Symposium review: Defining a pathway to climate neutrality for US dairy cattle production.

The US dairy industry has made substantial gains in reducing the greenhouse gas emission intensity of a gallon of milk. At the same time, consumer and investor interest for improved environmental benefits or reduced environmental impact of food production continues to grow. Following a trend of increasing greenhouse gas emission commitments for businesses across sectors of the economy, the US dairy industry has committed to a goal of net zero greenhouse gas emissions by 2050. The Paris Climate Accord's goal is to reduce warming of the atmosphere to less than 1.5 to 2°C based on preindustrial levels, which is different from emission goals of historic climate agreements that focus on emission reduction targets. Most of the emissions that account for the greenhouse gas footprint of a gallon of milk are from the short-lived climate pollutant CH4, which has a half-life of approximately 10 yr. The relatively new accounting system Global Warming Potential Star and the unit CO2 warming equivalents gives the industry the appropriate metrics to quantify their current and projected warming impact on future emissions. Incorporating this metric into potential future emissions pathways can allow the industry to understand the magnitude of emissions reductions needed to no longer contribute additional warming. Deterministic modeling was performed across the dairy industry's emission areas of enteric fermentation, manure management, feed production, and other upstream emissions necessary for dairy production. By reducing farm-level absolute emissions by 23% based on current levels, there is the opportunity for the US dairy industry to realize climate neutrality within the next few decades.

Chronic prepartum light-dark phase shifts in cattle disrupt circadian clocks, decrease insulin sensitivity and mammary development, and are associated with lower milk yield through 60 days postpartum.

Circadian and metabolic systems are interlocked and reciprocally regulated. To determine if the circadian system regulates glucose homeostasis and mammary development, the function of the circadian system was disrupted by exposing cattle to chronic light-dark cycle phase shifts from 5 wk before expected calving (BEC) to parturition. Multiparous Holstein cows were exposed to 16 h of light and 8 h of dark (CON, n = 8) or phase shifting (PS, n = 8) the light cycle 6 h every 3 d beginning 35 d BEC. After calving, both treatments were exposed to CON lighting. Mammary biopsies were taken at 21 d BEC and 21 d in milk (DIM), and histological analysis indicated PS treatment decreased the ratio of lumen to alveolar area and percentage of proliferating epithelial cells in the prepartum period. Intravenous glucose tolerance test was performed at 14 d BEC and 7 DIM by administering 50% dextrose. Blood glucose, ß-hydroxybutyrate, insulin, and nonesterified fatty acids were consequently measured over 3 h. At 14 d BEC no treatment differences were observed in baseline glucose or insulin. Treatment had no effect on blood glucose or glucose area under the curve at 14 d BEC and 7 DIM. Insulin area under the curve was higher in PS versus CON at 14 d BEC and 7 DIM. The PS cows produced less milk than CON cows through 60 DIM (40.3 vs. 42.6 kg/d). Exposure to chronic light-dark PS in late gestation decreased mammary development and increased insulin resistance in periparturient cows, which may have caused subsequent lower milk yield.

Triiodothyronine regulates angiogenic growth factor and cytokine secretion by isolated human decidual cells in a cell-type specific and gestational age-dependent manner.

