Cell colonies were stained with crystal violet, wells were photographed, and colonies were counted with ImageJ software (National Institutes of Health; n = 3). characteristics in GH4 cells in vitro and in vivo. These data demonstrate that activated MAPK promotes differentiation and is not sufficient to drive tumorigenesis, suggesting that pituitary lactotrope tumor cells have the ability to evade the tumorigenic fate that is often associated with Ras/MAPK activation. Prolactin-secreting lactotrope cells and GH-secreting somatotrope cells are derived from the common pituitary-specific transcription factor-1 (Pit-1) lineage. In some species, including rat, a bifunctional somatolactotrope precursor cell gives rise to lactotropes and somatotropes (13). Somatolactotropes retain plasticity, allowing for rapid cell differentiation and expansion in response to physiological demands. Somatolactotropes differentiate into lactotropes during pregnancy and lactation, and into somatotropes in response to exercise (1,4). Despite the species-specific differences in lactotrope cell origin, conserved signaling pathways are critical for controlling lactotrope biology from Cruzain-IN-1 humans to rodents. Pituitary lactotropes are under tonic inhibition by hypothalamic dopamine, which acts via the dopamine D2 receptor (D2R) to inhibit cAMP/protein kinase A and MAPK signaling pathways to limit prolactin (PRL) production and secretion, lactotrope proliferation, and growth of prolactin-secreting adenomas (59). Dopamine also increases autocrine TGF-1 signaling to inhibit lactotrope proliferation (10,11). During pregnancy and lactation, dopaminergic inhibition is usually diminished by estradiol, allowing local growth factors from folliculostellate support cells to stimulate lactotropes, promoting lactotrope hyperplasia and a doubling in pituitary size (5,1214). Dysregulation of these pathways contributes to the tumorigenesis of prolactin-secreting adenomas, or prolactinomas (1416). Prolactinomas cause hypogonadism, infertility, osteoporosis, and tumor mass effects, and are the most common type of neuroendocrine tumor (17,18). The specific signaling pathways that govern the lactotrope-specific phenotype and those that control lactotrope proliferation in both physiological and pathological lactotrope expansion are poorly comprehended. Uncontrolled activation of growth factor signaling pathways, such as the Ras/MAPK pathway, results in lactotrope hyperplasia with delayed but eventual benign adenoma formation in transgenic mice (15,19). Ras proteins are frequently mutated in human cancers, including the naturally occurring valine 12 (V12) mutation, which results in constitutive activation of Ras signaling (20,21). Ras is usually a critical effector of MAPK activation (19), and key regulators of lactotrope biology, such as TRH and vasoactive intestinal peptide, act via Ras to activate MAPK in somatolactotrope cells (2224). The precise role of MAPK signaling in lactotrope proliferation vs differentiation has been somewhat controversial. In vitro studies using rat pituitary somatolactotrope (GH3) or lactotrope (PR1) cell lines have shown that short-term (2496 h) MAPK pathway activation mediates cellular proliferation (12,25,26). By contrast, Cruzain-IN-1 long-term treatment of GH3 or GH4 rat pituitary somatolactotrope tumor cells over 47 days with epidermal growth factor (EGF), fibroblast growth factor (FGF)-4, or TRH result in a decreased GH4 Cruzain-IN-1 cell proliferation Rabbit Polyclonal to OR10A7 and enhanced differentiation to the lactotrope phenotype (2731). However, neither the pattern nor the peak of MAPK activation with long-term growth factor treatment has been reported. One study examined the differential effects of short-term vs long-term phosphorylated MAPK (pMAPK) activation, reporting that GH4 cells treated with FGF4 resulted in short-term pMAPK activation (<15 min) and increased cell proliferation, whereas GH4 cells treated with FGF2 resulted in prolonged pMAPK activation (>30 min) and little change in the cell number (32). Importantly, a persistent pattern of pMAPK activation has been shown to play a pivotal role in cellular differentiation in other endocrine tumors including thyroid carcinoma and pheochromocytoma (33,34). The specific role of MAPK signaling in durable lactotrope proliferation and differentiation, and whether activated pMAPK is sufficient for lactotrope proliferation and tumor formation remain unknown. Given that Ras mutations and persistently activated pMAPK are found in human tumors (35,36), including prolactinomas and other pituitary tumors (16,37,38), a better understanding of the causative role of MAPK in prolactinoma tumorigenesis is required. In this Cruzain-IN-1 study, we aimed to directly examine the role of persistent Ras/MAPK signaling in differentiation, proliferation, and tumorigenesis of Cruzain-IN-1 rat pituitary somatolactotrope GH4 cells. We stimulated Ras/MAPK signaling in a persistent, long-term manner (over 6 d) in GH4 cells using two distinct approaches: 1) a doxycycline-inducible, oncogenic V12Ras expression system; and 2) the addition of exogenous EGF. We found that long-term activation of the Ras/MAPK pathway over 6 days promotes differentiation of the bihormonal somatolactotrope GH4 precursor cell into a prolactin-secreting, lactotrope cell phenotype. Furthermore, we show that persistent activation of the Ras/MAPK pathway not only.