Undifferentiated thyroid carcinoma is one of the most aggressive human cancers with frequent RAS mutations. How mutations of the RAS gene contribute to undifferentiated thyroid cancer remains largely unknown. Mice harboring a potent dominant negative mutant thyroid hormone receptor β, TRβPV (Thrb(PV/PV)), spontaneously develop well-differentiated follicular thyroid cancer similar to human cancer. We genetically targeted the Kras(G12D) mutation to thyroid epithelial cells of Thrb(PV/PV) mice to understand how Kras(G12D) mutation could induce undifferentiated thyroid cancer in Thrb(PV/PV)Kras(G12D) mice. Thrb(PV/PV)Kras(G12D) mice exhibited poorer survival due to more aggressive thyroid tumors with capsular invasion, vascular invasion, and distant metastases to the lung occurring at an earlier age and at a higher frequency than Thrb(PV/PV) mice did. Importantly, Thrb(PV/PV)Kras(G12D) mice developed frequent anaplastic foci with complete loss of normal thyroid follicular morphology. Within the anaplastic foci, the thyroid-specific transcription factor paired box gene 8 (PAX8) expression was virtually lost and the loss of PAX8 expression was inversely correlated with elevated MYC expression. Consistently, co-expression of KRAS(G12D) with TRβPV upregulated MYC levels in rat thyroid pccl3 cells, and MYC acted to enhance the TRβPV-mediated repression of the Pax8 promoter activity of a distant upstream enhancer, critical for thyroid-specific Pax8 expression. Our findings indicated that synergistic signaling of KRAS(G12D) and TRβPV led to increased MYC expression. Upregulated MYC contributes to the initiation of undifferentiated thyroid cancer, in part, through enhancing TRβPV-mediated repression of the Pax8 expression. Thus, MYC might serve as a potential target for therapeutic intervention.

Polybrominated diphenyl ethers (PBDEs) have the potential to disturb the thyroid endocrine system, but little is known of such effects or underlying mechanisms of BDE-209 in fish. In the present study, bioconcentration and metabolism of BDE-209 were investigated in zebrafish embryos exposed at concentrations of 0, 0.08, 0.38 and 1.92 mg/L in water until 14 days post-fertilization (dpf). Chemical analysis revealed that BDE-209 was accumulated in zebrafish larvae, while also metabolic products were detected, including octa- and nona-BDEs, with nona-BDEs being predominant. The exposure resulted in alterations of both triiodothyronine (T3) and thyroxine (T4) levels, indicating thyroid endocrine disruption. Gene transcription in the hypothalamic-pituitary-thyroid (HPT) axis was further examined, and the results showed that the genes encoding corticotrophin-releasing hormone (CRH) and thyroid-stimulating hormone (TSHβ) were transcriptionally significantly up-regulated. Genes involved in thyroid development (Pax8 and Nkx2.1) and synthesis (sodium/iodide symporter, NIS, thyroglobulin, TG) were also transcriptionally up-regulated. Up-regulation of mRNA for thyronine deiodinase (Dio1 and Dio2) and thyroid hormone receptors (TRα and TRβ) was also observed. However, the genes encoding proteins involved in TH transport (transthyretin, TTR) and metabolism (uridinediphosphate-glucuronosyl-transferase, UGT1ab) were transcriptionally significantly down-regulated. Furthermore, protein synthesis of TG was significantly up-regulated, while that of TTR was significantly reduced. These results suggest that the hypothalamic-pituitary-thyroid axis can be evaluated to determine thyroid endocrine disruption by BDE-209 in developing zebrafish larvae.

OBJECTIVE

To correlate thyroid cancer genotype with histology and outcomes.

BACKGROUND

The prognostic significance of molecular signature in thyroid cancer (TC) is undefined but can potentially change surgical management.

METHODS

We reviewed a consecutive series of 1510 patients who had initial thyroidectomy for TC with routine testing for BRAF, RAS, RET/PTC, and PAX8/PPARG alterations. Histologic metastatic or recurrent TC was tracked for 6 or more months after oncologic thyroidectomy.