STUDY QUESTION: Does triiodothyronine (T3) regulate the secretion of angiogenic growth factors and cytokines by human decidual cells isolated from early pregnancy? SUMMARY ANSWER: T3 modulates the secretion of specific angiogenic growth factors and cytokines, with different regulatory patterns observed amongst various isolated subpopulations of human decidual cells and with a distinct change between the first and second trimesters of pregnancy. WHAT IS KNOWN ALREADY: Maternal thyroid dysfunction during early pregnancy is associated with complications of malplacentation including miscarriage and pre-eclampsia. T3 regulates the proliferation and apoptosis of fetal-derived trophoblasts, as well as promotes the invasive capability of extravillous trophoblasts (EVT). We hypothesize that T3 may also have a direct impact on human maternal-derived decidual cells, which are known to exert paracrine regulation upon trophoblast behaviour and vascular development at the uteroplacental interface. STUDY DESIGN, SIZE, DURATION: This laboratory-based study used human decidua from first (8-11 weeks; n = 18) and second (12-16 weeks; n = 12) trimester surgical terminations of apparently uncomplicated pregnancies. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary cultures of total decidual cells, and immunomagnetic bead-isolated populations of stromal-enriched (CD10+) and stromal-depleted (CD10-) cells, uterine natural killer cells (uNK cells; CD56+) and macrophages (CD14+) were assessed for thyroid hormone receptors and transporters by immunocytochemistry. Each cell population was treated with T3 (0, 1, 10, 100 nM) and assessments were made of cell viability (MTT assay) and angiogenic growth factor and cytokine secretion (immunomediated assay). The effect of decidual cell-conditioned media on EVT invasion through Matrigel(®) was evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: Immunocytochemistry showed the expression of thyroid hormone transporters (MCT8, MCT10) and receptors (TRα1, TRß1) required for thyroid hormone-responsiveness in uNK cells and macrophages from the first trimester. The viability of total decidual cells and the different cell isolates were unaffected by T3 so changes in cell numbers could not account for any observed effects. In the first trimester, T3 decreased VEGF-A secretion by total decidual cells (P < 0.05) and increased angiopoietin-2 secretion by stromal-depleted cells (P < 0.05) but in the second trimester total decidual cells showed only increased angiogenin secretion (P < 0.05). In the first trimester, T3 reduced IL-10 secretion by total decidual cells (P < 0.05), and reduced granulocyte macrophage colony stimulating factor (P < 0.01), IL-8 (P < 0.05), IL-10 (P < 0.01), IL-1ß (P < 0.05) and monocyte chemotactic protein -1 (P < 0.001) secretion by macrophages, but increased tumour necrosis factor-α secretion by stromal-depleted cells (P < 0.05) and increased IL-6 by uNK cells (P < 0.05). In contrast, in the second trimester T3 increased IL-10 secretion by total decidual cells (P < 0.01) but did not affect cytokine secretion by uNK cells and macrophages. Conditioned media from first trimester T3-treated total decidual cells and macrophages did not alter EVT invasion compared with untreated controls. Thus, treatment of decidual cells with T3 resulted in changes in both angiogenic growth factor and cytokine secretion in a cell type-specific and gestational age-dependent manner, with first trimester decidual macrophages being the most responsive to T3 treatment, but these changes in decidual cell secretome did not affect EVT invasion in vitro. LIMITATIONS, REASONS FOR CAUTION: Our results are based on in vitro findings and we cannot be certain if a similar response occurs in human pregnancy in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Optimal maternal thyroid hormone concentrations could play a critical role in maintaining a balanced inflammatory response in early pregnancy to prevent fetal immune rejection and promote normal placental development through the regulation of the secretion of critical cytokines and angiogenic growth factors by human decidual cells. Our data suggest that there is an ontogenically determined regulatory 'switch' in T3 responsiveness between the first and second trimesters, and support the notion that the timely and early correction of maternal thyroid dysfunction is critical in influencing pregnancy outcomes. STUDY FUNDING/COMPETING INTEREST(S): This study is funded by Wellbeing of Women (RG/1082/09 to S.Y.C., M.D.K., J.A.F., L.S.L., G.E.L.) and Action Medical Research - Henry Smith Charity (SP4335 to M.D.K., S.Y.C., L.S.L., J.A.F.). The authors have no conflicts of interest to disclose.

The pituitary tumor transforming gene in thyroid cancer.

The pituitary tumor transforming gene (PTTG) is a multifunctional proto-oncogene that is over-expressed in various tumors including thyroid carcinomas, where it is a prognostic indicator of tumor recurrence. PTTG has potent transforming capabilities in vitro and in vivo, and many studies have investigated the potential mechanisms by which PTTG contributes to tumorigenesis. As the human securin, PTTG is involved in critical mechanisms of cell cycle regulation, whereby aberrant expression induces aneuploidy. PTTG may further contribute to tumorigenesis through its role in DNA damage response pathways and via complex interactions with hormones and growth factors. Furthermore, PTTG over-expression negatively impacts upon the efficacy of radioiodine therapy in thyroid cancer, through repression of expression and function of the sodium iodide symporter. Given its various roles at all disease stages, PTTG appears to be an important oncogene in thyroid cancer. This review discusses the current knowledge of PTTG with particular focus on its role in thyroid cancer.

The expression of thyroid hormone transporters in the human fetal cerebral cortex during early development and in N-Tera-2 neurodifferentiation.

