Ursula Hopfner

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.

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...

Expression and localization of endothelin-1 and endothelin receptors in human meningiomas. Evidence for a role in tumoral growth.

In addition to its well-known homoeostatic actions in the cardiovascular system, ET-1 has been shown to constitute a potent growth regulatory peptide in various tissues. We have studied the expression of ET-1 and its receptors (ET-Ar and ET-Br) in human meningiomas (n = 35) as well as their involvement in cellular growth. By PCR of reverse-transcribed RNA we detected ET-1 mRNA in 91% (32 of 35), ET-Ar mRNA in 82% (29 of 35), and ET-Br mRNA in 42% (15 of 35) of human meningiomas examined. The localization of ET-1 mRNA, ET-Ar mRNA, and ET-1 peptide in tumoral cells was observed by in situ hybridization and immunohistochemistry, whereas ET-Br mRNA was expressed at low level only in cells belonging to blood vessels. In addition, we found that ET-1 stimulated [3H] thymidine incorporation in primary cell cultures of 20 meningiomas and that this effect could be blocked by BQ-123, a specific antagonist for ET-Ar. In contrast, RES-701-3, an antagonist of ET-Br, did not block the proliferative effect of ET-1. In conclusion, our data provide evidence that ET-1 constitutes an important growth factor for meningiomas acting via ET-Ar. We can hypothesize that ET-1, acting in concert with other growth factors and cytokines, is involved in the meningioma tumorigenesis.

Vascular Endothelial Growth Factor Production and Regulation in Rodent and Human Pituitary Tumor Cells in vitro

Angiogenesis, the formation of a new blood supply, is an essential step in tumorigenesis. Although vascular endothelial growth factor (VEGF) is known to be a very potent angiogenic factor in most solid tumors, little is known about its production and regulation in pituitary adenomas. We have investigated basal and stimulated VEGF production by rodent pituitary tumor cells (mouse corticotrope AtT20, rat lactosomatotrope GH3, mouse gonadotrope αT3-1 and mouse folliculostellate TtT/GF cells), and by hormone-inactive (27), corticotrope (9), lactotrope (3) and somatotrope (21) human pituitary adenoma cell cultures. All 4 pituitary cell lines secreted VEGF, which in the case of AtT20, GH3 and TtT/GF cells was inhibited by approximately 50% by dexamethasone. TtT/GF cells were the most responsive to the different stimuli used since basal values were augmented by pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), interleukin-6 (IL-6), transforming growth factor-α (TGF-α), IGF-I and the somatostatin analogue ocreotide. However, in GH3, AtT20 and αT3-1 cells, basal VEGF levels where not enhanced with any of the stimuli tested. The majority of the human adenomas tested (92%) basally secreted measurable VEGF which was inhibited by dexamethasone in most cases (84%). VEGF levels were increased in hormone inactive adenomas, somatotrope tumors and prolactinomas by TGF-α, PACAP-38, and 17β-estradiol, respectively. In conclusion, pituitary tumor cells are capable of producing VEGF which may be involved in tumoral angiogenesis. Our results concerning the suppression of VEGF by dexamethasone suggest that glucocorticoids may have anti-angiogenic properties and therefore therapeutic relevance for the treatment of pituitary adenomas.

Extracellular matrix components regulate ACTH production and proliferation in corticotroph tumor cells.

The extracellular matrix (ECM) conveys signals through membrane receptors called integrins producing changes in cell morphology, proliferation, differentiation and apoptosis. Previous studies suggest that the ECM plays an important role in pituitary physiology and tumorigenesis. In the present work we studied for the first time the effects of fibronectin, laminin, collagen I and collagen IV on hormone secretion and cell proliferation in the corticotroph tumor cell line AtT-20 and in normal pituitary cells, examining the signal transduction mechanisms that mediate these effects. ACTH production in AtT-20 cells was inhibited by fibronectin, laminin and collagen I. A reporter construct with the POMC promoter showed similar results, indicating that the effects of the ECM take place at the level of POMC gene transcription. In contrast, ACTH production was not significantly altered in normal pituitary cells. AtT-20 cell proliferation was stimulated by collagen IV and fibronectin, but inhibited by collagen I and laminin. In parallel, the cell morphology was modified by the ECM. We found that the production of reactive oxygen species mediate the effects of laminin and collagen IV. On the other hand, the effect of fibronectin was mimicked by beta1-integrin and Rho activation. These results show for the first time that the ECM controls ACTH biosynthesis and proliferation in corticotroph tumor cells and suggest a role for the ECM in corticotroph adenoma development.

Laminin inhibits lactotroph proliferation and is reduced in early prolactinoma development

Laminin is a component of the extracellular matrix (ECM) that regulates cell proliferation and hormone secretion. Here we describe the effects of laminin on prolactin secretion in normal and tumor cells and analyze laminin expression pattern during prolactinoma development. Prolactin secretion and cell proliferation were inhibited by laminin in GH3 cells. In contrast, no effect was observed in normal pituitary cells. Laminin showed a dynamic expression pattern during prolactinoma development, which was: (a) strong in normal pituitaries from wild type or dopamine D2 receptor deficient mice, (b) lower in pituitary hyperplasia and (c) markedly reduced in prolactinomas from D2R -/- mice. A similar gradual decrease in laminin was found by comparing normal human pituitaries, human pituitary hyperplasia and human prolactinomas. These results show dynamic changes of laminin expression during prolactinoma formation which, due to laminin action on PRL production and cell proliferation, indicate a possible role for laminin in prolactinoma development.

Cellular localization of endothelin receptor mRNAs (ETA and ETB) in brain tumors and normal human brain

Summary: We performed in situ hybridization (ISH), using radioactively labeled deoxyoligonucleotides, to localize endothelin (ET) receptor subtype mRNAs (ETA and ETB) in a series of meningiomas in comparison to normal human meninges. Our examination also addressed the cerebellum adjacent to the meninges investigated. Tumor cells of all meningiomas examined showed strong hybridization signals for ETA receptor mRNA, whereas for ETB receptor mRNA only weak hybridization signals could be detected in endothelial cells of tumor blood vessels and in some cell clusters. mRNAs for neither ET receptor were detectable in normal leptomeninges. In human cerebellum we found ETA receptor mRNA only in large blood vessels; ETB receptor mRNA was intensely expressed in Bergmann glial cells. Neither subtype was detectable in neurons.

Retinoic acid stimulates meningioma cell adhesion to the extracellular matrix and inhibits invasion

SummaryMeningiomas are tumours derived from the arachnoid and pia mater. During embryogenesis, these membranes develop from the migrating craniofacial neural crest. We have previously demonstrated that meningiomas have characteristic features of embryonic meninges. Craniofacial neural crest derivatives are affected during normal development and migration by retinoic acid. We speculated, therefore, that meningioma cell migration and invasion would be affected in a similar way. In this study we investigated the mechanisms of invasion and migration in meningiomas and the effects of retinoic acid (RA). We found that low doses of RA inhibit in vitro invasion in meningioma cells, without affecting cell proliferation or viability. The matrix metalloproteinases MMP-2 (72 kDa gelatinase) and MMP-9 (92 kDa gelatinase), which play a key role in invasion in other tumours, are not affected by RA. RA inhibits cell migration on collagen I and fibronectin. A possible mechanism for these effects is provided by the fact that RA strongly stimulates adhesion of meningioma cells to extracellular matrix substrates. As in vitro invasion, migration and decreased adhesion to the extracellular matrix correlate with the clinical manifestation of tumour invasion, we conclude that RA induces a non-invasive phenotype in meningioma cells.