Ileal mucosal bile acid absorption is increased in Cftr knockout mice

BACKGROUND: Excessive loss of bile acids in stool has been reported in patients with cystic fibrosis. Some data suggest that a defect in mucosal bile acid transport may be the mechanism of bile acid malabsorption in these individuals. However, the molecular basis of this defect is unknown. This study examines the expression of the ileal bile acid transporter protein (IBAT) and rates of diffusional (sodium independent) and active (sodium dependent) uptake of the radiolabeled bile acid taurocholate in mice with targeted disruption of the cftr gene. METHODS: Wild-type, heterozygous cftr (+/-) and homozygous cftr (-/-) mice were studied. Five one-cm segments of terminal ileum were excised, everted and mounted onto thin stainless steel rods and incubated in buffer containing tracer (3)H-taurocholate. Simultaneously, adjacent segments of terminal ileum were taken and processed for immunohistochemistry and Western blots using an antibody against the IBAT protein. RESULTS: In all ileal segments, taurocholate uptake rates were fourfold higher in cftr (-/-) and two-fold higher in cftr (+/-) mice compared to wild-type mice. Passive uptake was not significantly higher in cftr (-/-) mice than in controls. IBAT protein was comparably increased. Immuno-staining revealed that the greatest increases occurred in the crypts of cftr (-/-) animals. CONCLUSIONS: In the ileum, IBAT protein densities and taurocholate uptake rates are elevated in cftr (-/-) mice > cftr (+/-) > wild-type mice. These findings indicate that bile acid malabsorption in cystic fibrosis is not caused by a decrease in IBAT activity at the brush border. Alternative mechanisms are proposed, such as impaired bile acid uptake caused by the thick mucus barrier in the distal small bowel, coupled with a direct negative regulatory role for cftr in IBAT function

Probing the Functional Impact of Sequence Variation on p53-DNA Interactions Using a Novel Microsphere Assay for Protein-DNA Binding with Human Cell Extracts

The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation—including polymorphisms—and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks

Lymphangioleiomyomatosis

Review on Lymphangioleiomyomatosis, with data on clinics, and the genes involved

Erythropoietin-driven proliferation of cells with mutations in the tumor suppressor gene TSC2

Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction, resulting from proliferation of smooth-muscle-like cells, which have mutations in the tumor suppressor genes TSC1 or TSC2. Among 277 LAM patients, severe disease was associated with hypoxia and elevated red blood cell indexes that accompanied reduced pulmonary function. Because high red cell indexes could result from hypoxemia-induced erythropoietin (EPO) production, and EPO is a smooth muscle cell mitogen, we investigated effects of EPO in human cells with genetic loss of tuberin function, and we found that EPO increased proliferation of human TSC2−/−, but not of TSC2+/−, cells. A discrete population of cells grown from explanted lungs was characterized by the presence of EPO receptor and loss of heterozygosity for TSC2, consistent with EPO involvement. In LAM cells from lung nodules, EPO was localized to the extracellular matrix, supporting evidence for activation of an EPO-driven signaling pathway. Although the high red cell mass of LAM patients could be related to advanced disease, we propose that EPO, synthesized in response to episodic hypoxia, may increase disease progression by enhancing the proliferation of LAM cells

Airway gene transfer in mouse nasal-airways: importance of identification of epithelial type for assessment of gene transfer

Mouse nasal airways are often used for the assessment of both reporter and cystic fibrosis transmembrane conductance regulator (CFTR) gene transfer to respiratory epithelia. However, the mouse nasal cavity is lined by both olfactory (OE) and respiratory epithelium (RE). Previous gene transfer studies have suggested that OE may be more efficiently transduced by adenoviral vectors than RE. However, to provide data pertinent to CFTR gene transfer in humans, measurements of CFTR function in mice by transepithelial potential difference (TPD) should be directed towards respiratory rather than olfactory epithelium. We report a new technique to mark the position of the TPD sensing cannula tip in the mouse nasal cavity that permitted us to correlate TPD measurements with epithelial cell type. Using this technique, we found TPD values did not discriminate between respiratory and olfactory epithelia. We next assessed relationships between anatomic regions accessed by the TPD cannula and epithelial type. The frequently used insertion depth of approximately 5 mm from the nose tip predominantly recorded the TPD from anterior dorsal olfactory epithelium. Measurement of the TPD of respiratory epithelium in our study was maximized by insertion of the TPD cannula probe to 2.5 mm depth. Because TPD measurements are not sensitive to epithelial type, adequate control of position and TPD catheter insertion depth are required to ensure accurate estimation of CFTR gene transfer into the target RE in the mouse nasal cavity

Interferon γ Stimulates Accumulation of Gas Phase Nitric Oxide in Differentiated Cultures of Normal and Cystic Fibrosis Airway Epithelial Cells

BACKGROUND: Exhaled nitric oxide (NO) levels have been reported to be lower in patients with cystic fibrosis (CF) than in controls; however the mechanism(s) responsible and the effect on pathogenesis are unclear. The objective of these studies was to determine if the low levels of gas phase NO (gNO) could be reproduced in well-differentiated air–liquid interface (ALI) cultures of normal and CF cells. METHODS: Human bronchial epithelial (HBE) cells from CF and control tissues were cultured under ALI conditions that promote differentiation into a mostly ciliated, pseudostratified epithelium similar to that of the in vivo airway. Cultures were incubated in gas tight chambers and the concentration of gNO was measured using a Sievers nitric oxide analyzer. RESULTS: In CF and control cultures the level of accumulated gNO under baseline conditions was low (<20 ppb). Treatment with interferon gamma (IFNγ) induced iNOS expression and increased gNO significantly in differentiated cultures, while having no significant effect on undifferentiated cultures. Submersion of the apical surface with fluid drastically reduced the level of gNO. Importantly, the average level of gNO measured after IFNγ treatment of control cells (576 ppb) was threefold greater than that from CF cells (192 ppb). CONCLUSIONS: The results demonstrate that the lower level of exhaled NO observed in CF patients is reproduced in well-differentiated primary cultures of HBE cells treated with IFNγ, supporting the hypothesis that the regulation of NO production is altered in CF. The results also demonstrate that IFNγ treatment of differentiated cells results in higher levels of gNO than treatment of undifferentiated cells, and that a layer of fluid on the apical surface drastically reduces the amount of gNO, possibly by limiting the availability of oxygen