The Pax gene family, encoding transcription factors, has been classified into four subfamilies according to their genomic organization, the sequences of the paired domains (PD) and the expression pattern. Pax1 and Pax9 constitute one subfamily, Pax2, Pax5 and Pax8 another, Pax3 and Pax7 another one and Pax4 and Pax6 the fourth subfamily. The PD exhibits DNA-binding activity, and is the most conserved functional motif in all Pax proteins. A high-resolution analysis of a PD structure has been performed [Xu et al., Cell 80 (1995) 639-650] and the DNA-binding characteristics of members of two Pax subfamilies (Pax2, Pax5 and Pax6) have been determined. Here, we have utilized a PCR-based selection approach to identify the DNA-binding sequences of the Pax3/PD, a member of a subfamily which has not yet been characterized. Comparison of the Pax3/PD-binding sequences with those of other PD proteins revealed both similarities and differences in the DNA-recognition sequence. This suggests that different Pax proteins can regulate the expression of the same target gene, but they can also regulate the expression of completely unrelated genes by binding to their DNA regulatory regions.

Thyroid hormones are essential for a variety of developmental and metabolic processes. Congenital hypothyroidism (CHT) results in severe defects in the development of different tissues, in particular brain. As an animal model for CHT, we studied Pax8(-/-) mice, which are born without a thyroid gland. We determined the expression of iodothyronine deiodinase D1 in liver and kidney, D2 in brain and pituitary, and D3 in brain, as well as serum T(4), T(3), and rT(3) levels in Pax8(-/-) vs. control mice during the first 3 wk of life. In control mice, serum T(4) and T(3) were undetectable on the day of birth (d 0) and increased to maximum levels on d 15. In Pax8(-/-) mice, serum T(4) and T(3) remained below detection limits. Serum rT(3) was high on d 0 in both groups and rapidly decreased in Pax8(-/-), but not in control mice. Hepatic and renal D1 activities and mRNA levels were low on d 0 and increased in control mice roughly parallel to serum T(4) and T(3) levels. In Pax8(-/-) mice, tissue D1 activities and mRNA levels remained low. Cerebral D2 activities were low on d 0 and increased to maximum levels on d 15, which were approximately 10-fold higher in Pax8(-/-) than in control mice. D2 mRNA levels were higher in Pax8(-/-) than in control mice only on d 21. Cerebral D3 activities and mRNA levels were high on d 0 and showed a moderate decrease between d 3 and 15, with values slightly lower in Pax8(-/-) than in control mice. One day after the injection of 200 ng T(4) or 20 ng T(3)/g body weight, tissue deiodinase activities and mRNA levels were at least partially restored toward control levels, with the exception of cerebral D3 activity. In conclusion, these findings show dramatic age and thyroid state-dependent changes in the expression of deiodinases in central and peripheral tissues of mice during the first 3 wk of life.

The vertebrate inner ear develops from a thickening of the embryonic ectoderm, adjacent to the hindbrain, known as the otic placode. All components of the inner ear derive from the embryonic otic placode. Sox proteins form a large class of transcriptional regulators implicated in the control of a variety of developmental processes. One member of this family, Sox9, is expressed in the developing inner ear, but little is known about the early function of Sox9 in this tissue. We report the functional analysis of Sox9 during development of Xenopus inner ear. Sox9 otic expression is initiated shortly after gastrulation in the sensory layer of the ectoderm, in a bilateral patch of cells immediately adjacent to the cranial neural crest. In the otic placode, Sox9 colocalizes with Pax8 one of the earliest gene expressed in response to otic placode inducing signals. Depletion of Sox9 protein in whole embryos using morpholino antisense oligonucleotides causes a dramatic loss of the early otic placode markers Pax8 and Tbx2. Later in embryogenesis, Sox9 morpholino-injected embryos lack a morphologically recognizable otic vesicle and fail to express late otic markers (Tbx2, Bmp4, Otx2 and Wnt3a) that normally exhibit regionalized expression pattern throughout the otocyst. Using a hormone inducible inhibitory mutant of Sox9, we demonstrate that Sox9 function is required for otic placode specification but not for its subsequent patterning. We propose that Sox9 is one of the key regulators of inner ear specification in Xenopus.

