Vera Chesnokova

Senescence Mediates Pituitary Hypoplasia and Restrains Pituitary Tumor Growth

Understanding factors subserving pituitary cell proliferation enables understanding mechanisms underlying uniquely benign pituitary tumors. Pituitary tumor-transforming gene (Pttg) deletion results in pituitary hypoplasia, low pituitary cell proliferation rates, and rescue of pituitary tumor development in Rb(+/-) mice. Pttg(-/-) pituitary glands exhibit ARF/p53/p21-dependent senescence pathway activation evidenced by up-regulated p19, cyclin D1, and Bcl-2 protein levels and p53 stabilization. High pituitary p21 levels in the absence of PTTG were associated with suppressed cyclin-dependent kinase 2 activity, Rb phosphorylation, and cyclin A expression, all required for cell cycle progression. Although senescence-associated beta-galactosidase was enhanced in Pttg-deficient pituitary glands, telomere lengths were increased. DNA damage signaling pathways were activated and aneuploidy was evident in the Pttg-deficient pituitary, triggering senescence-associated genes. To confirm the p21 dependency of decreased proliferation and senescence in the Pttg-null pituitary, mouse embryonic fibroblast (MEF) colony formation was tested in wild-type, Pttg(-/-), Rb(+/-), Rb(+/-)Pttg(-/-), and Rb(+/-)Pttg(-/-)p21(-/-) cells. Rb(+/-)Pttg(-/-) MEFs, unlike Rb(+/-) cells, failed to produce colonies and exhibited high levels of senescence. p21 deletion from Rb(+/-)Pttg(-/-) MEFs enhanced anchorage-independent cell growth, accompanied by a marked decrease in senescence. As cell proliferation assessed by bromodeoxyuridine incorporation was higher in Rb(+/-)Pttg(-/-)p21(-/-) relative to Rb(+/-)Pttg(-/-) pituitary glands, p21-dependent senescence provoked by Pttg deletion may underlie pituitary hypoplasia and decreased tumor development in Rb(+/-)Pttg(-/-) mice.

Leukemia Inhibitory Factor Modulates Interleukin-1β-Induced Activation of the Hypothalamo-Pituitary-Adrenal Axis1

We have shown that leukemia inhibitory factor (LIF) is expressed in corticotroph cells and stimulates POMC gene expression and ACTH secretion in vivo and in vitro. We therefore examined the regulation of in vitro and in vivo pituitary LIF expression by cytokines known to stimulate the hypothalamo-pituitary-adrenal axis. In the corticotroph cell line AtT-20/D16v-F2, recombinant murine interleukin-1β (IL-1β; 0.1–10.0 ng/ml) caused a 5- to 10-fold increase in LIF messenger RNA (mRNA) levels. LIF mRNA expression was induced as early as 1 h, peaked at 2 h, and still persistently elevated above the baseline after 8 h. This effect of IL-1β on LIF mRNA expression was abolished by preincubation with human IL-1 receptor antagonist (100 ng/ml) or antimurine IL-1β antibody (10 μg/ml). Tumor necrosis factor-α (20 ng/ml) only modestly increased LIF mRNA, but was synergistic with IL-1β (up to 2.5-fold). In contrast, IL-2 and IL-6 did not alter LIF mRNA. In C57BL/6 mice, ip injection of 100 ng IL-1β increased plasma ACTH...

Leukemia Inhibitory Factor Mediates the Hypothalamic Pituitary Adrenal Axis Response to Inflammation1