RESULTS

Papillary thyroid cancer (PTC) was diagnosed in 97% of patients and poorly differentiated/anaplastic TC in 1.1%. Genetic alterations were detected in 1039 (70%); the most common mutations were BRAFV600E (644/1039, 62%), and RAS isoforms (323/1039, 31%). BRAFV600E-positive PTC was often conventional or tall cell variant (58%), with frequent extrathyroidal extension (51%) and lymph node metastasis (46%). Conversely, RAS-positive PTC was commonly follicular variant (87%), with infrequent extrathyroidal extension (4.6%) and lymph node metastasis (5.6%). BRAFV600E and RET/PTC-positive PTCs were histologically similar. Analogously, RAS and PAX8/PPARG-positive PTCs were histologically similar. Compared with RAS or PAX8/PPARG-positive TCs, BRAFV600E or RET/PTC-positive TCs were more often associated with stage III/IV disease (40% vs 15%, P < 0.001) and recurrence (10% vs 0.7%, P < 0.001; mean follow-up 33 ± 21 mo). Distant metastasis was highest in patients with RET/PTC-positive TC (10.8%, P = 0.02).

CONCLUSIONS

In this large study of prospective mutation testing in unselected patients with TC, molecular signature was associated with distinctive phenotypes including cancers, with higher risks of both distant metastasis and early recurrence. Preoperative genotype provides valuable prognostic data to appropriately inform surgery.

Differentiated thyroid cancer (DTC) is the most common endocrinological malignancy. There are several histological variants such as papillary and follicular thyroid carcinoma. Many patients with well-differentiated subtypes of DTC are cured by surgery alone or with radioiodine, while poorly differentiated types usually have a worse prognosis. The aggressiveness of thyroid tumors is closely linked to specific gene alterations. Several diagnostic and prognostic molecular markers such as BRAF and RAS point mutations; RET/PTC and PAX8/PPARγ gene rearrangements; MAPK, PI3K, p53, Wnt-beta catenin, HIF1α and NF-kappaB signaling pathways; microRNA profiles and aberrant methylation have been demonstrated in more than 70% of DTC. Diagnostic use of these molecular markers may be optimized for identifying higher risks of mortality, tumor recurrence and metastatic potential. Understanding the molecular biology of thyroid cancers can be an important avenue for diagnosis and treatment of radioiodine-refractory or inoperable DTC patients with novel molecular targeted therapeutic agents.

The retinoblastoma protein (RB)-E2F1 pathway has a central role in regulating the cell cycle. Several PAX proteins (tissue-specific developmental regulators), including PAX8, interact with the RB protein, and thus regulate the cell cycle directly or indirectly. Here, we report that PAX8 expression is frequent in renal cell carcinoma, bladder, ovarian and thyroid cancer cell lines, and that silencing of PAX8 in cancer cell lines leads to a striking reduction in the expression of E2F1 and its target genes, as well as a proteasome-dependent destabilization of RB protein, with the RB1 mRNA level remaining unaffected. Cancer cells expressing PAX8 undergo a G(1)/S arrest and eventually senesce following PAX8 silencing. We demonstrate that PAX8 transcriptionally regulates the E2F1 promoter directly, and E2F1 transcription is enhanced after RB depletion. RB is recruited to the PAX8-binding site, and is involved in PAX8-mediated E2F1 transcription in cancer cells. Therefore, our results suggest that, in cancer, frequent and persistent expression of PAX8 is required for cell growth control through transcriptional activation of E2F1 expression and upregulation of the RB-E2F1 pathway.

Renal hypoxia occurs in AKI of various etiologies, but adaptation to hypoxia, mediated by hypoxia-inducible factor (HIF), is incomplete in these conditions. Preconditional HIF activation protects against renal ischemia-reperfusion injury, yet the mechanisms involved are largely unknown, and HIF-mediated renoprotection has not been examined in other causes of AKI. Here, we show that selective activation of HIF in renal tubules, through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO), protects from rhabdomyolysis-induced AKI. In this model, HIF activation correlated inversely with tubular injury. Specifically, VHL deletion attenuated the increased levels of serum creatinine/urea, caspase-3 protein, and tubular necrosis induced by rhabdomyolysis in wild-type mice. Moreover, HIF activation in nephron segments at risk for injury occurred only in VHL-KO animals. At day 1 after rhabdomyolysis, when tubular injury may be reversible, the HIF-mediated renoprotection in VHL-KO mice was associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal, as demonstrated by quantitative PCR, pathway enrichment analysis, and immunohistochemistry. In conclusion, a HIF-mediated shift toward improved energy supply may protect against acute tubular injury in various forms of AKI.

