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 factorscytokines. 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-4Smad4 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 17beta-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 beta transforming growth factor family have important roles.     

Reversal of endogenous dopamine receptor silencing in pituitary cells augments receptor-mediated apoptosis

Dopamine (DA)-agonist targeting of the DA D(2) receptor (D2R) in prolactinomas is the first-line treatment choice for suppression of prolactin and induction of tumor shrinkage. Resistance to DA agonists seems to be related to receptor number. Using the MMQ and GH3 pituitary cell lines, that either do or do not express D2R, respectively, we explored the epigenetic profile associated with the presence or absence of D2R in these cells lines. These studies led us to explore pharmacological strategies designed to restore receptor expression and thereby potentially augment DA agonist-mediated apoptosis. We show in GH3 cells that the D2R harbors increased CpG island-associated methylation and enrichment for histone H3K27me3. Conversely, MMQ cells and normal pituitaries show enrichment for H3K9Ac and barely detectable H3K27me3. Coculture of GH3 cells with the demethylating agent zebularine and the histone deacetylase inhibitor trichostatin A was responsible for a decrease in CpG island methylation and enrichment for the histone H3K9Ac mark. In addition, challenge of GH3 cells with zebularine alone or coculture with both agents led to expression of endogenous D2R in these cells. Induced expression D2R in GH3 cells was associated with a significant increase in apoptosis indices to challenge with either DA or bromocriptine. Specificity of a receptor-mediated response was established in coincubations with specific D2R antagonist and siRNA approaches in GH3 cell and D2R expressing MMQ cell lines. These studies point to the potential efficacy of combined treatment with epigenetic drugs and DA agonists for the medical management of different pituitary tumor subtypes, resistant to conventional therapies.     

Somatostatin receptor subtypes mRNA in TSH-secreting pituitary adenomas: a case showing a dramatic reduction in tumor size during short octreotide treatment

TSH-secreting adenoma is a rare pituitary adenoma, and the expression levels of the specific subtypes of somatostatin receptors (sstr) mRNAs have remained obscure. To determine the quantitative expression of the sstr1-5 mRNAs in TSH-secreting adenomas that may be related to the efficacy of treatment with a somatostatin analogue, expression of the sstr1-5 mRNAs was examined and compared in TSH-secreting adenomas and other pituitary adenomas. The pituitary adenomas were obtained at transsphenoidal surgery from 4 cases of TSH-secreting adenoma, including 1 patient showing a significant shrinkage of the tumor size after only 10 days of octreotide treatment, 2 patients without tumor size reduction and 1 patient without treatment, and 5 GH-secreting adenomas, 6 prolactinomas, 5 nonfunctioning adenomas, 4 ACTH-secreting adenomas and normal pituitaries at autopsy from 4 normal subjects. In comparison to the normal pituitary, sstr2A>sstr1>sstr5>sstr3 mRNAs were expressed in the TSH-secreting adenomas examined. No expression of sstr2B or sstr4 mRNA was observed. The expression level of sstr2 mRNA was significantly higher than those in normal pituitary, prolactinomas, ACTH-secreting and nonfunctioning pituitary adenomas. The patient with marked shrinkage of the tumor showed the highest expression of both sstr2 and sstr5 mRNAs among all the cases of pituitary adenoma. A TSH-secreting tumor without shrinkage showed a similar expression level of sstr2 mRNA. These findings demonstrated that TSH-secreting adenomas express sstr1, 2A, 3 and 5 mRNAs, predominantly sstr2A, and in addition to the expression of sstr2 mRNA, the expression level of sstr5 mRNA may be a factor affecting the tumor shrinkage by somatostatin analogues against TSH-secreting adenomas.     

17Beta-hydroxysteroid dehydrogenase type 1, 2, 3, and 4 expression and enzyme activity in human anterior pituitary adenomas

