Birgit Mentrup

Endocrine disruptors and the thyroid gland--a combined in vitro and in vivo analysis of potential new biomarkers.

Background There is growing evidence that, in addition to the reproductive system, the hypothalamic–pituitary–thyroid axis is a target of endocrine-disrupting compounds (EDCs). However, this is not reflected adequately in current screening and assessment procedures for endocrine activity that to date determine only general parameters of thyroid function. Objective and Methods We used several in vitro and ex vivo assays in an attempt to identify suitable biomarkers for antithyroid action testing a selected panel of putative EDCs. Results In vitro we detected stimulation or inhibition of iodide uptake into FRTL-5 rat thyroid cells, inhibition of thyroid hormone binding to transthyretin, agonistic or antagonistic effects in a thyroid hormone receptor–dependent reporter assay, and inhibition of thyroid peroxidase using a novel assay system based on human recombinant thyroperoxidase that might be suitable for routine screening for potential EDCs. In rats, chronic application of several EDCs led to changes in thyroid morphology, alterations of thyrotropin and thyroid hormone serum levels as well as alterations in peripheral thyroid hormone–regulated end points such as malic enzyme and type I 5′-deiodinase activity. Conclusions As the effects of EDCs do not reflect classic mechanisms of hormone-dependent regulation and feedback, we believe multitarget and multimodal actions of EDCs affect the hypothalamic–pituitary–thyroid axis. These complex effects require a diverse approach for screening, evaluation, and risk assessment of potential antithyroid compounds. This approach involves novel in vitro or cell-based screening assays in order to assess thyroid hormone synthesis, transport, metabolism, and action as well as in vivo assays to measure thyroid hormone–regulated tissue-specific and developmental end points in animals.

Selenoproteins of the thyroid gland: expression, localization and possible function of glutathione peroxidase 3

Abstract The thyroid gland has an exceptionally high selenium content, even during selenium deficiency. At least 11 selenoproteins are expressed, which may be involved in the protection of the gland against the high amounts of H2O2 produced during thyroid hormone biosynthesis. As determined here by in situ hybridization and Northern blotting experiments, glutathione peroxidases (GPx) 1 and 4 and selenoprotein P were moderately expressed, occurring selectively in the follicular cells and in leukocytes of germinal follicles of thyroids affected by Hashimotos thyroiditis. Selenoprotein 15 was only marginally expressed and distributed over all cell types. GPx3 mRNA was exclusively localized to the thyrocytes, showed the highest expression levels and was down-regulated in 5 of 6 thyroid cancer samples as compared to matched normal controls. GPx3 could be extracted from thyroidal colloid by incubation with 0.5% sodium dodecyl sulfate indicating that this enzyme is (i) secreted into the follicular lumen and (ii) loosely attached to the colloidal thyroglobulin. These findings are consistent with a role of selenoproteins in the protection of the thyroid from possible damage by H2O2. Particularly, GPx3 might use excess H2O2 and catalyze the polymerization of thyroglobulin to the highly cross-linked storage form present in the colloid.

A complex DNA-repeat structure within the Selenoprotein P promoter contains a functionally relevant polymorphism and is genetically unstable under conditions of mismatch repair deficiency

Epidemiological data, animal studies and interventional studies provide evidence for a potential chemopreventive effect of selenium during development of colorectal cancer. The human glycoprotein Selenoprotein P (SeP) contains up to 50% of plasma selenium content. SeP is expressed in the gastrointestinal tract and the liver, where its expression is downregulated by various proinflammatory cytokines (Il1β, TGFβ, IFNγ). Previously, we have demonstrated dramatically reduced SeP expression in human colon adenomas. Here, we have identified a complex (A)4-C-(A)4-GG-(A)8-GCT-(TC)5-(T)17 (bp −429 to bp − 477) repeat structure within the SeP promoter and we have analysed this regulatory DNA sequence with respect to polymorphisms, genomic instability and functional relevance to promoter activity. As opposed to the (TC)5 variant we identified a novel (TC)3 polymorphism within this repeat in the general population, which conferred significantly reduced basal promoter activity to reporter gene constructs in HepG2 cells. Allelic distribution of this (TC)n element was similar in colon carcinoma patients and healthy controls. Additionally, we observed genetic instability within the (T)17 repeat motif in colon cancers of the mutator phenotype. This instability of the (T)17 repeat had no effect on basal promoter activity in reporter gene assays. In conclusion, we characterised a complex repeat structure within the SeP promoter that may be of functional relevance to SeP gene expression. Further studies on the effect of different SeP promoter genotypes on SeP protein expression and disease susceptibility are needed.

