טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAdler Lior
SubjectMolecular Mechanisms of Epithelial Cell-Specific
Expression and Regulation of the Human Anion
Exchanger (Pendrin) Gene
DepartmentDepartment of Medicine
Supervisor Professor Israel Zelikovic
Full Thesis textFull thesis text - English Version


Abstract

Pendrin, a Cl-/anion exchanger encoded by the gene PDS, is highly expressed in the kidney, thyroid and inner-ear epithelia and is essential for bicarbonate-secretion, iodide-accumulation and endolymph ion balance, respectively. Mutations in human PDS (hPDS) cause Pendred syndrome, characterized by sensorineural-deafness and goiter. This study aimed to define promoter regulatory elements essential for renal, thyroid and inner-ear epithelial-specific expression of hPDS, to explore the effect of ambient pH, aldosterone and Cl-, known modulators of acid-base and electrolyte transport, on hPDS promoter activity and to investigate the epithelial-specific expression/regulation of PDS in wild-type and transgenic mice. Endogenous pendrin mRNA and protein were detected in renal HEK293, thyroid LA2 and inner-ear VOT36 epithelial cells, but not in the fibroblast cells, NIH3T3. A 4.2kb hPDS 5'-flanking DNA sequence and a set of eleven 5'-deletion products, were cloned into luciferase reporter vectors and transiently transfected into the above cells. Distinct orientation-dependent differences in expression and activity of deduced positive/negative regulatory elements within the hPDS promoter between HEK293, LA2 and VOT36 were demonstrated, whereas only basal activity was detected in NIH3T3. Acidic pH decreased and alkaline pH increased hPDS promoter activity in transfected HEK293 and VOT36, but not in LA2. Aldosterone diminished hPDS promoter activity in transfected HEK293, but not in LA2 and VOT36. These ambient pH- and aldosterone-induced effects were narrowed-down to ~100bp segments on the hPDS promoter. High chloride concentration decreased and low chloride concentration increased hPDS promoter activity in transfected HEK293 and LA2, specifically influencing the proximal 2kb region of the hPDS promoter. Real-time PCR analysis of HEK293 showed an acidic pH-, and aldosterone-induced decrease and an alkaline pH-induced increase in endogenous pendrin mRNA. Pendrin mRNA and protein expression was decreased in kidneys of acid/chloride- or chloride-loaded mice and increased in kidneys of alkali-loaded mice. In vivo photon-luminescence of generated transgenic mice containing the proximal 2kb 5'-flanking region of hPDS driving the expression of Luc+-reporter-gene, showed transgene localization in the thyroidal region.

In conclusion, interplay between positive and negative regulatory elements on the 5'-flanking region of the pendrin gene likely determines renal, thyroid and inner-ear cell-specific expression of this gene. Pendrin-mediated action in the kidney, thyroid and inner-ear is differentially regulated at the transcriptional level by systemic pH, aldosterone, and Cl-. The effects of these modulators on pendrin activity occur at the DNA, RNA, and protein levels. The generated transgenic animals may help elucidate the transcriptional effect of various physiological/pathophysiological factors on the pendrin gene.