Role of Caveolae and miRNAs in Regulation of Urinary Bladder Function

Abstract: The urinary bladder is responsible for storage and expulsion of urine. Proper urination depends on an ability of the bladder to adjust to increasing volume and to contract during micturition. A disturbance of bladder contractility may affect continence. In the present thesis I have used genetic, biochemical and physiological approaches to examine the role of caveolae and mircoRNAs in bladder function. Caveolae are 60-80 nm large membrane invaginations present in a variety of cell types including smooth muscle. A classical view is that caveolae organize the cell membrane and thereby regulate cell signalling. The proteins caveolin-1 and cavin-1 are essential for biogenesis of caveolae in many cell types. The first aim of this thesis was to address the significance of caveolae for detrusor function by using caveolin-1 and cavin-1 knockout mice. Deletion of either caveolin-1 or cavin-1 led to lack of caveolae, reduced muscarinic and depolarisation-induced contraction and impaired neuro-effector transmission. Lack of cavin-1 was moreover associated with bladder hypertrophy. In vivo micturition patterns were however largely similar, arguing in favour of compensatory mechanisms during normal voiding. In all, my findings show that the phenotypic overlap of two distinct knockout models, both of which lack detrusor caveolae, is considerable. This establishes an important role of these organelles for detrusor function. MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression by mRNA degradation and translational repression. The biogenesis of most miRNAs depends on the enzyme Dicer. To investigate the role of miRNAs in the urinary bladder I used two animal models: 1) smooth muscle-specific Dicer knockout mice and 2) surgically induced bladder outlet obstruction, BOO. Depletion of miRNAs resulted in altered micturition pattern, impaired contractility and reduced twitches in response to electrical field stimulation. Dicer deletion was associated with reduced expression of L-type Ca2+ channels and reduced expression of the differentiation markers desmin and calponin. Partial outlet obstruction, on the other hand, resulted in altered expression of 50 miRNAs, supporting a role of this class of RNAs in bladder pathology. Most impressive was a five to ten-fold increase in expression of miR-132/212. Transcription factor binding site analysis pointed to the involvement of Ahr and Creb in miR-132/212 induction. Targets of these miRNAs correlated inversely with the miR-132/212 levels. Overexpression of antimirs and mimics for miR-132/212 caused reciprocal changes in cell proliferation. Taken together, my findings demonstrate that miRNAs play a key role for normal detrusor function and argue that they regulate gene expression and cell proliferation following obstruction.