17Beta-hydroxysteroid dehydrogenase (17betaHSD) isoforms reversibly catalyze the final step in the formation of estradiol (E2) from estrone (E1) and the formation of testosterone from androstenedione. We have investigated 17betaHSD type 1, 2, 3, and 4 gene expression and 17betaHSD estrogenic activity in human anterior pituitary adenomas. 17BetaHSD messenger ribonucleic acid (mRNA) expression was studied by RT-PCR in 42 pituitary tumors and 3 normal pituitaries, 17betaHSD activity was studied in 11 tumors and 17betaHSD type 1 was immunolocalized in vitro in 6 tumors. 17BetaHSD type 1 gene expression was detected in 34 of 42 adenomas in all tumor subtypes; 17betaHSD type 2 mRNA was detected in 18 of 42 adenomas, but not in prolactinomas; 17betaHSD type 3 mRNA was detected in 12 of 42 adenomas, but not in corticotropinomas; 17betaHSD type 4 was expressed in 20 of 42 adenomas by all adenoma subtypes. Reversible 17betaHSD activity was found in 9 of 11 adenomas, and 17betaHSD type 1 immunopositivity was cytoplasmically distributed in all 6 adenomas in vitro. All 4 17betaHSD isoforms are variably expressed in human anterior pituitary adenomas, which also show 17betaHSD enzyme activity, suggesting that 17betaHSD may play an important role in regulating the local cellular levels of estradiol.     

The release of leptin and its effect on hormone release from human pituitary adenomas

BACKGROUND: Leptin is the protein product of the obese gene, known to play an important role in body energy balance. The leptin receptor exists in numerous isoforms, the long isoform being the major form involved in signal transduction. Leptin expression has recently been demonstrated in the human pituitary, both in normal tissue and in pituitary adenomas. The long isoform of the leptin receptor has also been shown to be present in pituitary adenomas; however, contrasting results have been obtained regarding its expression in the normal human pituitary. AIM: The aim of this study was (i) to investigate the presence and pattern of distribution of leptin mRNA and the long isoform of its receptor mRNA in the normal pituitary and in different types of pituitary adenomas with RT-PCR; (ii) to study leptin secretion from human pituitary tumours in culture and (iii) to assess in vitro pituitary hormone release following stimulation with human leptin. RESULTS: Leptin receptor long isoform expression was detected in 2/4 GH-secreting adenomas, 12/17 non-functioning adenomas, 5/9 ACTH-secreting adenomas, 1/2 prolactinomas, 2/2 FSH-secreting adenomas and 5/5 normal pituitaries. The receptor long isoform did not segregate with any particular tumour type, and varying levels of expression were detected between the tissues studied. Leptin mRNA was detected at a low level of expression in 2/7 GH-secreting adenomas, 9/14 non-functioning adenomas, 2/3 ACTH-secreting adenomas, 1/3 prolactinomas and 1/3 FSH-secreting adenomas. We were unable to detect leptin mRNA in any of the five normal pituitaries removed at autopsy; however, immunostaining of a non-tumorous pituitary adjacent to an adenoma removed at transsphenoidal surgery showed scattered leptin positive cells. Culture of pituitary adenomas showed that 16/47 released leptin into the incubation media. Leptin release did not correlate with tumour type or with any of the other pituitary hormones released. In vitro leptin stimulation of pituitary tumours caused stimulation of FSH and alpha-subunit secretion from a non-functioning adenoma and TSH secretion from a somatotroph adenoma. CONCLUSION: We conclude that not only is leptin stored within the pituitary, but it may also be released from pituitary cells and modulate other pituitary hormone secretion. Pituitary leptin may therefore be a novel paracrine regulator of pituitary function.     

Epigenetic change in pituitary tumorigenesis

Throughout the genome CpG dinucleotides are found at one-fifth of their expected frequency and their rarity is further marked by the fact that 70% are methylated. In contrast, CpG islands (CGI), found associated with the promoters of many genes, have maintained their expected frequency of this dinucleotide, and remain unmethylated. Inappropriate methylation of CGIs is associated with histone deacetylation and gene silencing, while methylation of CpGs outside of CGIs is associated with significantly higher mutation rates. Methylation of CGIs is a frequent event in numerous tumour types including those that arise within the pituitary gland. Several studies now show highly frequent methylation of the p16 gene that is significantly associated with loss of cognate protein and that appears to be an early change in pituitary tumorigenesis. Collectively, studies show that somatotrophinomas are an infrequent target for p16 CGI methylation. However, in this pituitary tumour subtype, loss of pRb is associated with either CGI methylation or micro-deletion within the protein-pocket binding domain. As in other tumour types loss of p16 or RB1 appear to be mutually exclusive events in non-functional adenomas and somatotrophinomas respectively. Investigation of the Death Associated Protein Kinase gene shows that loss of its protein (DAPK), a pro-apoptotic molecule, in pituitary tumours is also associated with either methylation or deletion within its associated CGI. In the case of DAPK, however, these changes segregate with invasive pituitary tumours irrespective of tumour subtype. Methylation represents a positive signal that can be detected with exquisite sensitivity; in addition, this change targets multiple genes that show tumour type specificity. Taken together, the detection of DNA methylation changes, using either a panel of predefined marker-islands, or CGI arrays, provides the opportunity to generate "methylation profiles". This new knowledge will increase our understanding of tumour biology and could ultimately aid medical management in these different tumour types, including those of pituitary origin.     