Pax8-/- mice do not develop thyroid follicular structures and thus provide an ideal animal model to study the consequences of congenital hypothyroidism. Despite their athyroidism, Pax8-/- mice survive up to postnatal day 21 (P21). No auditory brain stem responses (ABR) to sound could be recorded in these animals at 130 dB SPL, even at P21, when hearing reaches adult sensitivity in control mice. Abnormalities in the outer and middle ear structures were found in a considerable percentage of Pax8-/- animals. Maturation of the inner ear appeared delayed by about 1 week with respect to euthyroid controls. Hearing of adult Pax8-/- mice could be nearly normalized by early postnatal substitution with thyroxine (T(4)), but structural and functional restoration of hearing was incomplete. Even when T(4) substitution was initiated at P1, ABR thresholds, measured at 6 weeks of age or more, were increased by about 20 dB, and each day of delay in the start of T(4) substitution resulted in an additional threshold loss of about 4 dB. The most prominent structural deficit in Pax8-/- animals in which T(4) substitution was started at P8 or later was an abnormally thick tectorial membrane. In these late-substituted animals, disarray of stereovilli from inner and outer hair cells was observed and also outer hair cell loss was found, predominantly in the basal part of the cochlea. The degree of structural disorder increased the later T(4) substitution was initiated. The structural and functional consequences of postnatal athyroidism observed in Pax8-/- mice are largely in agreement with and extend those data obtained from hypothyroid animal models in which hypothyroidism was induced by goitrogenic agents (methimazole, propylthiouracil) or animal models with disrupted genes for the TSH receptor or the thyroid hormone receptors. The hearing loss and also the recovery effect by T(4) substitution in Pax8-/- mice is larger than that in the other models. Although Pax8-/- mice are born by euthyroid Pax8+/- dams, the Pax8-/- phenotype could not be completely restored by immediate postnatal T(4) substitution, indicating that some deficits are the consequence of prenatal T(4) deficiency of the offspring.

Thyroid transcription factor 1 (TTF1), thyroid transcription factor 2 (TTF2) and paired box gene 8 (Pax8) are demonstrated to play a crucial role for the differentiation and organogenesis of thyroid follicular cells. Their roles in thyroid carcinogenesis are not very clear. Because dedifferentiation is a common process in thyroid carcinogenesis, thyroid-specific transcription factors seem also to be involving in thyroid carcinogenesis. The purpose of the present paper was to investigate their expression in a broad spectrum of follicular cell tumors in different degrees of differentiation, from well-differentiated benign follicular adenoma to anaplastic carcinoma. Medullary (C cell) carcinoma was also included in the investigation. Results of immunohistochemical staining showed that nuclear localization of these transcription factors was gradually decreased corresponding to the progressive dedifferentiation of thyroid tumors. Also, abnormal cytoplasmic accumulation of TTF2 and Pax8 was detected in many tumors samples, which may indicate a subtle regulation mechanism on the function of these transcription factors. In conclusion, abnormal expression of TTF1, TTF2 and Pax8 was closely related to thyroid tumorigenesis.

The thyroid gland is an organ primarily composed of endoderm-derived follicular cells. Although disturbed embryonic development of the thyroid gland leads to congenital hypothyroidism in humans and mammals, the underlying principles of thyroid organogenesis are largely unknown. In this study, we introduce zebrafish as a model to investigate the molecular and genetic mechanisms that control thyroid development. Marker gene expression suggests that the molecular pathways of early thyroid development are essentially conserved between fish and mammals. However during larval stages, we find both conserved and divergent features of development compared with mammals. A major difference is that in fish, we find evidence for hormone production not only in thyroid follicular cells, but also in an anterior non-follicular group of cells. We show that pax2.1 and pax8, members of the zebrafish pax2/5/8 paralogue group, are expressed in the thyroid primordium. Whereas in mice, only Pax8 has a function during thyroid development, analysis of the zebrafish pax2.1 mutant no isthmus (noi(-/-)) demonstrates that pax2.1 has a role comparable with mouse Pax8 in differentiation of the thyroid follicular cells. Early steps of thyroid development are normal in noi(-/-), but later expression of molecular markers is lost and the formation of follicles fails. Interestingly, the anterior non-follicular site of thyroid hormone production is not affected in noi(-/-). Thus, in zebrafish, some remaining thyroid hormone synthesis takes place independent of the pathway leading to thyroid follicle formation. We suggest that the noi(-/-) mutant serves as a new zebrafish model for hypothyroidism.

BACKGROUND

The diagnostic limitations of thyroid fine-needle aspiration (FNA), such as the indeterminate category, can be partially overcome by molecular analyses. However, until now, rearrangements have only been detected in fresh FNA material and the number of follicular thyroid carcinomas (FTCs) was rather low in previous studies. We aimed at investigating the impact of point mutations and rearrangement detection in a set of routine air-dried FNA smears with a higher percentage of FTCs.

