Eduardo Arzt

RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1α during Hypoxia

SUMO conjugation to proteins is involved in the regulation of diverse cellular functions. We have identified a protein, RWD-containing sumoylation enhancer (RSUME), that enhances overall SUMO-1, -2, and -3 conjugation by interacting with the SUMO conjugase Ubc9. RSUME increases noncovalent binding of SUMO-1 to Ubc9 and enhances Ubc9 thioester formation and SUMO polymerization. RSUME enhances the sumoylation of IkB in vitro and in cultured cells, leading to an inhibition of NF-kB transcriptional activity. RSUME is induced by hypoxia and enhances the sumoylation of HIF-1alpha, promoting its stabilization and transcriptional activity during hypoxia. Disruption of the RWD domain structure of RSUME demonstrates that this domain is critical for RSUME action. Together, these findings point to a central role of RSUME in the regulation of sumoylation and, hence, several critical regulatory pathways in mammalian cells.

Retinoic acid prevents experimental Cushing syndrome

Cushing syndrome is caused by an excess of adrenocorticotropic hormone (ACTH) production by neuroendocrine tumors, which subsequently results in chronic glucocorticoid excess. We found that retinoic acid inhibits the transcriptional activity of AP-1 and the orphan receptors Nur77 and Nurr1 in ACTH-secreting tumor cells. Retinoic acid treatment resulted in reduced pro-opiomelanocortin transcription and ACTH production. ACTH inhibition was also observed in human pituitary ACTH-secreting tumor cells and a small-cell lung cancer cell line, but not in normal cells. This correlated with the expression of the orphan receptor COUP-TFI, which was found in normal corticotrophs but not in pituitary Cushing tumors. COUP-TFI expression in ACTH-secreting tumor cells blocked retinoic acid action. Retinoic acid also inhibited cell proliferation and, after prolonged treatment, increased caspase-3 activity and induced cell death in ACTH-secreting cells. In adrenal cortex cells, retinoic acid inhibited corticosterone production and cell proliferation. The antiproliferative action and the inhibition of ACTH and corticosterone produced by retinoic acid were confirmed in vivo in experimental ACTH-secreting tumors in nude mice. Thus, we conclude that the effects of retinoic acid combine in vivo to reverse the endocrine alterations and symptoms observed in experimental Cushing syndrome.

Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk.

Pituitary tumor development involves clonal expansion stimulated by hormones and growth factors/cytokines. Using mRNA differential display, we found that the bone morphogenetic protein (BMP) inhibitor noggin is down-regulated in prolactinomas from dopamine D2-receptor-deficient mice. BMP-4 is overexpressed in prolactinomas taken from dopamine D2-receptor-deficient female mice, but expression of the highly homologous BMP-2 does not differ in normal pituitary tissue and prolactinomas. BMP-4 is overexpressed in other prolactinoma models, including estradiol-induced rat prolactinomas and human prolactinomas, compared with normal tissue and other pituitary adenoma types (Western blot analysis of 48 tumors). BMP-4 stimulates, and noggin blocks, cell proliferation and the expression of c-Myc in human prolactinomas, whereas BMP-4 has no action in other human pituitary tumors. GH3 cells stably transfected with a dominant negative of Smad4 (Smad4dn; a BMP signal cotransducer) or noggin have reduced tumorigenicity in nude mice. Tumor growth recovered in vivo when the Smad4dn expression was lost, proving that BMP-4/Smad4 are involved in tumor development in vivo. BMP-4 and estrogens act through overlapping intracellular signaling mechanisms on GH3 cell proliferation and c-myc expression: they had additive effects at low concentrations but not at saturating doses, and their action was inhibited by blocking either pathway with the reciprocal antagonist (i.e., BMP-4 with ICI 182780 or 17β-estradiol with Smad4dn). Furthermore, coimmunoprecipitation studies demonstrate that under BMP-4 stimulation Smad4 and Smad1 physically interact with the estrogen receptor. This previously undescribed prolactinoma pathogenesis mechanism may participate in tumorigenicity in other cells where estrogens and the type β transforming growth factor family have important roles.

Prenatal immune challenge alters the hypothalamic-pituitary-adrenocortical axis in adult rats.