Associations of neurological impairment with mutations in the thyroid hormone (TH) transporter, MCT8, and with maternal hypothyroxinaemia, suggest that THs are crucial for human fetal brain development. It has been postulated that TH transporters regulate the cellular supply of THs within the fetal brain during development. This study describes the expression of TH transporters in the human fetal cerebral cortex (7­20 weeks gestation) and during retinoic acid induced neurodifferentiation of the human N-Tera-2 (NT2) cell line, in triiodothyronine (T3) replete and T3-depleted media. Compared with adult cortex, mRNAs encoding OATP1A2, OATP1C1, OATP3A1 variant 2, OATP4A1, LAT2 and CD98 were reduced in fetal cortex at different gestational ages, whilst mRNAs encoding MCT8, MCT10, OATP3A1 variant 1 and LAT1 were similar. From the early first trimester, immunohistochemistry localised MCT8 and MCT10 to the microvasculature and to undifferentiated CNS cells. With neurodifferentiation, NT2 cells demonstrated declining T3 uptake, accompanied by reduced expressions of MCT8, LAT1, CD98 and OATP4A1. T3 depletion significantly reduced MCT10 and LAT2 mRNA expression at specific time points during neurodifferentiation but there were no effects upon T3 uptake, neurodifferentiation marker expression or neurite lengths and branching. MCT8 repression also did not affect NT2 neurodifferentiation. In conclusion, many TH transporters are expressed in the human fetal cerebral cortex from the first trimester, which could regulate cellular TH supply during early development. However, human NT2 neurodifferentiation is not dependent upon T3 or MCT8 and there were no compensatory changes to promote T3 uptake in a T3-depleted environment.

PTTG and PBF repress the human sodium iodide symporter.

The ability of the thyroid to accumulate iodide provides the basis for radioiodine ablation of differentiated thyroid cancers and their metastases. Most thyroid tumours exhibit reduced iodide uptake, although the mechanisms accounting for this remain poorly understood. Pituitary tumour transforming gene (PTTG) is a proto-oncogene implicated in the pathogenesis of thyroid tumours. We now show that PTTG and its binding factor PBF repress expression of sodium iodide symporter (NIS) messenger RNA (mRNA), and inhibit iodide uptake. This process is mediated at least in part through fibroblast growth factor-2. In detailed studies of the NIS promoter in rat FRTL-5 cells, PTTG and PBF demonstrated specific inhibition of promoter activity via the human upstream enhancer element (hNUE). Within this approximately 1 kb element, a complex PAX8-upstream stimulating factor 1 (USF1) response element proved critical both to basal promoter activity and to PTTG and PBF repression of NIS. In particular, repression by PTTG was contingent upon the USF1, but not the PAX8, site. Finally, in human primary thyroid cells, PTTG and PBF similarly repressed the NIS promoter via hNUE. Taken together, our data suggest that the reported overexpression of PTTG and PBF in differentiated thyroid cancer has profound implications for activity of the NIS gene, and hence significantly impacts upon the efficacy of radioiodine treatment.

The emerging role of pituitary tumour transforming gene (PTTG) in endocrine tumourigenesis.

It is now 10 years since PTTG was first cloned and isolated. Perhaps the major story of the intervening decade of work performed by numerous groups around the world is the sheer multifunctionality ascribed to this gene. PTTG has been implicated in mechanisms of gene transactivation, cell transformation, angiogenesis, metabolism, apoptosis, DNA repair, genetic instability and mitotic control, both in endocrine and non-endocrine settings. In the current review, we cast a critical eye over a decade of PTTG research within the field of endocrine neoplasia.

Functional consequences of the first reported mutations of the proto-oncogene PTTG1IP/PBF.