METHODS

RNA and DNA was extracted from 310 FNAs (164 indeterminate, 57 malignant, 89 benign) and corresponding formalin-fixed paraffin-embedded tissue (156 follicular adenomas [FAs], 32 FTCs, 44 papillary thyroid carcinomas [PTCs], 9 follicular variant PTCs, and 69 goiters). PAX8/PPARG and RET/PTC rearrangements were detected by qPCR, BRAF and RAS mutations by high-resolution melting PCR and by pyrosequencing.

RESULTS

Forty-seven mutations were detected in the FNAs: 22 BRAF, 13 NRAS, and 3 HRAS mutations, 8 PAX8/PPARG, and one RET/PTC-rearrangement. While the presence of a BRAF and RET/PTC mutation was associated with cancer in 100% of samples each, the presence of a RAS and PAX8/PPARG mutation was associated with cancer in only 12% and 50% of samples, respectively. In the indeterminate group 4 of 25 carcinomas were identified by molecular FNA screening, which increased the sensitivity from 67% (cytology alone) to 75% (cytology plus molecular screening).

CONCLUSIONS

Molecular screening for point mutations and rearrangements is feasible in air-dried FNAs. Although the impact of detecting point mutations and rearrangements in FNAs has most likely been overestimated in previous studies, molecular FNA analyses improve presurgical diagnostics. The detection of BRAF mutations in FNA may improve the choice of surgery and postsurgical treatment. Further data are necessary to elucidate the true impact of detecting RAS and PAX8/PPARG mutations in FNAs. The inclusion of additional rare somatic mutations and miRNA markers might further improve the impact of molecular FNA diagnostics.

PAX8 is a nuclear transcription factor with limited expression in normal and neoplastic tissues in a cell lineage-dependent manner. PAX8 has been detected in embryonic Müllerian ducts, human fallopian tubes, and ovarian carcinomas. However, little is known about its expression in other areas of the female genital tract. In this study, we used immunohistochemistry (IHC) to examine PAX8 expression in the normal uterine corpus and cervix, malignant tumors arising from these sites, and malignant effusions. We reported here that PAX8 was also detected in endometrial epithelial cells and endocervical glands, with a lower expression level in the latter, but not in the stromal cells of these areas. All endometrial carcinomas (N = 52) were positive for PAX8, whereas endocervical adenocarcinomas (N = 5) and uterine leiomyosarcomas (N = 3) were negative for PAX8. PAX8 was detected in 70% (22/31) and 68.8% (11/16) of metastatic carcinomas of the ovary and endometrium in serous effusions, respectively. No PAX8 was detected in carcinomas of nongynecologic origin or noncarcinomas (N = 71) in serous effusions except in one renal-cell carcinoma and one carcinoma of unknown primary in a woman. In addition, papillary serous carcinomas of the peritoneum (N = 10) were diffusely positive for PAX8, implying a Müllerian origin similar to malignant tumors in the female genital tract. Our findings suggest that PAX8 is an additional IHC marker for carcinomas of Müllerian origin hence we recommend including PAX8 for evaluation of malignant serous effusions in women, especially when tumors of Müllerian origin are in the differential diagnosis.

In patients, inactivating mutations in the gene encoding the thyroid hormone-transporting monocarboxylate transporter 8 (Mct8) are associated with severe mental and neurological deficits and disturbed thyroid hormone levels. The latter phenotype characterized by high T3 and low T4 serum concentrations is replicated in Mct8 knockout (ko) mice, indicating that MCT8 deficiency interferes with thyroid hormone production and/or metabolism. Our studies of Mct8 ko mice indeed revealed increased thyroidal T3 and T4 concentrations without overt signs of a hyperactive thyroid gland. However, upon TSH stimulation Mct8 ko mice showed decreased T4 and increased T3 secretion compared with wild-type littermates. Moreover, similar changes in the thyroid hormone secretion pattern were observed in Mct8/Trhr1 double-ko mice, which are characterized by normal serum T3 levels and normal hepatic and renal D1 expression in the presence of very low T4 serum concentrations. These data strongly indicate that absence of Mct8 in the thyroid gland affects thyroid hormone efflux by shifting the ratio of the secreted hormones toward T3. To test this hypothesis, we generated Mct8/Pax8 double-mutant mice, which in addition to Mct8 lack a functional thyroid gland and are therefore completely athyroid. Following the injection of these animals with either T4 or T3, serum analysis revealed T3 concentrations similar to those observed in Pax8 ko mice under thyroid hormone replacement, indicating that indeed increased thyroidal T3 secretion in Mct8 ko mice represents an important pathogenic mechanism leading to the high serum T3 levels.