1,25-Dihydroxyvitamin D3 Treatment Delays Cellular Aging in Human Mesenchymal Stem Cells while Maintaining Their Multipotent Capacity

1,25-dihydroxyvitamin D3 (1,25D3) was reported to induce premature organismal aging in fibroblast growth factor-23 (Fgf23) and klotho deficient mice, which is of main interest as 1,25D3 supplementation of its precursor cholecalciferol is used in basic osteoporosis treatment. We wanted to know if 1,25D3 is able to modulate aging processes on a cellular level in human mesenchymal stem cells (hMSC). Effects of 100 nM 1,25D3 on hMSC were analyzed by cell proliferation and apoptosis assay, β-galactosidase staining, VDR and surface marker immunocytochemistry, RT-PCR of 1,25D3-responsive, quiescence- and replicative senescence-associated genes. 1,25D3 treatment significantly inhibited hMSC proliferation and apoptosis after 72 h and delayed the development of replicative senescence in long-term cultures according to β-galactosidase staining and P16 expression. Cell morphology changed from a fibroblast like appearance to broad and rounded shapes. Long term treatment did not induce lineage commitment in terms of osteogenic pathways but maintained their clonogenic capacity, their surface marker characteristics (expression of CD73, CD90, CD105) and their multipotency to develop towards the chondrogenic, adipogenic and osteogenic pathways. In conclusion, 1,25D3 delays replicative senescence in primary hMSC while the pro-aging effects seen in mouse models might mainly be due to elevated systemic phosphate levels, which propagate organismal aging.

Retinoic acid-mediated down-regulation of ENO1/MBP-1 gene products caused decreased invasiveness of the follicular thyroid carcinoma cell lines.

Retinoic acid (RA) acts as an anti-proliferative and redifferentiation agent in the therapy of thyroid carcinoma. Our previous studies demonstrated that pretreatment of follicular thyroid carcinoma cell lines FTC-133 and FTC-238 resulted in decreased in vitro proliferation rates and reduced tumor cell growth of xenotransplants. In addition to the previous results, we found that RA led to decreased vitality and invasiveness of FTC-133 and FTC-238 cells as they reacted with reduction of intracellular ATP levels and number of migrated cells respectively. However, the molecular mechanisms by which RA mediates these effects are not well understood. Two-dimensional (2D) screening of the proteins related to ATP metabolism and western blot analysis revealed alpha-enolase (ENO1) to be down-regulated in FTC-133 and FTC-238 cells after RA treatment. 2D gel detection and mass spectrometric analysis revealed that ENO1 existed as three separate protein spots of distinct pIs (ENO1-A1-A3). Comparative 2D difference gel electrophoresis analysis of fluorescently labeled protein samples of RA-treated and untreated FTC-133 demonstrated a selective down-regulation of ENO1-A1 which we identified as a phosphoprotein. RA caused the dephosphorylation of ENO1-A1. Both, RA-mediated and specific knock-down of ENO1/MBP-1 resulted in the reduction of MYC oncoprotein, and simultaneously decreased proliferation rates of FTC-133 and FTC-238 cell lines. In summary, the RA-mediated down-regulation of the ENO1 gene products and MYC oncoprotein provides a novel molecular mechanism facilitating the anti-proliferative effect of RA in human thyroid carcinoma cells and suggests new pathways for supportive RA therapies.