Pituitary tumor-transforming gene 1-binding factor (PTTG1IP; PBF) is a multifunctional glycoprotein, which is overexpressed in a wide range of tumours, and significantly associated with poorer oncological outcomes, such as early tumour recurrence, distant metastasis, extramural vascular invasion and decreased disease-specific survival. PBF transforms NIH 3T3 fibroblasts and induces tumours in nude mice, while mice harbouring transgenic thyroidal PBF expression show hyperplasia and macrofollicular lesions. Our assumption that PBF becomes an oncogene purely through increased expression has been challenged by the recent report of mutations in PBF within the Catalogue of Somatic Mutations in Cancer (COSMIC) database. We therefore sought to determine whether the first 10 PBF missense substitutions in human cancer might be oncogenic. Anisomycin half-life studies revealed that most mutations were associated with reduced protein stability compared to wild-type (WT) PBF. Proliferation assays narrowed our interest to two mutational events which significantly altered cell turnover: C51R and R140W. C51R was mainly confined to the endoplasmic reticulum while R140W was apparent in the Golgi apparatus. Both C51R and R140W lost the capacity to induce cellular migration and significantly reduced cell invasion. Colony formation and soft agar assays demonstrated that, in contrast to WT PBF, both mutants were unable to elicit significant colony formation or anchorage-independent growth. However, C51R and R140W retained the ability to repress radioiodide uptake, a functional hallmark of PBF. Our data reveal new insight into PBF function and confirm that, rather than being oncogenic, mutations in PBF are likely to be passenger effects, with overexpression of PBF the more important aetiological event in human cancer.

Elevated PTTG and PBF predicts poor patient outcome and modulates DNA damage response genes in thyroid cancer.

The proto-oncogene PTTG and its binding partner PBF have been widely studied in multiple cancer types, particularly thyroid and colorectal, but their combined role in tumourigenesis is uncharacterised. Here, we show for the first time that together PTTG and PBF significantly modulate DNA damage response (DDR) genes, including p53 target genes, required to maintain genomic integrity in thyroid cells. Critically, DDR genes were extensively repressed in primary thyrocytes from a bitransgenic murine model (Bi-Tg) of thyroid-specific PBF and PTTG overexpression. Irradiation exposure to amplify p53 levels further induced significant repression of DDR genes in Bi-Tg thyrocytes (P=2.4 × 10-4) compared with either PBF- (P=1.5 × 10-3) or PTTG-expressing thyrocytes (P=NS). Consistent with this, genetic instability was greatest in Bi-Tg thyrocytes with a mean genetic instability (GI) index of 35.8±2.6%, as well as significant induction of gross chromosomal aberrations in thyroidal TPC-1 cells following overexpression of PBF and PTTG. We extended our findings to human thyroid cancer using TCGA data sets (n=322) and found striking correlations with PBF and PTTG expression in well-characterised DDR gene panel RNA-seq data. In addition, genetic associations and transient transfection identified PBF as a downstream target of the receptor tyrosine kinase-BRAF signalling pathway, emphasising a role for PBF as a novel component in a pathway well described to drive neoplastic growth. We also showed that overall survival (P=1.91 × 10-5) and disease-free survival (P=4.9 × 10-5) was poorer for TCGA patients with elevated tumoural PBF/PTTG expression and mutationally activated BRAF. Together our findings indicate that PBF and PTTG have a critical role in promoting thyroid cancer that is predictive of poorer patient outcome.

Pituitary tumor transforming gene (PTTG) stimulates thyroid cell proliferation via a vascular endothelial growth factor/kinase insert domain receptor/inhibitor of DNA binding-3 autocrine pathway.

CONTEXT: Vascular endothelial growth factor (VEGF) exerts its biological effects by binding to the tyrosine kinase receptors VEGF receptor type 1 (VEGFR1/Flt-1) and VEGFR2 (Flk-1/KDR). Kinase insert domain receptor (KDR) is the critical receptor controlling proliferation and migration of endothelial cells and has been shown to be expressed in some nonendothelial cells. We recently reported that the proangiogenic pituitary tumor transforming gene (PTTG) stimulates VEGF and up-regulates inhibitor of DNA binding-3 (ID3), an important gene in VEGF-dependent angiogenesis. OBJECTIVE: Our objective was to test whether VEGF, ID3, and KDR confer a PTTG-mediated effect on thyroid cell growth. DESIGN: Gene expression, MAPK stimulation, and cell proliferation were assessed in follicular thyroid cancer FTC133 cells. Gene expression and clinical associations were determined in 21 normal and 38 tumorous thyroid specimens (nine follicular and 29 papillary). RESULTS: ID3 correlated with VEGF mRNA expression in our series of thyroid cancers, which also showed up-regulated KDR mRNA. Stimulation of FTC133 cells with exogenous VEGF enhanced ID3 expression, which could be abrogated by the KDR-specific inhibitor ZM323881, suggesting that VEGF regulation of ID3 is KDR dependent. PTTG significantly correlated with KDR mRNA expression in our thyroid cancer cohort and up-regulated KDR and VEGF expression in FTC133 cells. Finally, cells transfected with PTTG demonstrated increased cell proliferation and phosphorylation of MAPK, which was abrogated by ZM323881. CONCLUSIONS: We report the presence of a VEGF/KDR/ID3-dependent autocrine pathway in FTC133 thyroid cells. By up-regulating both VEGF and KDR expression, we propose a novel PTTG-mediated proliferative pathway that may be critical to thyroid cancer growth and progression.