Thyroid carcinoma is the most common endocrine malignancy, and its incidence is continuing to increase. Most thyroid carcinomas contain one of several known driver mutations, such as the Val600Glu substitution in B-Raf, Ras mutations, RET gene fusions, or PAX8-PPARG gene fusions. The PAX8-PPARG gene fusion results in the production of a Pax-8-PPAR-γ fusion protein (PPFP), which is found in approximately one-third of follicular thyroid carcinomas, as well as some follicular-variant papillary thyroid carcinomas. In vitro and in vivo evidence indicates that PPFP is an oncoprotein. Although specific mechanisms of action remain to be defined, PPFP is considered to act as a dominant-negative inhibitor of wild-type PPAR-γ and/or as a unique transcriptional activator of subsets of PPAR-γ-responsive and Pax-8-responsive genes. Detection of the fusion transcript in thyroid nodule biopsy specimens can aid clinical decision-making when cytological findings are indeterminate. The PPAR-γ agonist pioglitazone is highly therapeutic in a transgenic mouse model of PPFP-positive thyroid carcinoma, suggesting that PPAR-γ agonists might be beneficial in patients with PPFP-positive thyroid carcinomas.

TGFβ signaling plays a central role in the development of acute and chronic kidney diseases. Previous in vivo studies involved systemic alteration of TGFβ signaling, however, limiting conclusions about the direct role of TGFβ in tubular cell injury. Here, we generated a double transgenic mouse that inducibly expresses a ligand-independent constitutively active TGFβ receptor type 1 (TβR1) kinase specifically in tubular epithelial cells, with expression restricted by the Pax8 promoter. In this model, activation of TGFβ signaling in the tubular epithelium alone was sufficient to cause AKI characterized by marked tubular cell apoptosis and necrosis, oxidative stress, dedifferentiation and regenerative cell proliferation, reduced renal function, and interstitial accumulation of inflammatory cells. This tubular injury was associated with mitochondrial-derived generation of reactive oxygen species (ROS), but cell damage and apoptosis were partially independent of mitochondrial-derived ROS. TβR1 signaling-induced tubular injury also associated with significant leukocyte infiltration consisting of F4/80(+) macrophages, CD11c(+) F4/80(+) dendritic cells, CD11c(+) F4/80(-) Ly6C(high) dendritic cells/monocytes, and T cells. Inhibition of mitochondrial-derived ROS significantly reduced accumulation of CD11c(+) F4/80(+) dendritic cells and T cells, suggesting a role for ROS in the activation and recruitment of the adaptive immune response to tubular injury. Taken together, these results suggest that TGFβ signaling in the tubular epithelium alone is sufficient to cause acute tubular injury and inflammation; therefore, TGFβ may be a mechanistic link between acute injury and chronic progression of kidney disease.

Nephrons comprise a blood filter and an epithelial tubule that is subdivided into proximal and distal segments, but what directs this patterning during kidney organogenesis is not well understood. Using zebrafish, we found that the HNF1β paralogues hnf1ba and hnf1bb, which encode homeodomain transcription factors, are essential for normal segmentation of nephrons. Embryos deficient in hnf1ba and hnf1bb did not express proximal and distal segment markers, yet still developed an epithelial tubule. Initiating hnf1ba/b expression required Pax2a and Pax8, but hnf1ba/b-deficient embryos did not exhibit the expected downregulation of pax2a and pax8 at later stages of development, suggesting complex regulatory loops involving these molecules. Embryos deficient in hnf1ba/b also did not express the irx3b transcription factor, which is responsible for differentiation of the first distal tubule segment. Reciprocally, embryos deficient in irx3b exhibited downregulation of hnf1ba/b transcripts in the distal early segment, suggesting a segment-specific regulatory circuit. Deficiency of hnf1ba/b also led to ectopic expansion of podocytes into the proximal tubule domain. Epistasis experiments showed that the formation of podocytes required wt1a, which encodes the Wilms' tumor suppressor-1 transcription factor, and rbpj, which encodes a mediator of canonical Notch signaling, downstream or parallel to hnf1ba/b. Taken together, these results suggest that Hnf1β factors are essential for normal segmentation of nephrons during kidney organogenesis.