BACKGROUND

Enhancer of zeste homolog 2 (EZH2) is a histone lysine methyltransferase belonging to the polycomb group protein family. Overexpression of EZH2 has been found in several human malignancies including hematological and solid tumors.

OBJECTIVE

In this study we investigated the expression levels of EZH2 and its polycomb group protein partners in thyroid carcinoma tissues with different degrees of malignancy to identify potential new therapeutic targets for anaplastic thyroid carcinoma (ATC).

RESULTS

We show that high EZH2 expression levels are characteristic of undifferentiated ATC, whereas no significant changes were observed in well-differentiated papillary and follicular thyroid carcinomas as compared with normal thyroid. Knockdown of EZH2 in ATC cell lines results in cell growth inhibition, loss of anchorage-independent growth, migration, and invasion properties. Moreover, we demonstrate that EZH2 directly controls differentiation of ATC cells by silencing the thyroid specific transcription factor paired-box gene 8 (PAX8).

CONCLUSIONS

EZH2 is specifically overexpressed in ATC, and it directly contributes to transcriptional silencing of PAX8 gene and ATC differentiation.

Anaplastic thyroid carcinoma can be difficult to diagnose because it does not show thyroid differentiation morphologically or immunohistochemically. Depending on the histologic variant, anaplastic thyroid carcinoma may be confused with sarcoma or squamous cell carcinoma of the head and neck. PAX8 is a transcription factor expressed in normal and neoplastic thyroid follicular epithelium and only a few other tissues. This restricted expression suggests that PAX8 staining could be useful when dealing with spindled or squamoid tumors of the neck. The purposes of this study were to determine the frequency of PAX8 staining in anaplastic thyroid carcinoma and to evaluate PAX8 immunohistochemistry as a means of distinguishing its squamoid variant from head and neck squamous cell carcinoma. PAX8 immunohistochemical staining was performed on 34 anaplastic thyroid carcinomas and 118 head and neck squamous cell carcinomas. PAX8 staining was present in 26 (76%) anaplastic thyroid carcinomas including 16 (100%) of 16 squamoid variants, 7 (58%) of 12 giant cell/pleomorphic variants, and 3 (50%) of 6 spindled variants. All head and neck squamous cell carcinomas were negative for PAX8. PAX8 expression is often retained in anaplastic thyroid carcinomas including the squamoid variant, but it is not expressed in head and neck squamous cancers. PAX8 staining is an excellent marker for carcinomas of follicular epithelial origin, including those carcinomas that are undifferentiated in other respects. The tissue specificity of PAX8 expression may be useful in resolving the differential diagnosis of anaplastic thyroid carcinoma such as the distinction between its squamoid variant and squamous cell carcinoma of the head and neck.

The presence of differentiated thyroid cells in thyroid cancer is critical for the antitumor response to radioactive iodide treatment, and loss of the differentiated phenotype is a key hallmark of iodide-refractory metastatic disease. The role of microRNAs (miRNA) in fine-tuning gene expression has become a major regulatory mechanism by which developmental and pathologic processes occur. In this study, we performed next-generation sequencing and expression analysis of eight papillary thyroid carcinomas (PTC) to comprehensively characterize miRNAs involved in loss of differentiation. We found that only a small set of abundant miRNAs is differentially expressed between PTC tissue and normal tissue from the same patient. In addition, we integrated computational prediction of potential targets and mRNA sequencing and identified a master miRNA regulatory network involved in essential biologic processes such as thyroid differentiation. Both mature products of mir-146b (miR-146b-5p and -3p) were among the most abundantly expressed miRNAs in tumors. Specifically, we found that miR-146b-3p binds to the 3'-untranslated region of PAX8 and sodium/iodide symporter (NIS), leading to impaired protein translation and a subsequent reduction in iodide uptake. Furthermore, our findings show that miR-146b and PAX8 regulate each other and share common target genes, thus highlighting a novel regulatory circuit that governs the differentiated phenotype of PTC. In conclusion, our study has uncovered the existence of a miR-146b-3p/PAX8/NIS regulatory circuit that may be exploited therapeutically to modulate thyroid cell differentiation and iodide uptake for improved treatment of advanced thyroid cancer.

