Loretta L. Collins

Deficits in Motor Coordination with Aberrant Cerebellar Development in Mice Lacking Testicular Orphan Nuclear Receptor 4

ABSTRACT Since testicular orphan nuclear receptor 4 (TR4) was cloned, its physiological function has remained largely unknown. Throughout postnatal development, TR4-knockout (TR4−/−) mice exhibited behavioral deficits in motor coordination, suggesting impaired cerebellar function. Histological examination of the postnatal TR4−/− cerebellum revealed gross abnormalities in foliation; specifically, lobule VII in the anterior vermis was missing. Further analyses demonstrated that the laminations of the TR4−/− cerebellar cortex were changed, including reductions in the thickness of the molecular layer and the internal granule layer, as well as delayed disappearance of the external granule cell layer (EGL). These lamination irregularities may result from interference with granule cell proliferation within the EGL, delayed inward migration of postmitotic granule cells, and a higher incidence of apoptotis. In addition, abnormal development of Purkinje cells was observed in the postnatal TR4−/− cerebellum, as evidenced by aberrant dendritic arborization and reduced calbindin staining intensity. Expression of Pax-6, Sonic Hedgehog (Shh), astrotactin (Astn), reelin, and Cdk-5, genes correlated with the morphological development of the cerebellum, is reduced in the developing TR4−/− cerebellum. Together, our findings suggest that TR4 is required for normal cerebellar development.

Loss of TR4 Orphan Nuclear Receptor Reduces Phosphoenolpyruvate Carboxykinase-Mediated Gluconeogenesis

OBJECTIVE—Regulation of phosphoenolpyruvate carboxykinase (PEPCK), the key gene in gluconeogenesis, is critical for glucose homeostasis in response to quick nutritional depletion and/or hormonal alteration. RESEARCH DESIGN/METHODS AND RESULTS— Here, we identified the testicular orphan nuclear receptor 4 (TR4) as a key PEPCK regulator modulating PEPCK gene via a transcriptional mechanism. TR4 transactivates the 490-bp PEPCK promoter-containing luciferase reporter gene activity by direct binding to the TR4 responsive element (TR4RE) located at −451 to −439 in the promoter region. Binding to TR4RE was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Eliminating TR4 via knockout and RNA interference (RNAi) in hepatocytes significantly reduced the PEPCK gene expression and glucose production in response to glucose depletion. In contrast, ectopic expression of TR4 increased PEPCK gene expression and hepatic glucose production in human and mouse hepatoma cells. Mice lacking TR4 also display reduction of PEPCK expression with impaired gluconeogenesis. CONCLUSIONS—Together, both in vitro and in vivo data demonstrate the identification of a new pathway, TR4 → PEPCK → gluconeogenesis → blood glucose, which may allow us to modulate metabolic programs via the control of a new key player, TR4, a member of the nuclear receptor superfamily.

2,3,7,8-Tetracholorodibenzo-p-Dioxin Exposure Disrupts Granule Neuron Precursor Maturation in the Developing Mouse Cerebellum

The widespread environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been linked to developmental neurotoxicity associated with abnormal cerebellar maturation in both humans and rodents. TCDD mediates toxicity via binding to the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of xenobiotic metabolizing enzymes and growth regulatory molecules. Our previous studies demonstrated that cerebellar granule neuron precursor cells (GNPs) express transcriptionally active AhR during critical developmental periods. TCDD exposure also impaired GNP proliferation and survival in vitro. Therefore, this study tested the hypothesis that TCDD exposure disrupts cerebellar development by interfering with GNP differentiation. In vivo experiments indicated that TCDD exposure on postnatal day (PND) 6 resulted in increased expression of a mitotic marker and increased thickness of the external granule layer (EGL) on PND10. Expression of the early differentiation marker TAG-1 was also more pronounced in postmitotic, premigratory granule neurons of the EGL, and increased apoptosis of GNPs was observed. On PND21, expression of the late GNP differentiation marker GABA(A alpha 6) receptor (GABAR(A alpha 6)) and total estimated cell numbers were both reduced following exposure on PND6. Studies in unexposed adult AhR(-/-) mice revealed lower GABAR(A alpha 6) levels and DNA content. In vitro studies showed elevated expression of the early differentiation marker p27/Kip1 and the GABAR(A alpha 6) in GNPs following TCDD exposure, and the expression patterns of proteins related to granule cell neurite outgrowth, beta III-tubulin and polysialic acid neural cell adhesion molecule, were consistent with enhanced neuroblast differentiation. Together, our data suggest that TCDD disrupts a normal physiological role of AhR, resulting in compromised GNP maturation and neuroblast survival, which impacts final cell number in the cerebellum.