Pituitary tumor-transforming gene regulates multiple downstream angiogenic genes in thyroid cancer.

CONTEXT: Pituitary tumor-transforming gene (PTTG) is a multifunctional protein involved in several tumorigenic mechanisms, including angiogenesis. PTTG has been shown to promote angiogenesis, a key rate-limiting step in tumor progression, by up-regulation of fibroblast growth factor-2 and vascular endothelial growth factor. OBJECTIVE: To investigate whether PTTG regulates other angiogenic genes in thyroid cells, we performed angiogenesis-specific cDNA arrays after PTTG transfection. Two of the genes [inhibitor of DNA binding-3 (ID3) and thrombospondin-1 (TSP-1)] which showed differential expression in primary thyroid cells were validated in vitro and in vivo. RESULTS: TSP-1 showed a 2.5-fold reduction and ID3 showed a 3.5-fold induction in expression in response to PTTG overexpression in vitro. Conversely, suppression of PTTG with small interfering RNA was associated with a 2-fold induction of TSP-1 and a 2.2-fold reduction in ID3 expression. When we examined TSP-1 and ID3 expression in 34 differentiated thyroid cancers, ID3 was significantly increased in tumors compared with normal thyroid tissue. Furthermore, ID3 expression was significantly higher in follicular thyroid tumors than in papillary tumors. Although mean TSP-1 expression was not altered in cancers compared with normal thyroids, we observed a significant independent association between TSP-1 expression and early tumor recurrence, with recurrent tumors demonstrating 4.2-fold lower TSP-1 expression than normal thyroid tissues. CONCLUSION: We have identified ID3 and TSP-1 as two new downstream targets of PTTG in thyroid cancer. We propose that PTTG may promote angiogenesis by regulating the expression of multiple genes with both pro- and antiangiogenic properties and may thus be a key gene in triggering the angiogenic switch in thyroid tumorigenesis.

Monocarboxylate transporter 8 expression in the human placenta: the effects of severe intrauterine growth restriction.

Thyroid hormones (THs) are essential for normal fetal development, with even mild perturbation in maternal thyroid status in early pregnancy being associated with neurodevelopmental delay in children. Transplacental transfer of maternal THs is critical, with increasing evidence suggesting a role for 3,3',5-tri-iodothyronine (T3) in development and function of the placenta itself, as well as in development of the central nervous and other organ systems. Intrauterine growth restriction (IUGR) is associated with fetal hypothyroxinaemia, a factor that may contribute to neurodevelopmental delay. The recent description of monocarboxylate transporter 8 (MCT8) as a powerful and specific TH membrane transporter, and the association of MCT8 mutations with profound neurodevelopmental delay, led us to explore MCT8 expression in placenta. We describe the expression of MCT8 in normal human placenta throughout gestation, and in normal third-trimester placenta compared with that associated with IUGR using quantitative reverse transcriptase PCR. MCT8 mRNA was detected in placenta from early first trimester, with a significant increase with advancing gestation (P=0.007). In the early third trimester, MCT8 mRNA was increased in IUGR placenta compared with normal samples matched for gestational age (P<0.05), but there was no difference between IUGR and normal placenta in the late third trimester. Western immunoblotting findings in IUGR and normal placentae were in accord with mRNA data. MCT8 immunostaining was demonstrated in villous cytotrophoblast and syncytiotrophoblast as well as extravillous trophoblast cells from the first trimester onwards with increasingly widespread immunoreactivity seen with advancing gestation. In conclusion, expression of MCT8 in placenta from early gestation is compatible with an important role in TH transport during fetal development and a specific role in placental development. Altered expression in placenta associated with IUGR may reflect a compensatory mechanism attempting to increase T3 uptake by trophoblast cells.