The PAX8/PPARgamma (PPFP) fusion-oncogene is moderately specific for follicular thyroid carcinomas (FTC). It remains unknown whether this can be translated into improved diagnosis, classification, or outcome prediction. We studied a cohort of well-characterized follicular adenomas (FA), FTC, and Hurthle cell carcinomas (HCC) from patients with complete clinical follow-up, to determine whether PPARgamma immunohistochemistry (as a surrogate of PAX8/PPARgamma expression) helps to distinguish FA from FTC and to assess its diagnostic accuracy as an adjunct to frozen section. We also correlated PPARgamma staining with clinical outcomes to assess its role as a prognostic marker.PPARgamma staining was more common in FTC (31 of 54; 57%) than in HCC (one of 23; 4%) or FA (four of 31; 13%) (P < 0.000001). Adjunctive use of PPARgamma immunohistochemistry improved diagnostic sensitivity of intraoperative frozen section from 84% to 96% (P < 0.05) but reduced specificity from 100% to 90% (P < 0.05). PPARgamma staining was associated with favorable prognostic indicators (female gender, better tumor differentiation, and lesser risk of metastases).PPARgamma staining may be helpful in the differential diagnosis of FA, FTC, and HCC, particularly when diagnostic sensitivity of histomorphology is reduced (e.g. during intraoperative frozen section). PPARgamma staining also shows an association with favorable prognosis and may have a role in risk stratification.

Congenital hypothyroidism with thyroid dysgenesis (TD) is a frequent human condition characterized by elevated levels of TSH in response to reduced thyroid hormone levels. Congenital hypothyroidism is a genetically heterogeneous disease. In the majority of cases studied, no causative mutations have been identified and very often the disease does not show a Mendelian transmission. However, in approximately 5% of cases, it can be a consequence of mutations in genes encoding the TSH receptor or the transcription factors TITF1, FOXE1, or PAX8. We report here that in mouse models, the combination of partial deficiencies in the Titf1 and Pax8 genes results in an overt TD phenotype that is absent in either of the singly deficient, heterozygous mice. The disease is characterized by a small thyroid gland, elevated levels of TSH, reduced thyroglobulin biosynthesis, and high occurrence of hemiagenesis. The observed phenotype is strain specific, and the pattern of transmission indicates that at least two other genes, in addition to Titf1 and Pax8, are necessary to generate the condition. These results show that TD can be of multigenic origin in mice and strongly suggest that a similar pathogenic mechanism may be observed in humans.

Sleep is an essential state of decreased activity and alertness but molecular factors regulating sleep duration remain unknown. Through genome-wide association analysis in 446,118 adults of European ancestry from the UK Biobank, we identify 78 loci for self-reported habitual sleep duration (p < 5 × 10^-8; 43 loci at p < 6 × 10^-9). Replication is observed for PAX8, VRK2, and FBXL12/UBL5/PIN1 loci in the CHARGE study (n = 47,180; p < 6.3 × 10^-4), and 55 signals show sign-concordant effects. The 78 loci further associate with accelerometer-derived sleep duration, daytime inactivity, sleep efficiency and number of sleep bouts in secondary analysis (n = 85,499). Loci are enriched for pathways including striatum and subpallium development, mechanosensory response, dopamine binding, synaptic neurotransmission and plasticity, among others. Genetic correlation indicates shared links with anthropometric, cognitive, metabolic, and psychiatric traits and two-sample Mendelian randomization highlights a bidirectional causal link with schizophrenia. This work provides insights into the genetic basis for inter-individual variation in sleep duration implicating multiple biological pathways.

OBJECTIVE

(131)I therapy is regularly used following surgery as a part of thyroid cancer management. Despite an overall relatively good prognosis, recurrent or metastatic thyroid cancer is not rare. CD133-expressing cells have been shown to mark thyroid cancer stem cells that possess the characteristics of stem cells and have the ability to initiate tumours. However, no studies have addressed the influence of CD133-expressing cells on radioiodide therapy of the thyroid cancer. The aim of this study was to investigate whether CD133(+) cells contribute to the radioresistance of thyroid cancer and thus potentiate future recurrence and metastasis.

METHODS

Thyroid cancer cell lines were analysed for CD133 expression, radiosensitivity and gene expression.