We investigated whether non-abortive maternal infections would compromise fetal brain development and alter hypothalamic-pituitary-adrenocortical (HPA) axis functioning when adult. To study putative teratogenic effects of a T cell-mediated immune response versus an endotoxic challenge, 10-d-pregnant rats received a single intraperitoneal injection of 5 x 10(8) human red blood cells (HRBC) or gram-negative bacterial endotoxin (Escherichia coli LPS: 30 micrograms/kg). The adult male progeny (3 mo old) of both experimental groups showed increased basal plasma corticosterone levels. In addition, after novelty stress the HRBC group, but not the LPS group, showed increased ACTH and corticosterone levels. Both groups showed substantial decreases in mineralocorticoid (MR) and glucocorticoid receptor (GR) levels in the hippocampus, a limbic brain structure critical for HPA axis regulation, whereas GR concentrations in the hypothalamus were unchanged and in anterior pituitary were slightly increased. HRBC and LPS indeed stimulated the maternal immune system as revealed by specific anti-HRBC antibody production and enhanced IL-1 beta mRNA expression in splenocytes, respectively. This study demonstrates that a T cell-mediated immune response as well as an endotoxic challenge during pregnancy can induce anomalies in HPA axis function in adulthood. Clinically, it may be postulated that disturbed fetal brain development due to prenatal immune challenge increases the vulnerability to develop mental illness involving inadequate responses to stress.

PATHOPHYSIOLOGICAL ROLE OF THE CYTOKINE NETWORK IN THE ANTERIOR PITUITARY GLAND

Recent evidence has demonstrated that cytokines and other growth factors act in the anterior pituitary gland. Using the traditional criteria employed to determine autocrine or paracrine functions our review shows that, in addition to their role as lymphocyte messengers, certain cytokines are autocrine or paracrine regulators of anterior pituitary function and growth. The cytokines known to regulate and/or be expressed in the anterior pituitary include the inflammatory cytokine family (IL-1 and its endogenous antagonist, IL-1ra; TNF-alpha, and IL-6), the Th1-cytokines (IL-2 and IFN-gamma), and other cytokines such as LIF, MIF, and TGF-beta. This review examines at the cellular, molecular, and physiological levels whether: (1) each cytokine alters some aspect of pituitary physiology; (2) receptors for the cytokine are expressed in the gland; and (3) the cytokine is produced in the anterior pituitary. Should physiological stimuli regulate pituitary cytokine production, this would constitute additional proof of their autocrine/paracrine role. In this context, we analyze in this review the current literature on the actions of cytokines known to regulate anterior pituitary hormone secretion, selecting the in vivo studies that support the direct action of the cytokine in the anterior pituitary. Further support for direct regulatory action is provided by in vitro studies, in explant cultures or pituitary cell lines. The cytokine receptors that have been demonstrated in the pituitary of several species are also discussed. The endogenous production of the homologous cytokines and the regulation of this expression are analyzed. The evidence indicating that cytokines also regulate the growth and proliferation of pituitary cells is reviewed. This action is particularly important since it suggests that intrinsically produced cytokines may play a role in the pathogenesis of pituitary adenomas. The complex cell to cell communication involved in the action of these factors is discussed.

Interleukin-2 and interleukin-2 receptor expression in human corticotrophic adenoma and murine pituitary cell cultures.

The production of IL-1 and IL-6 by pituitary cells has recently been demonstrated. In this study we investigated the expression of IL-2 and its receptor (IL-2R) by pituitary cells of different species. In Northern blots, a single hybridizing band of 1 kb, identical to that in normal stimulated lymphocytes, was obtained with specific IL-2 probes. In the mouse AT-20 pituitary tumor cell line, IL-2 mRNA expression was detected after stimulation with corticotropin-releasing hormone or phorbol myristate acetate. In human corticotrophic adenoma cells, basal IL-2 mRNA expression as well as IL-2 secretion were further stimulated by phorbol myristate acetate. Both adenoma and AtT-20 cells showed detectable amounts of IL-2R mRNA and by immunofluorescence, IL-2R membrane expression. In addition, dual immunofluorescence studies in rat anterior pituitary cells demonstrated colocalization of IL-2R with ACTH-positive cells and other cell types expressing the receptor. In addition to the action of lymphocyte-produced IL-2, this cytokine may have a paracrine or autocrine regulatory role within the pituitary. It remains to be established whether IL-2 production occurs in the normal pituitary or is intrinsic to the process of tumor development of these cells. IL-2 may be involved in the growth control of pituitary cells.