The in vitro effects of triiodothyronine on epidermal growth factor-induced trophoblast function.

The development of the human placenta involves a complex process of tightly regulated proliferation and invasion by extravillous trophoblast into the uterine decidua. Inadequate placentation is a feature of intrauterine growth restriction and other gestational pathology. There is some evidence that T(3) plays a role in the regulation of these processes and that T(3) may act synergistically with epidermal growth factor (EGF). The aim of this study was to define the expression of thyroid hormone receptors in extravillous trophoblast, elucidate the effects of T(3) on both proliferation and differentiation of human trophoblast cells of varying origins, and define the potential interaction between EGF and T(3) on these processes. Using immunohistochemistry, specific thyroid hormone receptor isoforms were localized in extravillous trophoblast in first- and second-trimester placental bed biopsies, indicating potential sensitivity to T(3). In studies of human trophoblast-derived cell lines and primary cultures of cytotrophoblast cells in vitro, T(3) and EGF exerted an antiproliferative effect on an extravillous-like cell line (SGHPL-4) but stimulated proliferation in JEG-3 choriocarcinoma cells. EGF enhanced survival of nonproliferative term primary cytotrophoblast cells and significantly enhanced invasion of fibrin gels by SGHPL-4 cells, an effect attenuated by T(3). Both T(3) and EGF also significantly enhanced SGHPL-4 motility. These results suggest that EGF and T(3) may act synergistically to regulate both proliferation and differentiated function of human trophoblast.

A potential role for PTTG/securin in the developing human fetal brain.

Human securin, known also as PTTG, has established oncogenic and cell cycle regulatory functions. PTTG/securin transforms cells in vitro, inhibits sister chromatid separation, and regulates secretion of fibroblast growth factor-2. FGF-2 is a key regulator of CNS development and PTTG/securin expression has been reported in murine fetal brain. We examined the expression and function of securin and FGF-2 in the developing human fetal brain and in a fetal neuronal cell line (NT 2). Securin expression was significantly reduced in first and second trimester fetal cerebral cortex compared with adult cerebral cortex, where immunocytochemistry revealed intense securin staining in neuronal cell bodies. FGF-2 protein was concordantly lower in fetal cortex, whereas pretranslational expression of PTTG binding factor (PBF) was not significantly altered in fetal brain compared with adult. PCNA expression demonstrated that high securin levels in adult cortex were associated with absent cell proliferation. In NT-2 cells, securin stimulated FGF-2 expression, which could be abrogated by a carboxyl-terminal mutation. Low transient expression of securin resulted in a significant proliferative effect, whereas high levels of securin expression inhibited cell turnover. We propose a potential role for human PTTG/securin in modulating cell proliferation and FGF-2 expression during human neurogenesis.

Vitamin D promotes human extravillous trophoblast invasion in vitro.

INTRODUCTION: Incomplete human extravillous trophoblast (EVT) invasion of the decidua and maternal spiral arteries is characteristic of pre-eclampsia, a condition linked to low maternal vitamin D status. It is hypothesized that dysregulated vitamin D action in uteroplacental tissues disrupts EVT invasion leading to malplacentation. METHODS: This study assessed the effects of the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3), and its precursor, 25-hydroxyvitamin D3 (25-D3), on primary human EVT isolated from first trimester pregnancies. Expression of EVT markers (cytokeratin-7, HLA-G), the vitamin D-activating enzyme (CYP27B1) and 1,25-D3 receptor (VDR) was assessed by immunocytochemistry. EVT responses following in vitro treatment with 1,25-D3 (0-10 nM) or 25-D3 (0-100 nM) for 48-60 h were assessed using quantitative RT-PCR (qRT-PCR) analysis of key target genes. Effects on EVT invasion through Matrigel(®) were quantified alongside zymographic analysis of secreted matrix metalloproteinases (MMPs). Effects on cell viability were assessed by measurement of MTT. RESULTS: EVT co-expressed mRNA and protein for CYP27B1 and VDR, and demonstrated induction of mRNA encoding vitamin D-responsive genes, 24-hydroxylase (CYP24A1) and cathelicidin following 1,25-D3 treatment. EVT could respond to 1,25-D3 and 25-D3, both of which significantly increased EVT invasion, with maximal effect at 1 nM 1,25-D3 (1.9-fold; p < 0.01) and 100 nM 25-D3 (2.2-fold; p < 0.05) respectively compared with untreated controls. This was accompanied by increased pro-MMP2 and pro-MMP9 secretion. The invasion was independent of cell viability, which remained unchanged. DISCUSSION: These data support a role for vitamin D in EVT invasion during human placentation and suggest that vitamin D-deficiency may contribute to impaired EVT invasion and pre-eclampsia.