RESULTS

The anaplastic thyroid cancer cell line ARO showed a higher percentage of CD133(+) cells and higher radioresistance. After γ-irradiation of the cells, the CD133(+) population was enriched due to the higher apoptotic rate of CD133(-) cells. In vivo (131)I treatment of ARO tumour resulted in an elevated expression of CD133, Oct4, Nanog, Lin28 and Glut1 genes. After isolation, CD133(+) cells exhibited higher radioresistance and higher expression of Oct4, Nanog, Sox2, Lin28 and Glut1 in the cell line or primarily cultured papillary thyroid cancer cells, and lower expression of various thyroid-specific genes, namely NIS, Tg, TPO, TSHR, TTF1 and Pax8.

CONCLUSIONS

This study demonstrates the existence of CD133-expressing thyroid cancer cells which show a higher radioresistance and are in an undifferentiated status. These cells possess a greater potential to survive radiotherapy and may contribute to the recurrence of thyroid cancer. A future therapeutic approach for radioresistant thyroid cancer may focus on the selective eradication of CD133(+) cells.

Mammalian retinas display an astonishing diversity in the spatial arrangement of their spectral cone photoreceptors, probably in adaptation to different visual environments. Opsin expression patterns like the dorsoventral gradients of short-wave-sensitive (S) and middle- to long-wave-sensitive (M) cone opsin found in many species are established early in development and thought to be stable thereafter throughout life. In mouse early development, thyroid hormone (TH), through its receptor TRβ2, is an important regulator of cone spectral identity. However, the role of TH in the maintenance of the mature cone photoreceptor pattern is unclear. We here show that TH also controls adult cone opsin expression. Methimazole-induced suppression of serum TH in adult mice and rats yielded no changes in cone numbers but reversibly altered cone patterns by activating the expression of S-cone opsin and repressing the expression of M-cone opsin. Furthermore, treatment of athyroid Pax8(-/-) mice with TH restored a wild-type pattern of cone opsin expression that reverted back to the mutant S-opsin-dominated pattern after termination of treatment. No evidence for cone death or the generation of new cones from retinal progenitors was found in retinas that shifted opsin expression patterns. Together, this suggests that opsin expression in terminally differentiated mammalian cones remains subject to control by TH, a finding that is in contradiction to previous work and challenges the current view that opsin identity in mature mammalian cones is fixed by permanent gene silencing.

Signaling by TSH through its receptor leads to the dissociation of trimeric G proteins into Galpha and Gbetagamma. Galphas activates adenylyl cyclase, which increases cAMP levels that induce several effects in the thyroid cell, including transcription of the sodium-iodide symporter (NIS) gene through a mechanism involving Pax8 binding to the NIS promoter. Much less is known about the function of Gbetagamma in thyroid differentiation, and therefore we studied their role in TSH signaling. Gbetagamma overexpression inhibits NIS promoter activation and reduces NIS protein accumulation in response to TSH and forskolin. Conversely, inhibition of Gbetagamma-dependent pathways increases NIS promoter activity elicited by TSH but does not modify forskolin-induced activation. Gbetagamma dimers are being released from the Gs subfamily of proteins, because cholera toxin mimics the effects elicited by TSH, whereas pertussis toxin has no effect on NIS promoter activity. We also found that TSH stimulates Akt phosphorylation in a phosphoinositide 3-kinase (PI3K)-dependent and cAMP-independent manner. This is mediated by Gbetagamma, because its overexpression or specific sequestration, respectively, increased or reduced phosphorylated Akt levels upon TSH stimulation. Gbetagamma sequestration increases NIS protein levels induced by TSH and Pax8 binding to the NIS promoter, which is also increased by PI3K inhibition. This is, at least in part, caused by Gbetagamma-mediated Pax8 exclusion from the nucleus that is attenuated when PI3K activity is blocked. These data unequivocally demonstrate that Gbetagamma released by TSH action stimulate PI3K, inhibiting NIS gene expression in a cAMP-independent manner due to a decrease in Pax8 binding to the NIS promoter.