The mesencephalic and metencephalic region (MMR) of the vertebrate central nervous system develops in response to signals produced by the isthmic organizer (IsO). We have previously reported that the LIM homeobox transcription factor Lmx1b is expressed within the chick IsO, where it is sufficient to maintain expression of the secreted factor wnt1. In this paper, we show that zebrafish express two Lmx1b orthologs, lmx1b.1 and lmx1b.2, in the rostral IsO, and demonstrate that these genes are necessary for key aspects of MMR development. Simultaneous knockdown of Lmx1b.1 and Lmx1b.2 using morpholino antisense oligos results in a loss of wnt1, wnt3a, wnt10b, pax8 and fgf8 expression at the IsO, leading ultimately to programmed cell death and the loss of the isthmic constriction and cerebellum. Single morpholino knockdown of either Lmx1b.1 or Lmx1b.2 has no discernible effect on MMR development. Maintenance of lmx1b.1 and lmx1b.2 expression at the isthmus requires the function of no isthmus/pax2.1, as well as Fgf signaling. Transient misexpression of Lmx1b.1 or Lmx1b.2 during early MMR development induces ectopic wnt1 and fgf8 expression in the MMR, as well as throughout much of the embryo. We propose that Lmx1b.1- and Lmx1b.2-mediated regulation of wnt1, wnt3a, wnt10b, pax8 and fgf8 maintains cell survival in the isthmocerebellar region.

Thyroid-like follicular carcinoma of the kidney is an extremely rare variant of renal cell carcinoma. Most previously reported cases presented as incidental small tumors confined to the kidney. Here we report a unique case in which the patient presented with flank pain and hematuria. Imaging studies demonstrated a large tumor in the right kidney and metastases to the lungs and retroperitoneal lymph nodes. Both the renal tumor and the sampled lung metastasis were composed almost entirely of follicles with dense, colloid-like material resembling thyroid follicular carcinoma. However, no lesion was found in the thyroid gland; and the patient's thyroid function test results were normal. The tumor cells were immunoreactive for PAX2 and PAX8 but lacked reactivity for thyroglobulin and thyroid transcription factor-1. To our knowledge, this is the first case of thyroid-like follicular carcinoma of the kidney to be initially associated with marked symptoms and widespread metastases, providing evidence that this rare variant of renal cell carcinoma can be clinically aggressive.

The prorenin receptor (PRR), a recently discovered component of the renin-angiotensin system, is expressed in the nephron in general and the collecting duct in particular. However, the physiological significance of nephron PRR remains unclear, partly due to developmental abnormalities associated with global or renal-specific PRR gene knockout (KO). Therefore, we developed mice with inducible nephron-wide PRR deletion using Pax8-reverse tetracycline transactivator and LC-1 transgenes and loxP flanked PRR alleles such that ablation of PRR occurs in adulthood, after induction with doxycycline. Nephron-specific PRR KO mice have normal survival to ∼1 yr of age and no renal histological defects. Compared with control mice, PRR KO mice had 65% lower medullary PRR mRNA and protein levels and markedly diminished renal PRR immunofluorescence. During both normal water intake and mild water restriction, PRR KO mice had significantly lower urine osmolality, higher water intake, and higher urine volume compared with control mice. No differences were seen in urine vasopressin excretion, urine Na(+) and K(+) excretion, plasma Na(+), or plasma osmolality between the two groups. However, PRR KO mice had reduced medullary aquaporin-2 levels and arginine vasopressin-stimulated cAMP accumulation in the isolated renal medulla compared with control mice. Taken together, these results suggest nephron PRR can potentially modulate renal water excretion.

To model the differentiation of thyroid epithelial cells, we examined embryoid bodies derived from undifferentiated murine embryonic stem cells treated with activin A to induce endoderm differentiation, the germ layer from which thyroid cells occur. The resulting endodermal cells were then further exposed to TSH and/or IGF-I for up to 21 d. Oct-4 and REX1 expression, required to sustain stem cell self-renewal and pluripotency, were appropriately down-regulated, whereas GATA-4, and alpha-fetoprotein, both endodermal-specific markers, increased as the embryonic stem cells were exposed to activin A. By d 5 culture, TSH receptor (TSHR) and sodium iodide symporter (NIS) gene and protein expression were markedly induced. Cells isolated by the fluorescence-activated cell sorter simultaneously expressed not only TSHR and NIS proteins but also PAX8 mRNA, an expression pattern unique to thyroid cells and expected in committed thyroid progenitor cells. Such expression continued until d 21 with no influence seen by the addition of TSH or IGF-I. The sequence of gene expression changes observed in these experiments demonstrated the emergence of definitive thyroid endoderm. The activin A induction of thyroid-specific markers, NIS and TSHR, occurred in the absence of TSH stimulation, and, therefore, the emergence of thyroid endoderm in vitro paralleled the emergence of thyroid cells in TSHR-knockout mice. Activin A is clearly a major regulator of thyroid endoderm.

