Acid-base transporters regulate intracellular pH (pHi) which affects both cellular proliferation and apoptosis. Thus, understanding the mechanisms of pHi regulation provides insight into processes critical to tissue regeneration and the potential for malignant transformation. Two genetic diseases, cystic fibrosis (CF) and congenital Cl- losing diarrhea (CLD) caused by mutations in HCO3 - transporters, exemplify this concept for intestinal epithelium by demonstrating pHi dysregulation, crypt hyperplasia and increased risk for gastrointestinal neoplasia. The proliferative compartment of the crypt is maintained through extracellular signaling by Wnt lipoglycoproteins but little is known about how this system is affected by pHi dysregulation. The long-term objectives of this application are to elucidate the transport mechanisms involved in pHi dysregulation in CF and CLD crypt epithelium, to demonstrate how this impacts Wnt signaling and to use this knowledge to reversibly manipulate intestinal regeneration for the prevention of intestinal mucositis, a common side-effect of chemotherapy resulting from crypt epithelial damage. Surprisingly little is known about pHi regulation in intact crypts because of their inaccessibility in native intestine and failure in primary culture. Recent breakthroughs in developmental biology provide a model of regenerating intestinal crypts in 3D gel culture that has been adapted for these studies of intestine from wild-type (WT), CF and CLD mice.
In Specific Aim 1, contributions of the anion channel Cftr and acid-base transporters to pHi regulation in WT, CF and CLD crypts will be determined under basal/challenged conditions and during the cell cycle using novel adaptations of electrophysiology, fluorescence microscopy and cell sorting techniques.
Specific Aim 2 will test two hypotheses about the effects of pHi dysregulation on Wnt signaling in CF and CLD crypts: 1) alkaline pHi near the plasma membrane facilitates binding of the Wnt transducer Disheveled (Dsh) to the Wnt receptor Frizzled (Fz) to increase Wnt-directed proliferation; and 2) alkaline pH in the CLD colonic mid-crypt inappropriately decreases hypoxia inducible factor 1alpha, thereby shifting Wnt signaling from cell migration to proliferation. These studies will use multi-photon confocal microscopy to measure submembrane pHi, immunological methods for Dsh-Fz binding and develop a Dsh-pHluorin transgenic mouse for simultaneous measurement of Dsh binding and submembrane pHi in live crypts.
Specific Aim 3 will explore pharmacological manipulation of Cftr and acid-base transporters to slow crypt proliferation during the damage phase and increase crypt proliferation during the repair phase of chemotherapy for prevention of mucositis. Regulation of Cftr will be emphasized because a number of orally-available potentiators and inhibitors of Cftr have been identified through high-throughput screening for cystic fibrosis treatment. These studies will provide new information on pathogenic processes contributing to the susceptibility of CF and CLD patients to intestinal neoplasia and introduce a therapeutic strategy for preventing chemotherapy-induced mucositis.
This research will identify the transport mechanisms regulating intracellular pH (pHi) in the intestinal crypt and how this impacts processes of proliferation/apoptosis, thereby potentially identifying new targets for neoplasia intervention. Further, the studies will specifically inform on pathogenic mechanisms contributing to the susceptibility of cystic fibrosis and congenital Cl- losing diarrhea patients to gastrointestinal neoplasia and, through manipulation of pHi regulation, develop a therapeutic strategy for reversibly controlling intestinal regeneration during injury-repair processes such as chemotherapy-induced intestinal mucositis.
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