Publikationsdatenbank

Candidates for alternative chloride conductance in cystic fibrosis (2011)

Art

Hochschulschrift

Autor

Braun, Josephine (WE 12)

Quelle

Berlin: Mensch und Buch Verlag, 2011 — 115 Seiten

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/5547

Kontakt

Institut für Tierpathologie

Robert-von-Ostertag-Str. 15
14163 Berlin
+49 30 838 62450
pathologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Cystic Fibrosis (CF) is a fatal autosomal recessive inherited genetic disease in humans caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes for a Cl- channel in secretory epithelia. Its absence or malfunction lead to altered regulation of Cl- secretion across epithelial membranes and a phenotype of severe pulmonary disease, pancreatic insufficiency, meconium ileus in newborns and other conditions. Interestingly, the CF phenotype has large variations. Even dizygous twins sharing most environmental influences show clinical variations. This suggests that factors other than CFTR modulate disease severity. One of these factors is a CFTRindependent, Ca2+-activated Cl- current (CaCC) which was demonstrated to ameliorate disease severity in the intestine, lungs and pancreas of CF mouse models and in part in humans. The molecules responsible for these CaCC may be potential therapeutical targets. However, its molecular identity is still unknown. First aim of the study was to investigate the tissue specific and cellular mRNA expression pattern of two new murine CLCA family members (mCLCA5 and mCLCA6) as important prerequisite for protein and functional analyses. Second aim was to characterize the cellular mRNA expression levels of select CaCC candidates and modulators and possibly transcriptional regulatory changes under conditions of CF. This study used laser capture microdissection (LCM) and immune electron microscopy to determine cellular localizations of mCLCA5 and mCLCA6. It used reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to quantify and compare the mRNA expression of 15 candidate modulators of CaCC in tissues relevant to CF (stomach, duodenum, jejunum, caecum, colon, pancreas, liver, lung, kidney) between two different CF mouse models and their wild type controls. One mouse model (cftrTgH(neoim)Hgu on BALB/c, DBA/2 and C57BL/6 backgrounds) exhibits residual cftr and a milder phenotype than the other mouse model (cftrtm1Cam on NMRI background) with the complete cftr knockout. Candidates tested were the Ca2+-activated Cl- channels mBEST1, mBEST2, mTTYH3, mTMEM16A, mTMEM16F, mTMEM16K, the Ca2+-activated Cltransporters mCLC-3B and mCLC-4, as well as six murine CLCA gene family members (mCLCA1 to -6), which encode proteins that evoke Ca2+-activated Clcurrents but do not form channels themselves. Additionally the Cl- channel SLC26A9 was tested. Similar to their homologs and orthologs within the CLCA gene family, mCLCA5 and mCLCA6 had their own distinct cellular localizations. mCLCA5 mRNA had a broad expression pattern whereas the protein was found exclusively in cytoplasmic granules of granular layer keratinocytes of stratified squamous epithelia suggesting a role in cornification and an inferior relevance in CF. mCLCA6 mRNA was exclusively expressed in enterocytes suggesting a role in transepithelial anion conductance and fluid secretion which may be of relevance in CF. Of all investigated candidates, only mCLCA6 and mCLCA3 were up-regulated in the caecum and stomach of the cftrTgH(neoim)Hgu DBA/2 mice respectively and only the tweety Cl- channel mTTYH3 was markedly down-regulated throughout the intestinal tract of the cftrtm1Cam NMRI mice. This supports mCLCA6 and mCLCA3 as potential mediators of “alternative” CaCC in CF but argues against a direct compensatory mechanism on transcriptional level in the lungs, pancreas or intestine. On the contrary, mTTYH3 may be linked to the more severe intestinal phenotype suggesting a negative modulatory effect. However, it cannot be excluded that any of the candidates tested are regulated on post-transcriptional level or by modulation of the protein activation status. Thus they may still play a role as modulators in the basic Clsecretory defect of CF and await further analyses.