Epithelial ovarian cancer presents mostly with serous, endometrioid or mucinous histology but is treated as a single disease. The development of histotype-specific therapy has been challenging because of the relative lack of studies attributing disrupted pathways to a distinct histotype differentiation. mTOR activation is frequently associated with poor prognosis in serous ovarian cancer, which is the most common and most deadly histotype. However, the mechanisms dysregulating mTOR in the pathogenesis of ovarian cancer are unknown. We detected copy number loss and correlated lower expression levels of LKB1, TSC1, TSC2 and PTEN tumor suppressor genes for upstream regulators of mTOR activity in up to 80% in primary ovarian serous tumor databases, with LKB1 allelic loss-predominant. Reduced LKB1 protein was usually associated with increased mTOR activity in both serous ovarian cancer cell lines and primary tumors. Conditional deletion of Lkb1 in murine ovarian surface epithelial (OSE) cells caused papillary hyperplasia and shedding but not tumors. Simultaneous deletion of Lkb1 and Pten, however, led to development of high-grade ovarian serous histotype tumors with 100% penetrance that expressed WT1, ERα, PAX8, TP53 and cytokeratin 8, typical markers used in the differential diagnosis of serous ovarian cancer. Neither hysterectomy nor salpingectomy interfered with progression of ovarian tumorigenesis, suggesting that neither uterine nor Fallopian tube epithelial cells were contributing to tumorigenesis. These results implicate LKB1 loss in the OSE in the pathogenesis of serous ovarian cancer and provide a compelling rationale for investigating the therapeutic potential of targeting LKB1 signaling in patients with this deadly disease.

OBJECTIVE

The aim of this article is to provide an update on the status of the clinical application of thyroid cancer biomarkers.

RESULTS

Our understanding of the tumor cell biology of thyroid cancer of follicular cell origin has improved and modern genomic technological tools are providing new data that may have clinical ramifications. The common somatic genetic changes in thyroid cancer of follicular cell origin (RET/PTC, NTRK, RAS, BRAF, PAX8-PPARgamma) are generally mutually exclusive, with distinct genotype-histologic subtype of thyroid cancer and genotype-phenotype associations observed. Mutation analysis in thyroid nodule fine needle aspiration biopsy has been applied to improve the diagnostic accuracy of fine needle aspiration biopsy and cytologic examination. Gene expression profiling studies have identified numerous diagnostic biomarkers of thyroid cancer that are beginning to be applied in fine needle aspiration biopsy samples to improve diagnosis. The BRAF mutation has recently been shown to be associated with disease aggressiveness, and as an independent prognostic biomarker.

CONCLUSIONS

There has been significant progress toward identifying biomarkers that could improve the accuracy of fine needle aspiration biopsy in the evaluation of patients with thyroid nodule and predicting disease aggressiveness. Future clinical trials evaluating the accuracy and cost-effectiveness of applying these biomarkers in the management of thyroid neoplasm should be considered.

Congenital hypothyroidism affects about 1:3000 to 1:4000 infants and may be caused by defects in thyroidal ontogeny or hormone synthesis. The impressive advances in molecular genetics led to the characterization of numerous genes that are essential for normal development and hormone production of the hypothalamic-pituitary-thyroid axis. Mutations in many of these genes now provide a molecular explanation for a subset of the sporadic and familial forms of congenital hypothyroidism. Defects in one of the multiple steps required for normal hormone synthesis account for about 10% of cases with congenital hypothyroidism. They are typically recessive and therefore more common in inbred families. In the vast majority of patients, congenital hypothyroidism is sporadic and associated with thyroid dysgenesis, a spectrum of developmental defects, which includes the absence of detectable thyroid tissue, ectopic tissue, and thyroid hypoplasia. The molecular defects known to date only explain a minority of these cases and include mutations in the paired box transcription factor PAX8, and the thyroid transcription factors TTF1 and TTF2. It is likely that a further subset of patients with thyroid dysgenesis have defects in other transacting proteins or elements of the signaling pathways controlling growth and function of thyrocytes. In other instances, thyroid dysgenesis may be a polygenic disease or have a multifactorial basis. Aside from providing fundamental insights into the ontogeny and the pathophysiology of the thyroid, the characterization of the molecular basis of congenital hypothyroidism may have growing importance for genetic testing and counseling in the future.

