Expression Pattern of Lysosomal Protective Protein/Cathepsin A: Implications for the analysis of hnman galactosialidosis

The lysosome represents a well characterized, membrane-contained intracellular digestive system. Iu this important organelle a battery of lysosomal hydro lases and accessory proteins work in concert on the step-wise conversion of macromolecular substrates into small biological building blocks, which are either reutHized by the cell 01' discarded. A failure of any of these enzymes to properly exert their hydrolytic activity results in the progressive accumulation of partially degraded· metabolltes that are retained ('stored') in the lysosome. The genetic disorders caused by a malfunction of the lysosomal system are collectively known as lysosomal storage disorders, aud are normally associated with a single enzyme deficiency. One known exception is the disease galactosialldosis which is due to partial or complete loss of activity of two glycosidases, acid p-D-galactosidase and N-acetyl-a-neuraminidase, because of a primary defect in the carboxypeptidase protective protein/cathepsin A (PPCA). The latter enzyme associates with both glycosidases soon after syntbesis, and is essential for their proper intracellular routing, lysosomal stability and activity. Aside from the protective function, PPCA has cathepsin Aldeamidase activity on a selected number ofneuropeptides. The aim of the experimental work presented in tbis thesis was to gain insigbts into the transcription regulation of the PPCA gene and the expression of PPCA mRNA and protein In mouse tissues. These studies have coutributed to the understanding of the phenotypic abnormalities in the murine model of galactosialldosis, which reflected to a large extent the distribution pattern of the protein in normal tissues. Given the fact that the observed pathology in galactosialldosis is iu part caused by tbe secondary neuraminidase deficiency, the isolation and characterization of the murine neuraminidase was instrumen

Changes in vasoactive pathways in congenital diaphragmatic hernia associated pulmonary hypertension explain unresponsiveness to pharmacotherapy

Background: Patients with congenital diaphragmatic hernia (CDH) have structural and functional different pulmonary vessels, leading to pulmonary hypertension. They often fail to respond to standard vasodilator therapy targeting the major vasoactive pathways, causing a high morbidity and mortality. We analyzed whether the expression of crucial members of these vasoactive pathways could explain the lack of responsiveness to therapy in CDH patients. Methods: The expression of direct targets of current vasodilator therapy in the endothelin and prostacyclin pathway was analyzed in human lung specimens of control and CDH patients. Results: CDH lungs showed increased expression of both ETA and ETB endothelin receptors and the rate-limiting Endothelin Converting Enzyme (ECE-1), and a decreased expression of the prostaglandin-I2 receptor (PTGIR). These data were supported by increased expression of both endothelin receptors and ECE-1, endothelial nitric oxide synthase and PTGIR in the well-established nitrofen-CDH rodent model. Conclusions: Together, these data demonstrate aberrant expression of targeted receptors in the endothelin and prostacyclin pathway in CDH already early during development. The analysis of this unique patient material may explain why a significant number of patients do not respond to vasodilator therapy. This knowledge could have important implications for the choice of drugs and the design of future clinical trials internationally

Pulmonary vascular development in congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a rare congenital anomaly characterised by a diaphragmatic defect, persistent pulmonary hypertension (PH) and lung hypoplasia. The relative contribution of these three elements can vary considerably in individual patients. Most affected children suffer primarily from the associated PH, for which the therapeutic modalities are limited and frequently not evidence based. The vascular defects associated with PH, which is characterised by increased muscularisation of arterioles and capillaries, start to develop early in gestation. Pulmonary vascular development is integrated with the development of the airway epithelium. Although our knowledge is still incomplete, the processes involved in the growth and expansion of the vasculature are beginning to be unravelled. It is clear that early disturbances of this process lead to major pulmonary growth abnormalities, resulting in serious clinical challenges and in many cases death in the newborn. Here we provide an overview of the current molecular pathways involved in pulmonary vascular development. Moreover, we describe the abnormalities associated with CDH and the potential therapeutic approaches for this severe abnormality

Ubiquitous expression of the rtTA2S-M2 inducible system in transgenic mice driven by the human hnRNPA2B1/CBX3 CpG island

Background: A sensitive, ubiquitously expressed tetracycline inducible system would be a valuable tool in mouse transgenesis. However, this has been difficult to obtain due to position effects observed at different chromosomal sites of transgene integration, which negatively affect expression in many tissues. The aim of this study was to test the utility of a mammalian methylation-free CpG island to drive ubiquitous expression of the sensitive doxycycline (Dox) inducible rtTA2S-M2 Tet-transactivator in transgenic mice. Results: An 8 kb genomic fragment from the methylation-free CpG island of the human hnRNPA2B1-CBX3 housekeeping gene locus was tested. In a number of transgenic mouse lines obtained, rtTA2S-M2 expression was detected in many tissues examined. Characterisation of the highest expressing rtTA2S-M2 transgenic mouse line demonstrated Dox-inducible GFP transgene expression in many tissues. Using this line we also show highly sensitive quantitative induction with low doses of Dox of an assayable plasma protein transgene under the control of a Tet Responsive Element (TRE). The utility of this rtTA2S-M2 line for inducible expression in mouse embryos was also demonstrated using a GATA-6 Tet-inducible transgene to show specific phenotypes in the embryonic lung, as well as broader effects resulting from the inducible widespread overexpression of the transgene. Conclusion: The ubiquitously expressing rtTA2S-M2 transgenic mouse line described here provides a very useful tool for studying the effects of the widespread, inducible overexpression of genes during embryonic development and in adult mic

Erythroid overexpression of C/EBPγ in transgenic mice affects γ-globin expression and fetal liver erythropoiesis