Activating mutations in RAS protooncogenes are associated with several different histotypes of thyroid cancer, including anaplastic thyroid carcinoma. The latter is the most aggressive cancer of the thyroid gland, showing little or no expression of the differentiated phenotype. Likewise, expression of viral RAS oncogenes in FRTL-5 rat thyroid cells mimics such loss of differentiation. We established FRTL-5 cell lines stably expressing constitutively active forms of RAS, either of viral (v-Ha-RAS or v-Ki-RAS) or cellular (H-RAS(V12)) origin and generated a tamoxifen-inducible RAS oncoprotein to analyze the timing of RAS effects on thyroid differentiation. In RAS-transformed FRTL-5 cells, we measured the expression of many thyroid-specific genes by real-time PCR and observed that a clear loss of differentiation was only obtained in the presence of high RAS oncogene expression. In contrast, TSH-independent growth appeared to be induced in the presence of both low and high levels of oncogenic RAS expression. We also showed that inhibition of differentiation is an early RAS-induced phenomenon. Finally, we demonstrated that only high doses of RAS oncogenes are able to inhibit the activity of Titf1 and Pax8, two transcription factors essential for the maintenance of thyroid differentiation, and that the homeodomain of Titf1 is a target of the inhibitory action of RAS. Our results represent the first evidence of a dose-dependent effect of RAS oncogenes on thyroid epithelial differentiation.

The embryonic kidney is a classic developmental model system for studying inductive tissue interactions that govern organogenesis. We report here that Wnt-6 is expressed in the ureter bud, and that cell lines expressing Wnt-6 induce nephrogenesis in vitro. Wnt-6 cells induce tubules with similar kinetics to spinal cord (SPC) and lead to induced expression of Pax2, Pax8, Sfrp2, and E-cadherin genes, early markers of tubulogenesis. Moreover, Wnt-6 signaling rescues tubulogenesis in mesenchyme separated from Wnt-4 mutant embryos and leads to activation of Wnt-4 transcription. Wnt-6 also induces a secondary axis in early Xenopus embryos. We conclude that Wnt-6 is a candidate for the ureter epithelium-derived signal that leads to activation of kidney tubulogenesis via Wnt-4.

Mutations in the paired domain transcription factor PAX8 are a rare cause of congenital hypothyroidism due to thyroid dysgenesis. We identified a novel and unique PAX8 mutation segregating in seven affected members of a three-generations family. The mutation replaces an invariant serine residue within helix 2 of the paired DNA-binding domain for phenylalanine. The mutant protein (PAX8-S48F) does not induce the thyroglobulin promoter in nonthyroid cells, but displays almost half of wild-type PAX8 activity in thyroid cells. PAX8-S48F shows no defect in expression, nuclear targeting, or DNA binding and retains the ability to synergize with thyroid transcription factor 1 (TTF-1, NKX2.1). However, we found that in nonthyroid cells, the acetylation-independent synergism with the general transcriptional adaptor p300 is completely abrogated, suggesting that PAX8-S48F may be unable to efficiently recruit p300. Reconstitution experiments in nonthyroid cells reveal that TTF-1 can partially rescue PAX8-S48F/p300 synergism and thus reproduce the situation in thyroid cells. These functional characteristics result in a dominant negative effect of PAX8-S48F on coexpressed wild-type PAX8 activity, which is not observed in paired domain mutations with DNA binding defect. Our results describe the first dominant negative missense mutation in a paired domain and provide evidence for a crucial role of the p300 coactivator in mediating the functional synergism between PAX8 and TTF-1 in thyroid-specific gene expression.

PAX5 is a nuclear transcription factor required for B cell development, and its expression was evaluated in upper aerodigestive malignancies and pancreatic cancer by immunoblotting. The PAX5 protein expression was relatively strong in small-cell lung cancer (SCLC, 11/12); however, its expression was not detected in non-SCLC (NSCLC, n=13), mesothelioma (n=7), pancreatic (n=6), esophageal (n=6) and head and neck cancer cell lines (n=12). In comparison, PAX8 and PAX3 expressions were absent or non-detectable in SCLC cell lines; however, PAX8 was expressed in most of the tested NSCLC cell lines (13/13) and also frequently in all the other cell lines. We also detected frequent expressions of PAX2 and PAX9 protein in the various cell lines. Utilizing neuroendocrine tumor samples, we found that the frequency as well as the average intensity of the expression of PAX5 increased from pulmonary carcinoid (9%, moderate and strong PAX5 expression, n=44), to large-cell neuroendocrine carcinoma (LCNC, 27%, n=11) to SCLC (33%, n=76). FISH analysis revealed no translocations of the PAX5 gene, but polyploidy in some SCLC tumor tissues (6/37). We determined that PAX5 could regulate the transcription of c-Met using luciferase-coupled reporter and chromatin immunoprecipitation analysis. In addition, the phospho-c-Met (active form) and PAX5 were both localized to the same intra-nuclear compartment in hepatocyte growth factor treated SCLC cells and interacted with each other. Finally, we determined the therapeutic translational potential of PAX5 using PAX5 knockdown SCLC cells in conjunction with Topoisomerase 1 (SN38) and c-Met (SU11274) inhibitors. Loss of endogenous PAX5 significantly decreased the viability of SCLC cells, especially when combined with SN38 or SU11274, and maximum effect was seen when both inhibitors were used. Therefore, we propose that PAX5 could be an important regulator of c-Met transcription and a potential target for therapy in SCLC.