During kidney development, Pax2 and Pax8 are expressed very early in the mammalian nephric duct and both precede the expression of receptor tyrosine kinase, c-Ret. However, in Pax2-/- mutant mice, expression of c-Ret is lost after embryonic day 10.5. As the Ret/Gdnf pathway is necessary for renal development and there is a temporal and spatial relationship of Pax2 and c-Ret expression in the developing genito-urinary system, we postulate that Pax2 is necessary for c-Ret expression in the developing kidney. In vitro, Pax2 protein is capable of physically interacting with a c-RET promoter, and both Pax2 and Pax8 can activate the expression of a reporter gene driven by the c-RET promoter. Compound heterozygous null mice (Pax2+/-: Ret+/-) display an increased incidence of unilateral and bilateral renal agenesis, and smaller kidneys with fewer nephrons. Furthermore, the expression of Gdnf is reduced 2-3-fold, whereas c-Ret expression is reduced 9-47-fold in Pax2 heterozygous embryonic kidneys as detected by real-time quantitative RT (QRT)-PCR. The data demonstrate that Pax2 plays an integral role in the initiation and maintenance of the Ret/Gdnf pathway by not only activating the ligand of the pathway, but by also enhancing the expression of the pathway receptor Ret. The effects of reduced Pax2 gene dosage are thus amplified resulting in a haploinsufficient phenotype.

The demonstration of the PAX8-PPAR(gamma) fusion oncogene in a subset of follicular thyroid tumors provides a new and promising starting point to dissect the molecular genetic events involved in the development of this tumor form. In the present study, we compared the gene expression profiles of follicular thyroid carcinomas (FTCs) bearing a PAX8-PPAR(gamma) fusion against FTCs that lack this fusion. Using unsupervised clustering and multidimensional scaling analyses, we show that FTCs possessing a PAX8-PPAR(gamma) fusion have a highly uniform and distinct gene expression signature that clearly distinguishes them from FTCs without the fusion. The PAX8-PPAR(gamma)(+) FTCs grouped in a defined cluster, where highly ranked genes were mostly associated with signal transduction, cell growth and translation control. Notably, a large number of ribosomal protein and translation-associated genes were concurrently underexpressed in the FTCs with the fusion. Taken together, our findings further support that follicular carcinomas with a PAX8-PPAR(gamma) rearrangement constitute a distinct biological entity. The current data represent one step to elucidate the molecular pathways in the development of FTCs with the specific PAX8-PPAR(gamma) fusion.

Renal cell carcinomas (RCCs) harboring the t(6;11)(p21;q12) translocation were first described in 2001 and recently recognized by the 2013 International Society of Urological Pathology Vancouver Classification of Renal Neoplasia. Although these RCCs are known to label for melanocytic markers HMB45 and Melan A and the cysteine protease cathepsin K by immunohistochemistry (IHC), a comprehensive IHC profile has not been reported. We report 10 new t(6;11) RCCs, all confirmed by break-apart TFEB fluorescence in situ hybridization. A tissue microarray containing 6 of these cases and 7 other previously reported t(6;11) RCCs was constructed and immunolabeled for 21 different antigens. Additional whole sections of t(6;11) RCC were labeled with selected IHC markers. t(6;11) RCC labeled diffusely and consistently for cathepsin K and Melan A (13 of 13 cases) and almost always at least focally for HMB45 (12 of 13 cases). They labeled frequently for PAX8 (14 of 23 cases), CD117 (10 of 14 cases), and vimentin (9 of 13 cases). A majority of cases labeled at least focally for cytokeratin Cam5.2 (8 of 13 cases) and CD10 and RCC marker antigen (10 of 14 cases each). In contrast to a prior study's findings, only a minority of cases labeled for Ksp-cadherin (3 of 19 cases). The median H score (product of intensity score and percentage labeling) for phosphorylated S6, a marker of mTOR pathway activation, was 101, which is high relative to most other RCC subtypes. In summary, IHC labeling for PAX8, Cam5.2, CD10, and RCC marker antigen supports classification of the t(6;11) RCC as carcinomas despite frequent negativity for broad-spectrum cytokeratins and EMA. Labeling for PAX8 distinguishes the t(6;11) RCC from epithelioid angiomyolipoma, which otherwise shares a similar immunoprofile. CD117 labeling is more frequent in the t(6;11) RCC compared with the related Xp11 translocation RCC. Increased pS6 expression suggests a possible molecular target for the uncommon t(6;11) RCCs that metastasize.