Lipopolysaccharide Directly Stimulates the Intrapituitary Interleukin-6 Production by Folliculostellate Cells via Specific Receptors and the p38α Mitogen-Activated Protein Kinase/Nuclear Factor-κB Pathway1

Bacterial lipopolysaccharide (LPS) activates the immune system and induces increases in peripheral cytokines, which, in turn, affect the endocrine system. In particular, LPS-induced cytokines stimulate the hypothalamic-pituitary-adrenal axis to increase levels of antiinflammatory-acting glucocorticoids. In the present work, we show that LPS directly stimulates interleukin (IL)-6 release by mouse pituitary folliculostellate (FS) TtT/GF tumor cells and FS cells of mouse pituitary cell cultures. The stimulatory effect of LPS was strongly enhanced in the presence of serum, suggesting that LPS is only fully active as a complex with LPS-binding protein (LBP). Both TtT/GF cells and mouse pituitaries expressed CD14, which binds the LPS/LBP complex, and Toll-like receptor type 4, which induces LPS signals. LPS increased phospoinositol turnover in TtT/GF cells and induced phosphorylation of p38α mitogen-activated protein kinase and the inhibitor (IκB) of nuclear factor-κ B. Nuclear factor-κ B was activated by LPS i...

The Corticotropin-Releasing Hormone Network and the Hypothalamic-Pituitary-Adrenal Axis: Molecular and Cellular Mechanisms Involved

Corticotropin-releasing hormone (CRH) plays a key role in adjusting the basal and stress-activated hypothalamic-pituitary-adrenal axis (HPA). CRH is also widely distributed in extrahypothalamic circuits, where it acts as a neuroregulator to integrate the complex neuroendocrine, autonomic, and behavioral adaptive response to stress. Hyperactive and/or dysregulated CRH circuits are involved in neuroendocrinological disturbances and stress-related mood disorders such as anxiety and depression. This review describes the main physiological features of the CRH network and summarizes recent relevant information concerning the molecular mechanism of CRH action obtained from signal transduction studies using cells and wild-type and transgenic mice lines. Special focus is placed on the MAPK signaling pathways triggered by CRH through the CRH receptor 1 that plays an essential role in CRH action in pituitary corticotrophs and in specific brain structures. Recent findings underpin the concept of specific CRH-signaling pathways restricted to specific anatomical areas. Understanding CRH action at molecular levels will not only provide insight into the precise CRH mechanism of action, but will also be instrumental in identifying novel targets for pharmacological intervention in neuroendocrine tissues and specific brain areas involved in CRH-related disorders.

Molecular mechanisms and Th1/Th2 pathways in corticosteroid regulation of cytokine production

We focus on how the IL-1 system, T-helper1 (Th1) or Th2 cytokines and glucocorticoids, converge to give a unified physiological response. Glucocorticoids inhibit IL-1 and IL-1 receptor antagonist (IL-1ra) expression, Th1 cytokines stimulate both and Th2 cytokines stimulate IL-1ra and inhibit IL-1. Thus, during the Th1 response there is a window for IL-1 inflammatory activity, absent during the Th2 response. We also study the interactions among glucocorticoid and cytokine transcriptional activity. Glucocorticoids inhibit cytokine-induced transcription factors (NFkB, AP1) and cytokines enhance glucocorticoid receptor (GR) transcriptional activity, thus reciprocally fine tuning immunological control mechanisms.

Corticotropin-releasing hormone activates ERK1/2 MAPK in specific brain areas.

Corticotropin-releasing hormone (CRH) coordinates hormonal and behavioral responses to stress. The mitogen-activated protein kinase extracellular signal-related kinase 1/2 (ERK1/2) mediates several functions in different forebrain structures and recently has been implicated in CRH signaling in cultured cells. To study in vivo CRH-mediated activation of central ERK1/2, we investigated the expression pattern of the phosphorylated ERK1/2(p-ERK1/2) in the mouse brain after intracerebroventricular CRH injections. As shown by immunohistochemistry and confocal microscopy analysis, CRH administration increased p-ERK1/2 levels specifically in the CA3 and CA1 hippocampal subfields and basolateral complex of the amygdala, both structures related to external environmental information processing and behavioral aspects of stress. Other regions such as hypothalamic nuclei and the central nucleus of the amygdala, also related to central CRH system but involved in the processing of the ascending visceral information and neuroendocrine-autonomic response to stress, did not show CRH-mediated ERK1/2 activation. To dissect the involvement of CRH receptor 1 (CRHR1) and CRHR2, we used conditional knockout mice in which Crhr1 is inactivated in the anterior forebrain and limbic structures. The conditional genetic ablation of Crhr1 inhibited the p-ERK1/2 increase, underlining the involvement of CRHR1 in the CRH-mediated activation. These findings underscore the fact that CRH activates p-ERK1/2 through CRHR1 only in selected brain regions, pointing to a specific role of this pathway in mediating behavioral adaptation to stress.