CpG island methylation as an early event during adenoma progression in carcinogenesis of sporadic colorectal cancer

BACKGROUND AND AIMS: CpG island methylation is present in various tumors, including colorectal carcinomas, and is thought to be an important mechanism in carcinogenesis. We evaluated the methylation status of primary colorectal tumors to determine its role in the adenoma to carcinoma sequence. METHODS: The methylation status of APC, THBS1, MGMT, hMLH1 and GSTP1, as determined by methylation specific PCR (MSP), and microsatellite instability (MSI) using three mononucleotide markers were assessed in 40 colorectal adenomas and 36 adenocarcinomas. The correlations of methylation status and MSI with the clinicopathologic parameters of the tumors were determined. RESULTS: Of the 40 adenomas, 24 (60%) were methylated at one or more loci, and 12 (30%) at two or more loci (CpG island methylation phenotype-high, CIMP-H). Of 36 carcinomas, 27 (75%) were methylated at one or more loci and 11 (30.5%) at two or more loci (CIMP-H). THBSI was the most frequently methylated locus in both adenomas (n = 19, 47.5%) and carcinomas (n = 16, 44.4%). Overall, methylation status of adenomas and carcinomas did not differ significantly (P = 0.53), nor did the methylation status of individual genes. For adenomas, size (P = 0.049) and histologic classification of the villous components (P = 0.018) were each associated with methylation status. For carcinomas, however, no clinicopathologic variable was related to methylation status. MSI was detected in three adenomas (7.5%) and five carcinomas (13.9%), and was closely correlated with hMLH1 methylation (P < 0.001). CONCLUSIONS: In colorectal tumors, CpG island methylation of tumor suppressor genes appears to be common and may be involved in the progression of adenomas.     

Expression of the transferrin receptor in human anterior pituitary adenomas is confined to gonadotrophinomas

OBJECTIVE  The human pituitary gland is affected selectively by conditions associated with iron deposition, but the mechanisms for this are unknown. In this study we have determined whether the transferrin receptor, which mediates iron uptake by cells, could be detected immunocytochemically in human pituitary adenomas in vitro .
PATIENTS  Data were derived from 35 patients undergoing transsphenoidal surgery and included 13 with clinically non-functioning adenomas, 15 with acromegaly, 4 prolactinomas, 2 patients with Cushing's disease and one patient with Nelson's syndrome.
MEASUREMENTS  Transferrin receptor immunopositivity was determined for each adenoma in dispersed cell culture using a specific monoclonal antibody.
RESULTS  Eight of 13 clinically functionless adenomas showed immunopositive transferrin receptor expression, whilst adenomas from 15 patients with acromegaly, 4 prolactinomas, 2 Cushing's syndrome and one patient with Nelson's syndrome were negative. The eight transferrin receptor positive tumours were gonadotrophinomas and accounted for eight of the nine tumours which secreted and immunostained for FSH; all eight also secreted and immunostained for LH.
CONCLUSIONS  These findings may reflect a special requirement for iron by gonadotrophin secreting cells in comparison to other pituitary cell types and this could underlie the reasons why in the normal pituitary these cells are especially susceptible to malfunction in iron overload syndromes such as genetic haemochromatosis and β-thalassaemia.     

Expression of somatostatin receptors on human pituitary adenomas in vivo and ex vivo.

The distribution and biologic activity of somatostatin receptor subtypes (SSTR) in pituitary adenomas is not clarified, especially regarding clinically non-functioning adenomas (NFPA). We therefore characterized SSTR in human pituitary adenomas by combining molecular biology and in vivo scintigraphy. Co-expression of gonadotropin-releasing hormone receptor (GnRH-R) mRNA was also assessed to see whether this feature was associated with adenoma subtype and SSTR status. Pituitary tumor biopsies were obtained during transsphenoidal adenomectomy from 21 patients (11 NFPA, 7 acromegalics, 2 prolactinomas, 1 Cushing's disease). Expression of mRNA encoding the 5 known SSTR subtypes and the GnRH-R was determined by RT-PCR. Twelve patients also underwent a pre-operative somatostatin receptor scintigraphy. Most adenomas (no.=18) expressed mRNA for more than one SSTR. SSTR2 mRNA was expressed in 18 cases, whereas SSTR4 was absent in all but one. SSTR3 was frequently expressed in NFPAs. Somatostatin receptor scintigraphy was positive in most cases, and with a significantly higher uptake index in GH-producing adenomas all of which expressed SSTR2 mRNA. The uptake index appeared to be related to receptor density rather than tumor volume. Expression of GnRH-R mRNA was found in both NFPAs and GH-producing adenomas and was not significantly associated with a particular SSTR subtype population. In conclusion: 1) the distribution of SSTR is not significantly different between NFPA and GH-producing adenomas; and 2) somatostatin receptor scintigraphy reveals a higher uptake in GH-producing adenomas which is not significantly related to either SSTR distribution or tumor volume.     

