Cell Polarity and Water Transport in Thyroid Epithelial Cells in Separated Follicles in Suspension Culture

Separated thyroid follicles maintained in suspension culture can be used to study the properties of thyroid epithelium in the virtual absence of other cell types and to study the effect of extracellular materials on the follicles. They can be prepared by enzymatic separation of thyroid into single cells followed by reaggregation of the epithelial cells and also by collagenase treatment of thyroids to release follicles and sheets of epithelia that can be separated from other materials by differential filtration. The follicles can exist with normal orientation or inverted (inside out). The follicles are inverted in the presence of high serum concentrations (5%) but can have normal orientation when embedded in a type I collagen gel, even at high serum concentrations. When normally oriented follicles invert, the polarity of the epithelial cells reverses while they are connected to neighbors. During inversion, bipolar cells are observed having microvilli- bearing surfaces at both lumen and medium. Inverted follicles can revert to normal orientation when embedded in collagen gel. Various functional properties of normally oriented follicles are similar to those of follicles in vitro. However, inverted follicles do not concentrate iodide, although they synthesize thyroglobulin and secrete it into the medium. Mutants are available in established cell lines. They have functional and organizational properties that differ from those of normal cells and demonstrate a lack of coupling between functional properties and organization. Inverted follicles transport water from medium into the lumen, although at rates somewhat less than MDCK cells

The Paired Domain-containing Factor Pax8 and the Homeodomain-containing Factor TTF-1 Directly Interact and Synergistically Activate Transcription

Pax genes encode for transcription factors essential for tissue development in many species. Pax8, the only member of the family expressed in the thyroid tissue, is involved in the morphogenesis of the gland and in the transcriptional regulation of thyroid-specific genes. TTF-1, a homeodomain-containing factor, is also expressed in the thyroid tissue and has been demonstrated to play a role in thyroid-specific gene expression. Despite the presence of Pax8 and TTF-1 also in a few other tissues, the simultaneous expression of the two transcription factors occurs only in the thyroid, supporting the idea that Pax8 and TTF-1 might cooperate to influence thyroid-specific gene expression. In this report, we describe a physical and functional interaction between these two factors. The fusion protein GST-Pax8 is able to bind TTF-1 present in thyroid or in non-thyroid cell extracts, and by using bacterial purified TTF-1 we demonstrate that the interaction is direct. By co-immunoprecipitation, we also show that the interaction between the two proteins occurs in vivo in thyroid cells. Moreover, Pax8 and TTF-1 when co-expressed in HeLa cells synergistically activate Tg gene transcription. The synergism requires the N-terminal activation domain of TTF-1, and deletions of Pax8 indicate that the C-terminal domain of the protein is involved. Our results demonstrate a functional cooperation and a physical interaction between transcription factors of the homeodomain-containing and of the paired domain-containing gene families in the regulation of tissue-specific gene expression

Mitochondrial Abnormalities in Down Syndrome: Pathogenesis, Effects and Therapeutic Approaches

Down syndrome (DS) consists of a complex phenotype with constant features, such as mental retardation and hypotonia, and variable features, including heart defects and susceptibility to Alzheimer’s disease, type 2 diabetes, obesity and immune disorders. Overexpression of genes mapping to chromosome 21 (Hsa21) is directly or indirectly responsible for pathogenesis of DS phenotypic features, as overexpressed Hsa21 genes dysregulate several other genes mapping to different chromosomes. Many of these genes are involved in mitochondrial function. Recent studies highlight a link between mitochondrial dysfunction, consistently observed in DS subjects, and DS phenotype. In this review, we first provide a basic overview of mitochondrial alterations in DS in terms of mitochondrial bioenergetics, biogenesis and morphology. We then discuss how mitochondrial malfunction may contribute to the pathogenesis of clinical manifestations and how specific Hsa21 genes may cause the disruption of mitochondrial phenotype. Finally, we focus on drugs, which affect mitochondrial function and network to propose possible therapeutic approaches aimed at improving and/or preventing various aspects of the DS phenotype. Our working hypothesis is that correcting the mitochondrial defect might improve the neurological phenotype and prevent DS-associated pathologies, thus providing a better quality of life for DS individuals and their families

Caveolin Transfection Results in Caveolae Formation but Not Apical Sorting of Glycosylphosphatidylinositol (GPI)-anchored Proteins in Epithelial Cells

Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The “raft” hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI- anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI- anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42–53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI- anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes

The microRNA-Processing Enzyme Dicer Is Essential for Thyroid Function

Dicer is a type III ribonuclease required for the biogenesis of microRNAs (miRNAs), a class of small non-coding RNAs regulating gene expression at the post-transcriptional level. To explore the functional role of miRNAs in thyroid gland function, we generated a thyrocyte-specific Dicer conditional knockout mouse. Here we show that development and early differentiation of the thyroid gland are not affected by the absence of Dicer, while severe hypothyroidism gradually develops after birth, leading to reduced body weight and shortened life span. Histological and molecular characterization of knockout mice reveals a dramatic loss of the thyroid gland follicular architecture associated with functional aberrations and down-regulation of several differentiation markers. The data presented in this study show for the first time that an intact miRNAs processing machinery is essential for thyroid physiology, suggesting that deregulation of specific miRNAs could be also involved in human thyroid dysfunctions

Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy

<p>Abstract</p> <p>Background</p> <p>The Down syndrome phenotype has been attributed to overexpression of chromosome 21 (Hsa21) genes. However, the expression profile of Hsa21 genes in trisomic human subjects as well as their effects on genes located on different chromosomes are largely unknown. Using oligonucleotide microarrays we compared the gene expression profiles of hearts of human fetuses with and without Hsa21 trisomy.</p> <p>Results</p> <p>Approximately half of the 15,000 genes examined (87 of the 168 genes on Hsa21) were expressed in the heart at 18–22 weeks of gestation. Hsa21 gene expression was globally upregulated 1.5 fold in trisomic samples. However, not all genes were equally dysregulated and 25 genes were not upregulated at all. Genes located on other chromosomes were also significantly dysregulated. Functional class scoring and gene set enrichment analyses of 473 genes, differentially expressed between trisomic and non-trisomic hearts, revealed downregulation of genes encoding mitochondrial enzymes and upregulation of genes encoding extracellular matrix proteins. There were no significant differences between trisomic fetuses with and without heart defects.</p> <p>Conclusion</p> <p>We conclude that dosage-dependent upregulation of Hsa21 genes causes dysregulation of the genes responsible for mitochondrial function and for the extracellular matrix organization in the fetal heart of trisomic subjects. These alterations might be harbingers of the heart defects associated with Hsa21 trisomy, which could be based on elusive mechanisms involving genetic variability, environmental factors and/or stochastic events.</p

Overexpression of the Hsa21 Transcription Factor RUNX1 Modulates the Extracellular Matrix in Trisomy 21 Cells

Down syndrome is a neurodevelopmental disorder frequently characterized by other developmental defects, such as congenital heart disease. Analysis of gene expression profiles of hearts from trisomic fetuses have shown upregulation of extracellular matrix (ECM) genes. The aim of this work was to identify genes on chromosome 21 potentially responsible for the upregulation of ECM genes and to pinpoint any functional consequences of this upregulation. By gene set enrichment analysis of public data sets, we identified the transcription factor RUNX1, which maps to chromosome 21, as a possible candidate for regulation of ECM genes. We assessed that approximately 80% of ECM genes overexpressed in trisomic hearts have consensus sequences for RUNX1 in their promoters. We found that in human fetal fibroblasts with chromosome 21 trisomy there is increased expression of both RUNX1 and several ECM genes, whether located on chromosome 21 or not. SiRNA silencing of RUNX1 reduced the expression of 11 of the 14 ECM genes analyzed. In addition, collagen IV, an ECM protein secreted in high concentrations in the culture media of trisomic fibroblasts, was modulated by RUNX1 silencing. Attenuated expression of RUNX1 increased the migratory capacity of trisomic fibroblasts, which are characterized by a reduced migratory capacity compared to euploid controls

DiGeorge-like Syndrome in a Child with a 3p12.3 Deletion Involving MIR4273 Gene Born to a Mother with Gestational Diabetes Mellitus

Chromosome 22q11.2 deletion is the most common chromosomal alteration associated with DiGeorge syndrome (DGS), even though this is not the only underlying cause of DGS. In rare patients, mutations in a single gene, TBX1, have been described resulting in a DGS phenotype. Recently, it has been reported that at least part of the TBX1 mutant phenotype is due to excessive bone morphogenetic proteins (BMP) signaling. Evidence suggests that miRNA may modulate the expression of critical T-box transcriptional regulators during midface development and Bmp-signaling. We report on a 7-year-old Caucasian male born to a mother affected with gestational diabetes (GDM) who had a 371Kb-interstitial deletion of 3p12.3 identified by array CGH, involving the ZNF717, MIR1243 and 4273 genes. The child presented with a DiGeorge anomaly (DGA) associated with unilateral renal agenesis and language delay. The immunological evaluation revealed a severe reduction and impairment of T lymphocytes. FISH analysis and TBX1 sequencing were negative. Among the miRNA-4273 predicted target genes, we found BMP3, which is involved in several steps of embryogenesisincluding kidney and lung organogenesis and in insulin gene expression. Since DGA is not commonly found in newborns of diabetic mothers, we hypothesize that the pathogenesis of DGA associated with GDM is multifactorial, involving both genetic and/or epigenetic cofactors

Variegated silencing through epigenetic modifications of a large Xq region in a case of balanced X;2 translocation with Incontinentia Pigmenti-like phenotype

Molecular mechanisms underlying aberrant phenotypes in balanced X;autosome translocations are scarcely understood. We report the case of a de novo reciprocal balanced translocation X;2(q23;q33) presenting phenotypic alterations highly suggestive of Incontinentia Pigmenti (IP) syndrome, a genodermatosis with abnormal skin pigmentation and neurological failure, segregating as X-linked dominant disorder. Through molecular studies, we demonstrated that the altered phenotype could not be ascribed to chromosome microdeletions or to XIST-mediated inactivation of Xq24-qter. Interestingly, we found that the Xq24-qter region, which translocated downstream of the heterochromatic band 2q34, undergoes epigenetic silencing mediated by DNA methylation and histone alterations. Among the downregulated genes, we found the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase gamma (IKBKG/NEMO), the causative gene of IP. We hypothesize that a mosaic functional nullisomy of the translocated genes, through a Position Effect Variegation-like heterochromatization, might be responsible for the proband's phenotypic anomalies. Partial silencing of IKBKG may be responsible for the skin anomalies observed, thereby mimicking the IP pathological condition. In addition to its clinical relevance, this paper addresses fundamental issues related to the chromatin status and nuclear localization of a human euchromatic region translocated proximally to heterochromatin. In conclusion, the study provides new insight into long-range gene silencing mechanisms and their direct impact in human disease

Physiology, pathology and relatedness of human tissues from gene expression meta-analysis

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