor-15 (GDF15) is a stress-activated cytokine that regulates cell growth and inflammatory and stress responses. We previously reported the role and regulation of GDF15 in pituitary corticotrophs. Dexamethasone increases Gdf15 gene expression levels and production. GDF15 suppresses adrenocorticotropic hormone synthesis in pituitary corticotrophs and subsequently mediates the negative feedback effect of glucocorticoids. Here, we analyzed corticotropin-releasing factor (Crf) promoter activity in hypothalamic 4B cells transfected with promoter-driven luciferase reporter constructs. The effects of time and GDF15 concentration on Crf mRNA levels were analyzed using quantitative real-time polymerase chain reaction. Glial cell-derived neurotrophic factor family receptor α-like (GFRAL) protein is expressed in 4B cells. GDF15 increased Crf promoter activity and Crf mRNA levels in 4B cells. The protein kinase A and C pathways also contributed to the GDF15-induced increase in Crf gene expression. GDF15 stimulates GFRAL, subsequently increasing the phosphorylation of AKT, an extracellular signal-related kinase, and the cAMP response element-binding protein. Therefore, GDF15-dependent pathways may be involved in regulating Crf expression under stressful conditions in hypothalamic cells.

Growth differentiation factor-15 modulates adrenocorticotropic hormone synthesis in murine AtT-20 corticotroph cells.

Growth differentiation factor-15 (GDF15) is a stress-responsive cytokine that plays important roles in regulation of inflammatory responses, cell growth, and cell differentiation. However, the nature of these roles remains unclear. Here, we aimed to examine the regulatory effects of dexamethasone on Gdf15 expression in murine AtT-20 corticotroph cells. Human Gdf15 promoter-driven luciferase reporter constructs were transfected into corticotroph cells to analyze their promoter activity. The effects of time and concentration of dexamethasone on Gdf15 and proopiomelanocortin (Pomc) mRNA levels were assessed using quantitative real-time polymerase chain reaction. Dexamethasone induced Gdf15 transcription and mRNA levels as well as GDF15 production in transfected cells, whereas reduced the Pomc mRNA levels. GDF15 modulated adrenocorticotropic hormone (ACTH) synthesis, and the dexamethasone-mediated reduction in Pomc mRNA levels were partially relieved upon Gdf15 knockdown. We concluded that GDF15 modulated ACTH production in pituitary corticotrophs in an autocrine manner by suppressing Pomc expression and subsequently mediating the negative feedback effect of glucocorticoids, thereby contributing to pituitary stress response and homeostasis.

Involvement of histone deacetylase 1/2 in adrenocorticotropic hormone synthesis and proliferation of corticotroph tumor AtT-20 cells.

Cushing's disease is mainly caused by autonomous production of adrenocorticotropic hormone (ACTH) from pituitary adenomas. In our previous study, a histone deacetylase (HDAC) inhibitor, trichostatin A, inhibited cell proliferation and ACTH production via decreased pituitary tumor-transforming gene 1 (PTTG1) in AtT-20 mouse corticotroph tumor cells. In the present study, we examined the effects of romidepsin, a potent and selective HDAC1/2 inhibitor, on cell proliferation and ACTH synthesis. To elucidate further potential mechanisms of romidepsin, we examined the effects of HDAC1/2 on proopiomelanocortin (Pomc) and Pttg1 mRNA levels and cell proliferation. Small interfering RNA-mediated knockdown was used to decrease HDAC1 or 2. Romidepsin treatment decreased Pomc and Pttg1 mRNA levels, and cell proliferation. The drug also increased Hdac1 and decreased Hdac2 mRNA levels. Hdac1 knockdown decreased basal Pttg1 mRNA levels and cell proliferation, but not Pomc mRNA levels. Romidepsin treatment decreases ACTH synthesis in corticotroph tumor cells. Romidepsin suppresses cell proliferation via PTTG1. HDAC1 is also involved in the proliferation of corticotroph cells via PTTG1.

Inhibitory effects of a selective Jak2 inhibitor on adrenocorticotropic hormone production and proliferation of corticotroph tumor AtT20 cells.

PURPOSE: The primary cause of Cushing's disease is adrenocorticotropic hormone (ACTH)-producing pituitary adenomas. EGFR signaling induces POMC mRNA-transcript levels and ACTH secretion from corticotroph tumors. The Jak-STAT pathway is located downstream of EGFR signaling; therefore, a Jak2 inhibitor could be an effective therapy for EGFR-related tumors. In this study, we determined the effect of a potent and selective Jak2 inhibitor, SD1029, on ACTH production and proliferation in mouse AtT20 corticotroph tumor cells. MATERIALS AND METHODS: AtT20 pituitary corticotroph tumor cells were cultured after transfection with PTTG1- or GADD45ß-specific siRNA. Expression levels of mouse POMC, PTTG1, and GADD45ß mRNAs were evaluated using quantitative real-time polymerase chain reaction. ACTH levels were measured using ACTH ELISA. Western blot analysis was performed to examine protein expression of phosphorylated STAT3/STAT3. Viable cells and DNA fragmentation were measured using a cell-proliferation assay and cell-death detection ELISA, respectively. Cellular DNA content was analyzed using fluorescence-activated cell sorting. RESULTS: SD1029 decreased POMC and PTTG1 mRNA and ACTH levels, while increasing GADD45ß levels. The drug also decreased AtT20-cell proliferation and induced apoptosis, but did not alter cell-cycle progression. SD1029 also inhibited STAT3 phosphorylation. PTTG1 knockdown inhibited POMC mRNA levels and cell proliferation. However, combined treatment with PTTG1 knockdown and SD1029 had no additive effect on POMC mRNA levels or cell proliferation. GADD45ß knockdown inhibited the SD1029-induced decrease in POMC mRNA levels and also partially inhibited the decrease in cell proliferation. CONCLUSION: Both PTTG1 and GADD45ß may be responsible, at least in part, for the Jak2-induced suppression of ACTH synthesis and cell proliferation. Accordingly, therapies that target EGFR-dependent Jak2/STAT3 may have clinical applications for treating Cushing's disease.