The CCAAT boxes of the β-like globin genes interact with three proteins: NF-Y, GATA-1 and NFE-6. We demonstrate that NFE-6 contains C/EBPγ, and address its role in globin gene regulation by erythroid overexpression of C/EBPγ, and a dominant-negative form C/EBPγΔB, in mice. Elevated levels of C/EBPγ, but not C/EBPγΔB, increase expression of the (fetal) γ-globin relative to the (adult) β-globin gene. Interestingly, fetal liver erythropoiesis is ablated when the C/EBPγ and C/EBPγΔB levels are further increased in homozygous transgenics. We suggest that targeted expression of dominant-negative leucine zipper proteins is a generally applicable approach to ablate specific tissues in mice.</p

Erythroid overexpression of C/EBPγ in transgenic mice affects γ-globin expression and fetal liver erythropoiesis

The CCAAT boxes of the β-like globin genes interact with three proteins: NF-Y, GATA-1 and NFE-6. We demonstrate that NFE-6 contains C/EBPγ, and address its role in globin gene regulation by erythroid overexpression of C/EBPγ, and a dominant-negative form C/EBPγΔB, in mice. Elevated levels of C/EBPγ, but not C/EBPγΔB, increase expression of the (fetal) γ-globin relative to the (adult) β-globin gene. Interestingly, fetal liver erythropoiesis is ablated when the C/EBPγ and C/EBPγΔB levels are further increased in homozygous transgenics. We suggest that targeted expression of dominant-negative leucine zipper proteins is a generally applicable approach to ablate specific tissues in mice.</p

Erythroid overexpression of C/EBPγ in transgenic mice affects γ-globin expression and fetal liver erythropoiesis

The CCAAT boxes of the β-like globin genes interact with three proteins: NF-Y, GATA-1 and NFE-6. We demonstrate that NFE-6 contains C/EBPγ, and address its role in globin gene regulation by erythroid overexpression of C/EBPγ, and a dominant-negative form C/EBPγΔB, in mice. Elevated levels of C/EBPγ, but not C/EBPγΔB, increase expression of the (fetal) γ-globin relative to the (adult) β-globin gene. Interestingly, fetal liver erythropoiesis is ablated when the C/EBPγ and C/EBPγΔB levels are further increased in homozygous transgenics. We suggest that targeted expression of dominant-negative leucine zipper proteins is a generally applicable approach to ablate specific tissues in mice.</p

Differentiated type II pneumocytes can be reprogrammed by ectopic Sox2 expression

The adult lung contains several distinct stem cells, although their properties and full potential are still being sorted out. We previously showed that ectopic Sox2 expression in the developing lung manipulated the fate of differentiating cells. Here, we addressed the question whether fully differentiated cells could be redirected towards another cell type. Therefore, we used transgenic mice to express an inducible Sox2 construct in type II pneumocytes, which are situated in the distal, respiratory areas of the lung. Within three days after the induction of the transgene, the type II cells start to proliferate and form clusters of cuboidal cells. Prolonged Sox2 expression resulted in the reversal of the type II cell towards a more embryonic, precursor-like cell, being positive for the stem cell markers Sca1 and Ssea1. Moreover, the cells started to co-express Spc and Cc10, characteristics of bronchioalveolar stem cells. We demonstrated that Sox2 directly regulates the expression of Sca1. Subsequently, these cells expressed Trp63, a marker for basal cells of the trachea. So, we show that the expression of one transcription factor in fully differentiated, distal lung cells changes their fate towards proximal cells through intermediate cell types. This may have implications for regenerative medicine, and repair of diseased and damaged lungs

Hypoxia Inducible Factor 3α Plays a Critical Role in Alveolarization and Distal Epithelial Cell Differentiation during Mouse Lung Development

Lung development occurs under relative hypoxia and the most important oxygen-sensitive response pathway is driven by Hypoxia Inducible Factors (HIF). HIFs are heterodimeric transcription factors of an oxygen-sensitive subunit, HIFα, and a constitutively expressed subunit, HIF1β. HIF1α and HIF2α, encoded by two separate genes, contribute to the activation of hypoxia inducible genes. A third HIFα gene, HIF3α, is subject to alternative promoter usage and splicing, leading to three major isoforms, HIF3α, NEPAS and IPAS. HIF3α gene products add to the complexity of the hypoxia response as they function as dominant negative inhibitors (IPAS) or weak transcriptional activators (HIF3α/NEPAS). Previously, we and others have shown the importance of the Hif1α and Hif2α factors in lung development, and here we investigated the role of Hif3α during pulmonary development. Therefore, HIF3α was conditionally expressed in airway epithelial cells during gestation and although HIF3α transgenic mice were born alive and appeared normal, their lungs showed clear abnormalities, including a post-pseudoglandular branching defect and a decreased number of alveoli. The HIF3α expressing lungs displayed reduced numbers of Clara cells, alveolar epithelial type I and type II cells. As a result of HIF3α expression, the level of Hif2α was reduced, but that of Hif1α was not affected. Two regulatory genes, Rarβ, involved in alveologenesis, and Foxp2, a transcriptional repressor of the Clara cell specific Ccsp gene, were significantly upregulated in the HIF3α expressing lungs. In addition, aberrant basal cells were observed distally as determined by the expression of Sox2 and p63. We show that Hif3α binds a conserved HRE site in the Sox2 promoter and weakly transactivated a reporter construct containing the Sox2 promoter region. Moreover, Hif3α affected the expression of genes not typically involved in the hypoxia response, providing evidence for a novel function of Hif3α beyond the hypoxia response