Xp11 translocation cancers include Xp11 translocation renal cell carcinoma (RCC), Xp11 translocation perivascular epithelioid cell tumor (PEComa), and melanotic Xp11 translocation renal cancer. In Xp11 translocation cancers, oncogenic activation of TFE3 is driven by the fusion of TFE3 with a number of different gene partners; however, the impact of individual fusion variant on specific clinicopathologic features of Xp11 translocation cancers has not been well defined. In this study, we analyze 60 Xp11 translocation cancers by fluorescence in situ hybridization using custom bacterial artificial chromosome probes to establish their TFE3 fusion gene partner. In 5 cases RNA sequencing was also used to further characterize the fusion transcripts. The 60 Xp11 translocation cancers included 47 Xp11 translocation RCC, 8 Xp11 translocation PEComas, and 5 melanotic Xp11 translocation renal cancers. A fusion partner was identified in 53/60 (88%) cases, including 18 SFPQ (PSF), 16 PRCC, 12 ASPSCR1 (ASPL), 6 NONO, and 1 DVL2. We provide the first morphologic description of the NONO-TFE3 RCC, which frequently demonstrates subnuclear vacuoles leading to distinctive suprabasal nuclear palisading. Similar subnuclear vacuolization was also characteristic of SFPQ-TFE3 RCC, creating overlapping features with clear cell papillary RCC. We also describe the first RCC with a DVL2-TFE3 gene fusion, in addition to an extrarenal pigmented PEComa with a NONO-TFE3 gene fusion. Furthermore, among neoplasms with the SFPQ-TFE3, NONO-TFE3, DVL2-TFE3, and ASPL-TFE3 gene fusions, the RCCs are almost always PAX8 positive, cathepsin K negative by immunohistochemistry, whereas the mesenchymal counterparts (Xp11 translocation PEComas, melanotic Xp11 translocation renal cancers, and alveolar soft part sarcoma) are PAX8 negative, cathepsin K positive. These findings support the concept that despite an identical gene fusion, the RCCs are distinct from the corresponding mesenchymal neoplasms, perhaps due to the cellular context in which the translocation occurs. We corroborate prior data showing that the PRCC-TFE3 RCCs are the only known Xp11 translocation RCC molecular subtype that are consistently cathepsin K positive. In summary, our data expand further the clinicopathologic features of cancers with specific TFE3 gene fusions and should allow for more meaningful clinicopathologic associations to be drawn.

Retinoic acid (RA) has pleiotropic functions during embryogenesis. In zebrafish, increasing or blocking RA signaling results in enlarged or reduced otic vesicles, respectively. Here we elucidate the mechanisms that underlie these changes and show that they have origins in different tissues. Excess RA leads to ectopic foxi1 expression throughout the entire preplacodal domain. Foxi1 provides competence to adopt an otic fate. Subsequently, pax8, the expression of which depends upon Foxi1 and Fgf, is also expressed throughout the preplacodal domain. By contrast, loss of RA signaling does not affect foxi1 expression or otic competence, but instead results in delayed onset of fgf3 expression and impaired otic induction. fgf8 mutants depleted of RA signaling produce few otic cells, and these cells fail to form a vesicle, indicating that Fgf8 is the primary factor responsible for otic induction in RA-depleted embryos. Otic induction is rescued by fgf8 overexpression in RA-depleted embryos, although otic vesicles never achieve a normal size, suggesting that an additional factor is required to maintain otic fate. fgf3;tcf2 double mutants form otic vesicles similar to RA-signaling-depleted embryos, suggesting a signal from rhombomere 5-6 may also be required for otic fate maintenance. We show that rhombomere 5 wnt8b expression is absent in both RA-signaling-depleted embryos and in fgf3;tcf2 double mutants, and inactivation of wnt8b in fgf3 mutants by morpholino injection results in small otic vesicles, similar to RA depletion in wild type. Thus, excess RA expands otic competence, whereas the loss of RA impairs the expression of fgf3 and wnt8b in the hindbrain, compromising the induction and maintenance of otic fate.