Adipocyte differentiation is regulated largely through the actions of the peroxisome proliferator-activated receptor (PPAR) gamma nuclear receptor and the insulin signaling pathway. 3-phosphoinositide-dependent protein kinase-1 (PDK1) serves as a critical regulatory point in insulin signaling through its ability to phosphorylate the activation loop of several protein kinase families. The present study was undertaken to determine the interrelationships between the PDK1 and PPARgamma signaling pathways, and their association with adipocyte differentiation. Coexpression of PDK1 and PPARgamma1 in 293T cells stimulated PPARgamma response element-dependent reporter gene activity in either the presence or absence of ligand. PDK1-mediated stimulation of PPARgamma1 activity was comparable in magnitude to the coactivator activated in breast cancer-1, and was blocked by either the corepressor silencing mediator of retinoid and thyroid hormone receptor or dominant-negative PAX8-PPARgamma1. Heterologous Gal4-PPARgamma1 assays indicated that PDK1 interacted with the ligand binding domain, and physically associated with PPARgamma1; however, PDK1-mediated stimulation was not dependent on phosphorylation of PPARgamma1 by PDK1. PDK1 stimulatory activity was eliminated by mutation of the alpha-helical hydrophobic motifs in PDK1, L(268)XII, and V(313)XXLL, and expression of the alpha-helical region encompassing these motifs stimulated PPARgamma response element-dependent transcription. PDK1-PPARgamma interaction was confirmed by chromatin immunoprecipitation analysis of the lipoprotein lipase and adipocyte fatty acid-binding protein promoters. In cells expressing PDK1 and PPARgamma, binding to PPARgamma response elements occurred, which was enhanced by treatment with a PPARgamma agonist. Expression of PDK1 in 3T3-L1 or COMMA-1D mammary epithelial cells promoted adipocyte differentiation in the presence of a PPARgamma agonist that was comparable to the response of PPARgamma1-transfected cells in the presence of agonist; expression of PDK1 and PPARgamma resulted in a synergistic effect. Adipocyte differentiation in the presence of a PPARgamma agonist was markedly attenuated in PDK1 null cells. These results suggest that PDK1 can function as a PPARgamma1 coactivator independently of its catalytic activity and establishes an important mechanistic link between adipocyte differentiation and the insulin signaling pathway.

Thyroid-enriched transcription factors, Pax-8 and TTF-1, are involved in the thyroid-specific expression of thyroglobulin (TG) gene. Here we demonstrate redox regulation of both factors in vitro and in vivo. When analyzed by electrophoretic mobility shift assay (EMSA), oxidation with diamide abolished the DNA binding of Pax-8. Subsequent reduction with dithiothreitol (DTT) restored the binding. Thioredoxin (TRX), a cellular reducing catalyst, restored the binding more efficiently than DTT. When TTF-1 was oxidized with diamide, its binding was decreased and the TTF-1-DNA complex migrated faster on EMSA. DTT reversed these effects. These observations indicate that reduction is required for full DNA binding of Pax-8 and TTF-1 in vitro. We then examined whether TSH modulates their binding through redox regulation. Whole cell extracts were prepared from FRTL-5 cells at intervals after TSH treatment without reducing agents and subjected to EMSA. Pax-8 and TTF-1 binding activities were gradually increased during the initial 6 h after TSH. This increase was due to reduction of the factors, since treatment of the extracts with DTT masked the increase by enhancing their binding activities. These results suggest that TSH up-regulates the binding of Pax-8 and TTF-1, at least in part, by reducing the preexisting, oxidized forms in FRTL-5 cells. Northern analysis showed that the increase in TRX mRNA level by TSH in FRTL-5 cells was associated with the increase in the binding activities. Cotransfection of luciferase-reporter plasmid driven by TG promoter with Pax-8- and TRX-expressing plasmids into a heterologous cells revealed that TRX up-regulated the Pax8-mediated TG promoter activity. Taken together, the present study suggests that the redox regulation of Pax-8 and TTF-1 by TSH, probably through TRX, modulates the TG gene expression.