Spermatogenesis and Testis Development Are Normal in Mice Lacking Testicular Orphan Nuclear Receptor 2

ABSTRACT Early in vitro cell culture studies suggested that testicular orphan nuclear receptor 2 (TR2), a member of the nuclear receptor superfamily, may play important roles in the control of several pathways including retinoic acids, vitamin D, thyroid hormones, and ciliary neurotrophic factor. Here we report the surprising results showing that mice lacking TR2 are viable and have no serious developmental defects. Male mice lacking TR2 have functional testes, including normal sperm number and motility, and both male and female mice lacking TR2 are fertile. In heterozygous TR2+/− male mice we found that β-galactosidase, the indicator of TR2 protein expression, was first detected at the age of 3 weeks and its expression pattern was restricted mainly in the spermatocytes and round spermatids. These protein expression patterns were further confirmed with Northern blot analysis of TR2 mRNA expression. Together, results from TR2-knockout mice suggest that TR2 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. Alternatively, the roles of TR2 may be redundant and could be played by other close members of the nuclear receptor superfamily such as testicular orphan receptor 4 (TR4) or unidentified orphan receptors that share many similar functions with TR2. Further studies with double knockouts of both orphan nuclear receptors, TR2 and TR4, may reveal their real physiological roles.

DNA cytometry and chromosome 9 aberrations by fluorescence in situ hybridization of irrigation specimens from bladder cancer patients

OBJECTIVES To determine the sensitivity and specificity of combining fluorescence in situ hybridization (FISH) measurement of chromosome 9 and DNA cytometry of bladder irrigation specimens in the detection of bladder cancer. METHODS Bladder irrigation specimens were obtained from 37 normal control patients and 317 bladder cancer patients during cystoscopic examinations. Bladder cancer patients were sampled in the absence of observable tumor (256 specimens) and concurrently with tumor (204 specimens). Chromosome 9 copy number was determined on a cellular basis by FISH, and cellular DNA content was determined by Feulgen DNA staining and image cytometry. RESULTS Sensitivity of chromosome 9 FISH was 42% for all tumors and was not correlated to transitional cell carcinoma tumor grade, while the sensitivity of DNA cytometry was 55% and improved with increasing grade from 38% for grade 1 to 90% for grade 3 tumors. The results of FISH and DNA cytometry were combined, resulting in specificity of 92% and sensitivity of 69% for grade 1, 76% for grade 2, and 97% for grade 3 tumors. CONCLUSIONS The lack of increase with grade in the percentage of positive specimens by FISH supports the hypothesis that chromosome 9 aberrations are critical events in bladder tumorigenesis for many patients. These data demonstrate the presence of cells in irrigation specimens with specific genomic lesions of chromosome 9 and DNA content. Combining FISH on chromosome 9 and DNA cytometry provides an increase in sensitivity to transitional cell carcinoma over either test alone.

The p53/retinoblastoma-mediated repression of testicular orphan receptor-2 in the rhesus monkey with cryptorchidism.

Whereas the linkage of infertility to cryptorchidism, the failure of the testis to descend into the scrotum at birth, has been well documented, the detailed molecular mechanism remains unclear. Here we report that the testicular orphan receptor-2 (TR2) expression, which modulates many signal pathways, was completely repressed in the surgery-induced cryptorchidism of the rhesus monkey. Further studies link TR2 repression to the induction of p53 and results suggest that induced p53 could repress TR2 expression via the p53→p21→CDK→Rb→E2F signal pathway. In return, TR2 could also control the expression of p53 and Rb through the regulation of human papillomavirus 16 E6/E7 genes. Together, our data suggest a feedback control mechanism between TR2 and p53/Rb tumor suppressors, which might play important roles in male infertility associated with cryptorchidism.

CHROMOSOME 9 MONOSOMY BY FLUORESCENCE IN SITU HYBRIDIZATION OF BLADDER IRRIGATION SPECIMENS IS PREDICTIVE OF TUMOR RECURRENCE

PURPOSE Bladder irrigation specimens are effective for sampling the urothelium for detection of recurrent bladder cancer. These specimens can be evaluated by cytology or quantitative techniques. Proliferation and ploidy changes are readily detected using deoxyribonucleic acid (DNA) cytometry. Tumor associated chromosomal aberrations can be assayed using fluorescence in situ hybridization (FISH). The prognostic values of DNA cytometry, and chromosome 9 and 9p21 FISH on exfoliated cells from bladder irrigation specimens from 61 bladder cancer patients were evaluated. MATERIALS AND METHODS A total of 61 consecutive bladder irrigation specimens were obtained during cystoscopy. DNA cytometry was performed by image analysis. FISH was performed using a centromeric chromosome 9 probe and a cosmid contig (COSp16) probe to the CDKN2A/p16 tumor suppressor region of 9p21. Proportional hazards regression analysis was performed with statistical software to test the predictor variables of initial patient status (presence of tumor), COSp16 fraction (the proportion of COSp16 signals relative to centromeric probe signals), monosomic and hyperdisomic fractions of the chromosome 9 probe, and hyperdiploid fraction from DNA cytometry. Median time to recurrence was calculated using statistical software survival analysis. RESULTS Initial patient status and monosomy of chromosome 9 were predictive of bladder cancer recurrence (p <0.0001 and p = 0.0073, respectively). The 11 patients with chromosome 9 monosomy fractions greater than 15% and a visible tumor had a median time to recurrence of 105 days. In contrast, only 8 of the 25 patients with chromosome 9 monosomy fractions less than 15% and no visible tumor had recurrence within 560 days. Median time to recurrence was 185 days for 6 patients with chromosome 9 monosomy fractions greater than 15% and no visible tumor, and 225 for 19 with chromosome 9 monosomy fractions less than 15% and a visible tumor. Hyperdiploid fraction was suggestive but not predictive of bladder cancer recurrence (p = 0.078). COSp16 and hyperdisomic fractions were not predictive of bladder tumor recurrence (p = 0.11 and p = 0.30, respectively). CONCLUSIONS Chromosome 9 monosomy by FISH was predictive of bladder tumor recurrence. Furthermore, our findings support the hypothesis that losses of tumor suppressor genes on chromosome 9 are critical, perhaps initiating genetic events in bladder cancer.

