Integrins are a broad family of cell surface adhesion and signaling molecules which link the extracellular matrix (ECM) to the cytoskeleton of the cell. Integrins bind directly to ECM ligands on the extracellular surface and are linked to the actin cytoskeleton through multiple adaptor proteins and receptor tyrosine kinases on the plasma membrane. They play critical roles in all cell types and regulate cell migration, differentiation, proliferation and apoptosis. They are also force sensors and transduce mechanical stimuli into biochemical signals. This project explores the role of integrins and integrin-signaling pathways within the epithelium lining the bladder - the urothelium - where almost nothing is known of their function. Our primary hypothesis is that integrins represent the initial upstream mechanical sensor in the bladder and detect bladder filling through membrane stretch. In order to investigate this hypothesis we have created a conditional knockout of 21-integrin within the urothelium (21-cKO). Loss of 21-integrin knocks out all integrins within the umbrella and intermediate cells making the superficial layers of the epithelium integrin-null. Preliminary data indicates that the bladders of these mice release significantly less ATP into the lumen upon stretch, hyperactivate ion conductances upon stretch and exhibit detrusor hyperreflexia and detrusor-sphincter dyssynergia when measured by cystometry. These findings are strongly indicative of an obstructed bladder phenotype possibly due to inadequate urethral sphincter relaxation. Taken together these symptoms indicate a mechanical signaling deficit originating in the urothelium which leads to an obstructed bladder phenotype. An understanding of the signaling mechanisms employed by the bladder as it fills is essential in understanding many different bladder disorders, from overactive bladder to interstitial cystitis.
Specific aim 1 of our study will examine the repertoire of integrin binding partners in umbrella cells in vivo and will explore the mechanisms by which integrins regulate ATP secretion. ATP has been show to be an extremely important autocrine and paracrine mediator in the bladder and signals through purinergic receptors on both the luminal surface and in suburothelial layers e.g. on afferent nerve fibers.
Specific aim 2 will investigate how integrins interact with and regulate mechanosensory ion channels within the urothelium using selective inhibitors and ion substitution protocols to determine which ion channels regulated by integrins. The role of ion fluxes in regulating membrane trafficking and ATP release will also be studied using (i) capacitance changes in response to stretch to monitor membrane trafficking;and (ii) short circuit currents on bladders mounted in Ussing chambers.
Specific aim 3 will use cystometry in the presence of various pharmacological modulators of ion channels and receptors to investigate at the molecular level how integrins regulate filling and voiding behavior.
There are large numbers of people who for many different reasons - spinal cord injury, diabetes, cystitis - suffer from disorders of the bladder which can broadly be characterized as overactive bladder. They suffer greatly from embarrassing and debilitating symptoms of incontinence, urgency, pain and frequency which profoundly affect quality of life. This project will provide new knowledge of the mechanisms by which the bladder senses filling and responds and in so doing may suggest new therapeutic options for bladder disease.
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