BACKGROUND

We studied two sisters with congenital hypothyroidism and choreoathetosis but not respiratory distress.

OBJECTIVE

The aim of this study was to establish the genetic defect that causes this phenotype and study the molecular mechanisms of the pathology by means of functional analysis.

METHODS

Sequencing of DNA, expression vectors generation, EMSAs, transfections experiments as well as bioinformatics analysis were performed.

RESULTS

We found a new single deletion (825delC) in one allele of the TITF1/NKX2.1 gene. The mutation located in the C-terminal domain generates a nonsense thyroid transcription factor 1 (TTF1) protein, with 22 amino less and rich in positive charges. This protein shows diminished binding to DNA, does not interfere with wild-type (wt) TTF1 binding, and fails to activate reporter genes harboring the thyroglobulin (Tg), thyroperoxidase (TPO), or surfactant protein B (SP-B) promoters. In addition, the mutant (mut) protein has a dominant-negative effect on the transcriptional activity of wt TTF1 in a promoter-specific manner, inhibiting the transcription of Tg and TPO but not of SP-B. Using a Gal4 reporter system, we demonstrate that the mut protein is not transcriptionally active and does not likely compete with the wild type for coactivators. Interestingly, the mut protein impairs the wt capacity to synergize with paired box 8 (PAX8). This cooperation is necessary for Tg and TPO transcription but dispensable for SP-B expression.

CONCLUSIONS

These results are concordant with the phenotype of the two sisters studied and demonstrate a differential role for TTF1 in the different tissues in which it is expressed.

Vertebrate cranial placodes contribute vitally to development of sensory structures of the head. Amongst posterior placodes, the otic placode forms the inner ear whereas nearby epibranchial placodes produce sensory ganglia within branchial clefts. Though diverse in fate, these placodes show striking similarities in their early regulation. In zebrafish, both are initiated by localized Fgf signaling plus the ubiquitous competence factor Foxi1, and both express pax8 and sox3 in response. It has been suggested that Fgf initially induces a common otic/epibranchial field, which later subdivides in response to other signals. However, we find that otic and epibranchial placodes form at different times and by distinct mechanisms. Initially, Fgf from surrounding tissues induces otic expression of pax8 and sox3, which cooperate synergistically to establish otic fate. Subsequently, pax8 works with related genes pax2a/pax2b to downregulate otic expression of foxi1, a necessary step for further otic development. Additionally, pax2/8 activate otic expression of fgf24, which induces epibranchial expression of sox3. Knockdown of fgf24 or sox3 causes severe epibranchial deficiencies but has little effect on otic development. These findings clarify the roles of pax8 and sox3 and support a model whereby the otic placode forms first and induces epibranchial placodes through an Fgf-relay.

Neurogenesis is one of the most complex events in embryonic development. However, little information is available regarding the molecular events that occur during neurogenesis. To identify regulatory genes and underlying mechanisms involved in the differentiation of embryonic stem (ES) cells to neurons, gene expression profiling was performed using cDNA microarrays. In mouse ES cells, we compared the gene expression of each differentiated cell stage using a five-stage lineage selection method. Of 10,368 genes, 1633 (16%) known regulatory genes were differentially expressed at least 2-fold or greater at one or more stages. At stage 3, during which ES cells differentiate into neural stem cells, modulation of nearly 1000 genes was observed. Most of transcription factors (Otx2, Ebf-3, Ptx3, Sox4, 13, 18, engrailed, Irx2, Pax8, and Lim3), signaling molecules (Wnt, TGF, and Shh family members), and extracellular matrix/adhesion molecules (collagens, MAPs, and NCAM) were up-regulated. However, some genes which may play important roles in maintaining the pluripotency of ES cells (Kruppel-like factor 2, 4, 5, 9, myeloblast oncogene like2, ZFP 57, and Esg-1) were down-regulated. The many genes identified with this approach that are modulated during neurogenesis will facilitate studies of the mechanisms underlying ES cell differentiation, neural induction, and neurogenesis.