Thyroid hormone and estrogen receptor expression in normal pituitary and nonfunctioning tumors of the anterior pituitary.

Nonfunctioning tumors (NFTs) of the anterior pituitary often express elevated levels of the glycoprotein hormone alpha-subunit, which, under normal physiological conditions, is under negative feedback control by thyroid and gonadal steroid hormones. We postulate that inappropriately elevated levels of expression of alpha-subunit in the face of normal levels of these target organ hormones may reflect an abnormality of thyroid hormone receptors (TRs) and/or gonadal steroid receptors in NFTs. Using immunocytochemistry and Western blotting we have examined TR and estrogen receptor (ER) protein expression in normal human anterior pituitary glands and NFTs. Pretranslational expression of these receptors was examined using semiquantitative reverse transcriptase-PCR. Expression of all TR variant and ER proteins was reduced in pituitary tumors compared with that in normal pituitaries. The expression of messenger ribonucleic acids encoding the TR beta1 and TR beta2 isoforms and ER was also significantly reduced in tumors compared with normal tissues, although there was no difference between tumors and normals in the level of expression of TR alpha1 and alpha2 messenger ribonucleic acids. We suggest that reduced expression of TRs and ER may account for inappropriate expression of the glycoprotein hormone alpha-subunit gene in some NFTs and may contribute to uncontrolled tumor growth.

Thyroid receptor alpha1 and alpha2 mutations in nonfunctioning pituitary tumors.

We previously reported that nonfunctioning tumors of the anterior pituitary exhibit reduced expression of thyroid receptor (TR) alpha and beta isoforms, an observation that may account for abnormalities of T3-mediated negative regulation of the glycoprotein hormone common alpha-subunit. Reduced TR protein was associated with a parallel reduction in TRbeta messenger RNA (mRNA), although TRalpha1 and alpha2 mRNA levels were similar in nonfunctioning tumors and normal pituitaries. Because TRalpha shows aberrant posttranscriptional processing, and TRbeta is under ligand-dependent autoregulation, we hypothesized that aberrant TR expression in nonfunctioning tumors may reflect mutation in receptor coding and regulatory sequences, and therefore screened TRalpha mRNA and TRbeta T3 response elements and ligand binding domains for sequence anomalies. Screening TRalpha mRNA in 23 tumors and subsequently sequencing candidate fragments identified one silent change from published sequences and three novel missense mutations, two in the common TRalpha region (ser45ile and lys370asn) and one that was alpha2 specific (ser377leu). TRbeta response elements failed to show any differences from published sequences in 14 nonfunctioning tumors. Sequencing of TRbeta ligand binding domains were also identical to wild type in 23 nonfunctioning tumors. The functional significance of the novel TRalpha mutations is unknown; definition of mutant TR action may provide insight into the role of TRs in the growth control of pituitary cells.

Placental iodothyronine deiodinase expression in normal and growth-restricted human pregnancies.

We have described the expression of specific iodothyronine deiodinase mRNAs (using quantitative RT-PCR) and activities in normal human placentas throughout gestation and compared our findings to those in placentas from pregnancies affected by intrauterine growth restriction (IUGR). The predominant deiodinase expressed in placenta was type III (D3); type II (D2) was also present. In general terms, the activities of the enzymes D2 and D3 (and mRNAs encoding these enzymes) were higher earlier in gestation (<28 wk) than at term and displayed an inverse relationship with the duration of gestation (P < 0.05). Comparison of the relative expressions of mRNAs encoding D2 and D3 as well as their activities in placentas associated with IUGR (early and late gestational groups) with findings from normal placentas of similar gestational ages revealed no significant differences. Immunolocalization of D2 and D3 in syncytiotrophoblast (including syncytial sprouts) and cytotrophoblast of human placentas was demonstrated at both early and late gestation. Treatment of primary cultures of term cytotrophoblast cells in vitro with increasing doses of T(3) (1, 10, and 100 nM) resulted in increased expression of mRNAs encoding both D2 and D3 at 100-nM concentrations (P < 0.01) compared with control. Experiments with JEG-3 choriocarcinoma cells demonstrated a similar effect on D3 mRNA at 10 and 100 nM T(3) (P < 0.01). The demonstrated changes in iodothyronine deiodinase expression in the placenta across pregnancy are likely to contribute to regulation of the thyroid hormone supply to the developing fetus. The lack of difference in deiodinase expression in normal placentas and those found in IUGR argues against placental deiodinases being responsible for the hypothyroxemia in circulating fetal thyroid hormones observed in this condition.