Clinical Aspects of Hypophosphatasia: An Update

Hypophosphatasia (HPP) is a heterogeneous rare inborn error of bone and mineral metabolism caused by mutations in the ALPL gene encoding the isoenzyme, tissue-nonspecific alkaline phosphatase (TNAP). These mutations result in a decreased level of TNAP activity and increased levels of its substrates, including inorganic pyrophosphate, pyridoxal-5′-phosphate and phosphoethanolamine. Clinical presentations are highly variable, ranging from stillbirth and absence of mineralization in severe disease to mild dental problems or osteopenia in adulthood. Further clinical symptoms include defective bone mineralization with bone deformities, recurrent fractures, chronic non-bacterial osteomyelitis, craniosynostosis, neonatal seizures, nephrocalcinosis, muscular hypotonia, failure to thrive and dental abnormalities with premature exfoliation of teeth and caries. Prognosis is very poor in severe perinatal forms with most patients dying from pulmonary complications of their skeletal disease but patients with mild phenotypes (adult form or Odonto-HPP) usually do not have a limitation in their life expectancy. Although TNAP is a ubiquitous enzyme, mostly known for its crucial role during mineralization of bone and teeth, its exact biological role in different human organs is still unclear, and the pathophysiology of symptoms due to TNAP deficiency in HPP are not understood in detail. Since inflammation and tissue destruction of the musculoskeletal system may occur in HPP, TNAP may also play an important role in controlling inflammatory processes. Recent investigations provide evidence that TNAP is also essentially involved in the development of the central nervous system and might contribute to multiple functions of the human brain. HPP can be diagnosed on clinical, biochemical and radiological criteria, and genetic testing confirms the diagnosis and is useful for genetic counseling. Since clinical symptoms are highly variable, patients should be followed up by a multidisciplinary team having experience in HPP treatment. Up to now, no curative treatment of HPP is available. Therefore, symptomatic treatment in particular with regard to pain, seizures and other metabolic phenomena is most important. However, recently, enzyme replacement therapy with a bone-targeted recombinant human TNAP molecule has been reported to improve bone mineralization, respiratory function and physical activity in severely affected infants with HPP, and further clinical trials are ongoing. Hopefully, this and other new therapeutic strategies may improve the prognosis and quality of life of patients with HPP and may contribute to our understanding of bone metabolism in general.

Functional characterization of a novel mutation localized in the start codon of the tissue-nonspecific alkaline phosphatase gene

Hypophosphatasia (HPP) is a rare inborn disease caused by different mutations in the tissue-nonspecific alkaline phosphatase (ALPL) gene. Previous studies showed that gene mutations could exhibit a dominant negative effect leading to a mild HPP phenotype in heterozygous carriers. In the present report we describe the clinical and functional studies of a novel mutation localized in the start codon of transcript variant 1 of the ALPL gene from a female adult heterozygous carrier. The mutation results in translation of an N-terminally truncated protein, which might be identical to the deduced protein from ALPL transcript variant 2. When overexpressed in HEK-293 cells it does not exhibit any enzymatic activity and has no significant effect on the wild type ALPL protein. Furthermore it is not attached to the cell membrane. Due to the loss of the signal peptide an intracellular misrouting and a premature degradation is obvious. Hence the new isoform deposited in the database does not produce an active protein as it is the case in the natural mutation of our patient. Since the mutation does not produce a dominant negative protein in heterozygous carriers, the clinical phenotype in our patient and her relatives is very mild with only unspecific myalgia. However the patient developed bone marrow edema of both femoral heads during lactation after delivery of a healthy child, indicating a risk to develop alterations of bone metabolism in challenge situations. Her sister complains of identical symptoms, her father shows distinct symptoms of odonto-hypophosphatasia. The question if or if not carriers of ALPL mutations in general or only with distinct genotypes can be symptomatic in normal life or in challenge situations requires systematic clinical studies.

Probenecid as a sensitizer of bisphosphonate-mediated effects in breast cancer cells