EMs患者在位子宫内膜hMLH1蛋白表达变化及其基因启动子CpG岛甲基化观察

目的观察子宫内膜异位症（EM s）患者在位子宫内膜组织中hMLH1蛋白的表达变化及hMLH1基因启动子CpG岛甲基化情况。方法采用免疫组化法分别检测52例（Ⅰ、Ⅱ、Ⅲ、Ⅳ期分别为9、16、12、15例）EM s患者及30例健康志原者（对照组）在位子宫内膜中的hMLH1,采用甲基化特异性聚合酶链反应（MSP）法检测两组在位子宫内膜hMLH1启动子CpG岛甲基化情况。分离hMLH1启动子CpG岛甲基化异常者在位内膜中的腺细胞和间质细胞,采用MSP志愿分别检测两种细胞hMLH1启动子CpG岛甲基化情况。结果 EM s组中hMLH1蛋白表达减弱共6例,其中Ⅰ、Ⅱ、Ⅲ、Ⅳ期分别为0、0、1、5例,其余样本hMLH1蛋白表达均呈阳性;对照组30例无hMLH1蛋白表达减弱者,hMLH1蛋白表达均表现为阳性。EM s组中hMLH1启动子CpG岛部分甲基化3例,其中Ⅰ、Ⅱ、Ⅲ、Ⅳ期分别为0、0、0、3例,余均表现为非甲基化;对照组30例均表现为hMLH1启动子CpG岛非甲基化。EM s组Ⅳ期患者与Ⅰ、Ⅱ、Ⅲ期患者及对照组相比,P均〈0.05。3例hMLH1启动子CpG岛表现为部分甲基化者子宫内膜组织中hMLH1蛋白表达均减弱,其腺细胞中hMLH1启动子区表现为半甲基化,间质细胞hMLH1启动子区未见明显甲基化条带。结论 EM s患者在位子宫内膜组织中存在hMLH1表达减弱及hMLH1启动子CpG岛部分甲基化,主要集中在腺细胞,hMLH1异常与严重EM s的发病有关。     

Runx3基因甲基化及Runx3蛋白表达在结肠癌发生中的意义

目的:评价Runx3 基因启动子区CpG 岛甲基化状态及Runx3 蛋白表达在结肠癌发生中的作用及临床意义.方法:应用DNA 甲基化特异性PCR(MSP) 技术检测结肠癌、结肠腺瘤、正常结肠黏膜Runx3 基因CpG 岛甲基化状态,应用免疫组织化学发法检测Runx3 蛋白的表达.结果:MSP 方法检测发现Runx3 基...     

Should aip gene screening be recommended in family members of FIPA patients with R16H variant?

Germline mutations of aryl-hydrocarbon-receptor interacting protein (AIP) are associated with pituitary adenoma predisposition. They occur in 20 % of familial isolated pituitary adenoma (FIPA) and in about 3–5 % of sporadic pituitary adenomas, especially in early onset somatotropinomas and prolactinomas. Our aim was to evaluate the clinical and genetic features of a large Italian FIPA family, where an AIP variant was identified. AIP direct sequencing from genomic DNA was carried out in 16 available family members. AIP R16H carriers also underwent magnetic resonance imaging and hormonal assessments. AIP mutations were also searched in 16 patients with sporadic growth hormone-secreting pituitary adenoma and in 6 unrelated patients in whom pituitary adenoma was excluded. We found an AIP R16H variation in two family members harbouring a pituitary adenoma and in 6 unaffected family members. No AIP mutation was found neither in growth hormone-secreting pituitary adenoma patients, nor in the unrelated patients without pituitary adenoma. We report a FIPA family harbouring an AIP R16H change, supporting the hypothesis that the latter represents a variant of unknown significance.