The pleiotropic cytokine leukemia inhibitory factor (LIF) is expressed in murine hypothalamus and pituitary and increases POMC gene transcription and ACTH secretion in vitro and in vivo. As hypothalamic pituitary adrenal (HPA) axis activation during inflammation is an important protective mechanism, we determined whether LIF stimulates the HPA inflammatory stress response. Two experimental models were employed: sc injection of complete Freund’s adjuvant (CFA) and im administration of turpentine. Hypothalamic LIF gene expression was increased up to 5 days after CFA, and up to 24 h after turpentine. LIF induction was concordant with elevated plasma ACTH and corticosterone levels and pituitary POMC messenger RNA (mRNA) expression. Pituitary levels of LIF-inducible signaling inhibitor (SOCS 3) mRNA were stimulated 3-fold after CFA and turpentine treatment. In contrast, in LIF knockout mice (LIFKO) pituitary POMC mRNA levels and plasma ACTH and corticosterone responses to both inflammatory challenges were markedly lower than in wild-type (WT) animals. Injection of exogenous LIF (5 mg) to turpentine-treated LIFKO mice induces POMC gene expression. These results indicate that LIF is an essential component for the neuroendocrine response to inflammatory processes. (Endocrinology 141: 4032–4040, 2000) L inhibitory factor (LIF), a member of the common cytokine family comprising oncostatin M, IL 6, IL 11, ciliary neutropic factor, and cardiotropin 1 (1, 2, 3) was initially described for its ability to induce differentiation of murine M1 leukemia cells (4, 5). LIF is now recognized to have pleiotropic actions regulating metabolism, growth, and differentiation (6) and is required for neuronal response to injury (7). LIF is produced as a component of the host response to inflammatory stimuli and is an early mediator of the inflammatory cytokine response (8). During inflammation, LIF is markedly increased locally and induces IL 1 and IL 6 cytokine expression in a variety of peripheral tissues such as hematopoietic, epithelial, and connective (9–11). Serum LIF levels rise progressively during lethal endotoxemia shock in mice, and prior administration of LIF protects against lethality in a doseand time-dependent manner (11). We recently demonstrated pituitary LIF gene expression in human fetal (12) and adult pituitary tissue (13) and in murine corticotrophs (12). LIF potently enhances CRH induction of POMC transcription, induces ACTH secretion in vitro (14) and in vivo (15), and is essential for psychological stressactivation of the hypothalamic pituitary adrenal (HPA) axis (15). LIF and LIF receptor are constitutively expressed in the normal murine hypothalamus and pituitary and are induced in response to lipopolysaccharide endotoxin (LPS) injection (16). Taken together, these data indicate that LIF is an inducible hypothalamo-pituitary proinflammatory cytokine that functions as either an autocrine or paracrine ACTH

Pituitary senescence: The evolving role of Pttg

Despite the high prevalence of pituitary adenomas they are invariably benign, indicative of unique intrinsic mechanisms controlling pituitary cell proliferation. Cellular senescence is characterized by a largely irreversible cell cycle arrest and constitutes a strong anti-proliferative response, which can be triggered by DNA damage, chromosomal instability and aneuploidy, loss of tumor suppressive signaling or oncogene activation. In vivo senescence is an important protective mechanism against cancer. Here we discuss prospective mechanisms underlying senescence-associated molecular pathways activated in benign pituitary adenomas. Both deletion and over-expression of pituitary tumor transforming gene (Pttg) promote chromosomal instability and aneuploidy. Pttg deletion abrogates tumor development by activating p53/p21-dependent senescence pathways. Abundant PTTG in GH-secreting pituitary adenomas also triggers p21-dependent senescence. Pituitary p21 may therefore safeguard against further chromosomal instability by constraining pituitary tumor growth. These observations point to senescence as a target for effective therapy for both tumor silencing and growth restraint towards development of pituitary malignancy.

Constitutive Somatostatin Receptor Activity Determines Tonic Pituitary Cell Response

Somatostatin (SRIF) binds G protein-coupled SRIF receptor subtypes (SST1, -2, -3, -4, and -5) to regulate cell secretion and proliferation. Hypothalamic SRIF inhibits pituitary growth hormone, thyroid stimulating hormone, and ACTH secretion. We tested SRIF-independent constitutive SST activity in AtT20 mouse pituitary corticotroph cells in which ACTH secretion is highly sensitive to SRIF action. Stable transfectants expressing SST2 or SST5 were sensitized to selective agonist action, and constitutive SST receptor activity was demonstrated by forskolin and pertussis toxin cAMP cell responses. Persistent constitutive SST activity decreased cell ACTH responses to CRH through decreased expression of CRH receptor subtype 1. Decreased dopamine receptor type 1 expression was associated with attenuated dopamine agonist action, whereas responses to isoproterenol were enhanced through increased beta2-adrenoreceptor expression. Thus, integrated pituitary cell ACTH regulation is determined both by phasic SRIF action, as well as by tonic constitutive SST activity, independently of SRIF.