BackgroundAnti-resorptive bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases. Clinical studies indicated a benefit in survival and tumor relapse in subpopulations of breast cancer patients receiving zoledronic acid, thus stimulating the debate about its anti-tumor activity. Amino-bisphosphonates in nM concentrations inhibit farnesyl pyrophosphate synthase leading to accumulation of isopentenyl pyrophosphate (IPP) and the ATP/pyrophosphate adduct ApppI, which induces apoptosis in osteoclasts. For anti-tumor effects μM concentrations are needed and a sensitizer for bisphosphonate effects would be beneficial in clinical anti-tumor applications. We hypothesized that enhancing intracellular pyrophosphate accumulation via inhibition of probenecid-sensitive channels and transporters would sensitize tumor cells for bisphosphonates anti-tumor efficacy.MethodMDA-MB-231, T47D and MCF-7 breast cancer cells were treated with BP (zoledronic acid, risedronate, ibandronate, alendronate) and the pyrophosphate channel inhibitors probenecid and novobiocin. We determined cell viability and caspase 3/7 activity (apoptosis), accumulation of IPP and ApppI, expression of ANKH, PANX1, ABCC1, SLC22A11, and the zoledronic acid target gene and tumor-suppressor KLF2.ResultsTreatment of MDA-MB-231 with BP induced caspase 3/7 activity, with zoledronic acid being the most effective. In MCF-7 and T47D either BP markedly suppressed cell viability with only minor effects on apoptosis. Co-treatment with probenecid enhanced BP effects on cell viability, IPP/ApppI accumulation as measurable in MCF-7 and T47D cells, caspase 3/7 activity and target gene expression. Novobiocin co-treatment of MDA-MB-231 yielded identical results on viability and apoptosis compared to probenecid, rendering SLC22A family members as candidate modulators of BP effects, whereas no such evidence was found for ANKH, ABCC1 and PANX1.ConclusionsIn summary, we demonstrate effects of various bisphosphonates on caspase 3/7 activity, cell viability and expression of tumor suppressor genes in breast cancer cells. Blocking probenecid and novobiocin-sensitive channels and transporters enhances BP anti-tumor effects and renders SLC22A family members as good candidates as BP modulators. Further studies will have to unravel if treatment with such BP-sensitizers translates into preclinical and clinical efficacy.

Exome sequencing identifies a nonsense mutation in Fam46a associated with bone abnormalities in a new mouse model for skeletal dysplasia

We performed exome sequencing for mutation discovery of an ENU (N-ethyl-N-nitrosourea)-derived mouse model characterized by significant elevated plasma alkaline phosphatase (ALP) activities in female and male mutant mice, originally named BAP014 (bone screen alkaline phosphatase #14). We identified a novel loss-of-function mutation within the Fam46a (family with sequence similarity 46, member A) gene (NM_001160378.1:c.469G>T, NP_001153850.1:p.Glu157*). Heterozygous mice of this mouse line (renamed Fam46aE157*Mhda) had significantly high ALP activities and apparently no other differences in morphology compared to wild-type mice. In contrast, homozygous Fam46aE157*Mhda mice showed severe morphological and skeletal abnormalities including short stature along with limb, rib, pelvis, and skull deformities with minimal trabecular bone and reduced cortical bone thickness in long bones. ALP activities of homozygous mutants were almost two-fold higher than in heterozygous mice. Fam46a is weakly expressed in most adult and embryonic tissues with a strong expression in mineralized tissues as calvaria and femur. The FAM46A protein is computationally predicted as a new member of the superfamily of nucleotidyltransferase fold proteins, but little is known about its function. Fam46aE157*Mhda mice are the first mouse model for a mutation within the Fam46a gene.

Recombinant Enzyme Replacement Therapy in Hypophosphatasia

Hypophosphatasia (HPP) is a rare monogenetic and multisystemic disease with involvement of different organs, including bone, muscle, kidney, lung, gastrointestinal tract and the nervous system. The exact metabolic mechanisms of the effects of TNAP deficiency in different tissues are not understood in detail. There is no approved specific treatment for HPP; therefore symptomatic treatment in order to improve the clinical features is of major interest. Enzyme replacement therapy (ERT) is a relatively new type of treatment based on the principle of administering a medical treatment replacing a defective or absent enzyme. Recently ERT with a bone targeted recombinant human TNAP molecule has been reported to be efficient in ten severely affected patients and improved survival of life threatening forms. These results are very promising especially with regard to the skeletal phenotype but it is unclear whether ERT also has beneficial effects for craniosynostosis and in other affected tissues in HPP such as brain and kidney. Long-term data are not yet available and further systematic clinical trials are needed. It is also necessary to establish therapeutic approaches to help patients who are affected by less severe forms of HPP but also suffer from a significant reduction in quality of life. Further basic research on TNAP function and role in different tissues and on its physiological substrates is critical to gain a better insight in the pathogenesis in HPP. This and further experiences in new therapeutic strategies may improve the prognosis and quality of life of patients with all forms of HPP.