The roles of testicular orphan nuclear receptor 4 (TR4) in cerebellar development

Since Testicular Receptor 4 (TR4) was cloned, efforts have been made to elucidate its physiological function. To examine the putative functions of TR4, the conventional TR4 knockout (TR4−/−) mouse model was generated. Throughout postnatal and adult stages, TR4−/− mice exhibited behavioral deficits in motor coordination, suggesting impaired cerebellar function. Histological examination of the postnatal and adult TR4−/− cerebellum revealed gross abnormalities in foliation. Further analyses demonstrated changes in the lamination of the TR4−/− cerebellar cortex, including reduction in the thickness of both the molecular layer (ML) and the internal granule layer (IGL). Analyses of the developing TR4−/− cerebellum indicate that the lamination irregularities observed may result from disrupted granule cell proliferation within the external granule cell layer (EGL), delayed inward migration of post-mitotic granule cells, and increased apoptosis during cerebellar development. In addition, abnormal development of Purkinje cells was observed in the postnatal TR4−/− cerebellum, as indicated by aberrant dendritic arborization. In postnatal, neuronal-specific TR4 knockout mice, architectural changes in the cerebellum were similar to those seen in TR4−/− animals, suggesting that TR4 function in neuronal lineages might be important for cerebellar morphogenesis, and that the effect on Purkinje cell development is likely mediated by changes elsewhere, such as in granule cells, or is highly dependent on developmental stage. Together, our findings from various TR4 knockout mouse models suggest that TR4 is required for normal cerebellar development and that failure to establish proper cytoarchitecture results in dysfunction of the cerebellum and leads to abnormal behavior.

Activation of the Aryl Hydrocarbon Receptor during Different Critical Windows in Pregnancy Alters Mammary Epithelial Cell Proliferation and Differentiation

Exposure to the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during pregnancy causes severe defects in mammary gland development and function; however, the underlying mechanism remains unclear. Alterations in epithelial cell proliferation, differentiation, and apoptosis during pregnancy-related mammary development can lead to failed lactogenesis. To determine which of these processes are affected and at what time periods, we examined proliferation, differentiation and apoptosis in mammary glands following exposure to TCDD during early, mid or throughout pregnancy. Although AhR activation throughout pregnancy did not cause early involution, there was a 50% decrease in cell proliferation, which was observed as early as the sixth day of pregnancy (DP). TCDD treatment on the day of impregnation only reduced development and proliferation in early and mid-pregnancy, followed by partial recovery by DP17. However, when AhR activation was delayed to DP7, developmental impairment was not observed in mid-pregnancy, but became evident by DP17, whereas proliferation was reduced at all times. Thus, early exposure to TCDD was neither necessary nor sufficient to cause persistent defects in lactogenesis. These varying outcomes in mammary development due to exposure at different times in pregnancy suggest there are critical windows during which AhR activation impairs mammary epithelial cell proliferation and differentiation.

TCDD exposure disrupts mammary epithelial cell differentiation and function

Mammary gland growth and differentiation during pregnancy is a developmental process that is sensitive to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is a widespread environmental contaminant and a potent ligand for the aryl hydrocarbon receptor (AhR). We demonstrate reduced beta-casein protein induction in mouse mammary glands and in cultured SCp2 mammary epithelial cells following exposure to TCDD. SCp2 cells exposed to TCDD also show reduced cell clustering and less alveolar-like structure formation. SCp2 cells express transcriptionally active AhR, and exposure to TCDD induces expression of the AhR target gene CYP1B1. Exposure to TCDD during pregnancy reduced expression of the cell adhesion molecule E-cadherin in the mammary gland and decreased phosphorylation of STAT5, a known regulator of beta-casein gene expression. These data provide morphological and molecular evidence that TCDD-mediated AhR activation disrupts structural and functional differentiation of the mammary gland, and present an in vitro model for studying the effects of TCDD on mammary epithelial cell function.