Diminished Pancreatic β-Cell Mass in Securin-Null Mice Is Caused by β-Cell Apoptosis and Senescence

Pituitary tumor transforming gene (PTTG) encodes a securin protein critical in regulating chromosome separation. PTTG-null (PTTG(-/-)) mice exhibit pancreatic beta-cell hypoplasia and insulinopenic diabetes. We tested whether PTTG deletion causes beta-cell senescence, resulting in diminished beta-cell mass. We examined beta-cell mass, proliferation, apoptosis, neogenesis, cell size, and senescence in PTTG(-/-) and WT mice from embryo to young adulthood before diabetes is evident. The roles of cyclin-dependent kinase inhibitors and DNA damage in the pathogenesis of diabetes in PTTG(-/-) mice were also addressed. Relative beta-cell mass in PTTG(-/-) mice began to decrease at 2-3 wk, whereas beta-cell proliferation rate was initially normal but decreased in PTTG(-/-) mice beginning at 2 months. Apoptosis was also much more evident in PTTG(-/-) mice. At 1 month, beta-cell neogenesis was robust in wild-type mice but was absent in PTTG(-/-) mice. In addition, the size of beta-cells became larger and macronuclei were prominent in PTTG(-/-) animals. Senescence-associated beta-galactosidase was also active in PTTG(-/-) beta-cells at 1 month. Cyclin-dependent kinase inhibitor p21 was progressively up-regulated in PTTG(-/-) islets, and p21 deletion partially rescued PTTG(-/-) mice from development of diabetes. mRNA array showed that DNA damage-associated genes were activated in PTTG(-/-) islets. We conclude that beta-cell apoptosis and senescence contribute to the diminished beta-cell mass in PTTG(-/-) mice, likely secondary to DNA damage. Our results also suggest that ductal progenitor beta-cells are exhausted by excessive neogenesis induced by apoptosis in PTTG(-/-) mice.

Pituitary Tumour-Transforming Gene (PTTG) and Pituitary Senescence

Pituitary tumours account for 15% of intracranial neoplasms and are benign monoclonal neoplasms that may be clinically silent or secrete hormones, including prolactin, growth hormone, adrenocorticotrophic hormone or, rarely, thyroid-stimulating hormone or gonadotrophins. These adenomas account for clinical infertility, growth disorders and hypercortisolism or metabolic dysfunctions associated with hypopituitarism. We explored the role of disordered pituitary cell proliferation control in the pathogenesis of these invariably benign adenomas, studying the mechanisms underlying pituitary aneuploidy, premature proliferative arrest (senescence), markers of cell proliferation and tumorigenesis in single, double or triply mutant transgenic mice with mutations of Rb, Pttg and/or p21. Our results provide further insights into the role of cell-cycle control and growth constraints on experimental and human pituitary tumours, which underlie their failure to progress to malignancy. These results improve our understanding of pituitary syndromes associated with infertility, growth disorders, hypercortisolism or adrenal, thyroid and gonadal failure due to abrogated pituitary function.

Leukemia inhibitory factor signaling is implicated in embrionic development of the HPA axis.

Leukemia inhibitory factor (LIF) is expressed in the hypothalamic‐pituitary adrenal (HPA) axis and stimulates pituitary POMC transcription. The role of LIF receptor signaling in HPA axis development was examined. Lifr −/− and Lifr +/+ fetuses were obtained by Cesarean section on E18.5. Despite a 3‐fold induction of hypothalamic CRH mRNA, pituitary POMC mRNA and RU486‐induced ACTH levels were decreased in Lifr −/− mice. High CRH may be caused by increased central TNFα and IL‐6 expression. Lifr −/− mice demonstrate elevated pituitary glucocorticoid (GR) and mineralocorticoid (MR) receptor mRNA and protein levels, indicating the importance of LIF signaling for HPA axis development.

Increased leptin and white adipose tissue hypoplasia are sexually dimorphic in Lif null/Igf‐I haploinsufficient mice

We previously showed cooperation of leukemia inhibitory factor (LIF) and insulin‐like growth factor I (IGF‐I) during development. Mice doubly deficient in LIF and IGF‐I died at birth. We now analyze the possible combined influence of both factors on postnatal growth. The haploinsufficiency of the Igf‐I gene on a Lif null background caused a marked reduction in body mass index and white adipose tissue only in female mice. These animals had increased leptin, increased serum IGF‐I and apparent substitution of white adipose tissue by brown adipose tissue. The complex interrelationships between LIF and IGF‐I in regulating weight thus involve sexually dimorphic effects on adipose tissue differentiation and circulating leptin.