Vascular endothelial growth factor, its receptor KDR/Flk-1, and pituitary tumor transforming gene in pituitary tumors.

Pituitary tumorigenesis is a poorly understood process involving dysregulation of the cell cycle, proliferation, and angiogenesis. The novel securin pituitary tumor transforming gene (PTTG) disrupts cell division and stimulates fibroblast growth factor (FGF)-2-mediated angiogenesis. We investigated expression of the angiogenic vascular endothelial growth factor (VEGF) and its receptor KDR/Flk-1 in 103 human pituitary tumors, and we assessed functional relationships between these genes in vitro. Nonfunctioning tumors (n = 81) demonstrated markedly raised VEGF mRNA (3.2-fold, P < 0.05) and protein concentrations, compared with normal pituitaries (n = 10). KDR was also highly induced in nonfunctioning tumors (14-fold, P < 0.001, n = 78) as well as in the whole cohort of pituitary tumors, compared with normal pituitary samples (14-fold, P < 0.0001, n = 100). In vitro, PTTG induced VEGF, but not KDR, expression in fetal neuronal NT2 cells (2.7-fold, P < 0.001, n = 8), MCF-7 breast carcinoma cells (1.9-fold, P = 0.03, n = 10), and choriocarcinoma JEG-3 cells (P = 0.0002, n = 8). A mutated PTTG construct that cannot be phosphorylated showed identical VEGF up-regulation (2.9-fold, P < 0.001, n = 8) in NT2 cells, compared with wild-type PTTG, but a further mutated construct with abrogation of the key protein:protein interaction domain of PTTG resulted in a significant reduction in VEGF stimulation, compared with wild-type (0.37-fold reduction, P < 0.001, n = 8). FGF-2 findings mirrored those of VEGF, although antibody depletion of secreted FGF-2 in the cell medium failed to influence VEGF up-regulation by PTTG. Overall, our findings implicate altered VEGF and KDR signaling in pituitary tumorigenesis, and we propose that PTTG stimulation of FGF-2 and VEGF expression in the presence of up-regulated growth factor receptors may account for angiogenic growth and progression of human pituitary tumors.

Pituitary tumor transforming gene and fibroblast growth factor-2 expression: potential prognostic indicators in differentiated thyroid cancer.

Differentiated thyroid cancers are the most common endocrine cancers, but there are no reliable molecular markers of prognosis. Pituitary tumor transforming gene (PTTG) plays several potential roles in tumor initiation and progression, including regulating mitosis and stimulating expression of fibroblast growth factor (FGF)-2. Increased expression of PTTG has been demonstrated in follicular thyroid lesions, and expression of this oncogene has been identified as a potential prognostic marker in pituitary adenomas and colon carcinomas. We assessed the expression of PTTG and FGF-2 and its receptor FGF-R-1 in 27 differentiated thyroid cancers, and we compared this with expression in 11 normal thyroids, 25 multinodular goiters, and 13 Graves' disease specimens. We also examined the relationship between gene expression and clinical markers of tumor behavior. PTTG and FGF-2 were overexpressed in thyroid carcinomas (9.5-fold increase, P = 0.003, and 5.0-fold increase, P < 0.001, respectively) compared with normal thyroid. Increased FGF-2 mRNA expression was independently associated with the findings of lymph node invasion (R(2) = 0.71; P < 0.001) and distant metastasis (R(2) = 0.55; P = 0.009) at tumor presentation, after taking into account known prognostic factors such as age and gender of the patient and size and type of the tumor. High PTTG expression was independently associated with tumor recurrence (R(2) = 0.64; P = 0.003). We conclude that PTTG and FGF-2 expression are potential prognostic markers (and perhaps therapeutic targets) for differentiated thyroid cancer.