Teaching: Gastrointestinal physiology and pharmacology
Cystic fibrosis, epithelial transport and stem cell biology. Dr. Clarke’s laboratory investigates abnormalities of acid-base transporters in cystic fibrosis (CF) and other genetic diseases as they relate to intestinal stem cell biology (ISCs) and differentiation/function of secretory cells. Studies of mice with gene-targeted deletion of CFTR (the cystic fibrosis gene) and other acid-base transporters such Na+/H+ and Cl–/HCO3– exchangers are performed using mice or regenerating murine or human intestinal organoids grown in 3D gel culture. As an anion channel, CFTR is responsible for epithelial fluid secretion which provides the proper environment for nutrient absorption and maintains normal viscoelastic properties of mucus. Functional activity of acid-base or nutrient transporters is measured in real time using fluorescence confocal or conventional microscopy and electrophysiological methods. Gene or protein expression is measured using quantitative real-time PCR, immunoblots, immunofluorescence and laser capture microdissection.
Currently, three major projects in the laboratory are funded by NIDDK, the Cystic Fibrosis Foundation (CFF) or CFF Therapeutics, Inc. (CFFT). NIDDK-funded projects investigate the role of CFTR, a cAMP-regulated Cl– and HCO3– ion channel, in down-regulating cell cycle dynamics and Wnt/b-catenin signaling in intestinal stem cells (ISCs). Loss of this regulation in CF results in hyperproliferation of ISCs which forms the platform for a six-fold increase in the incidence of gastrointestinal cancer in young CF patients. A second project investigates the acid-base transporters expressed in ISCs that determine intracellular pH (pHi). Manipulation of pHi is used to control ISC proliferation to offset “bystander” damage to ISCs resulting from therapeutic doses of chemotherapeutic reagents and radiation during cancer treatment. Two CFF-funded projects investigate goblet cell (mucus secreting) dysfunction in the CF intestine, i.e., mucoviscidosis. The goals of the first project are to determine the role of CFTR in goblet cell dysfunction and evaluate the impact of abnormal goblet cell antigen presentation to dendritic cells on CF immune status. The goal of the second project is produce a CF mouse that expresses a pH-sensitive fluorescent mucin to evaluate the abnormalities of mucus pH during processing and release. To facilitate the translational potential of the above CFF projects, a CFFT-funded project has the goal to develop a human CFTR “rescue” mouse model in which murine CFTR is replaced by the human ortholog of the gene. This humanized CFTR mouse will enable pharmacological testing and gene-editing strategies for correcting defective function of CFTR in CF patients or to combat diarrheal diseases in humans and animals that are caused by enterotoxigenic bacteria such a Vibrio cholerae.
Liu, J, Walker, NM, Ootani, A, Strubberg, AM, Clarke, LL. Defective goblet cell exocytosis contributes to murine cystic fibrosis-associated intestinal disease. J. Clin. Invest. 125: 1056-1068, 2015.
Liu, J, Walker, NM, Cook, MT, Ootani, A, Clarke, LL. Functional Cftr in crypt epithelium of organotypic enteroid cultures from murine small intestine. Am. J. Physiol. Cell Physiol. 302: C1492-C1503, 2012.
Alper, SL, Stewart, AK, Vandorpe, DH, Clark, JS, Horack, RZ, Simpson, JE, Walker, NM and Clarke, LL. Native and recombinant Slc26a3 (down-regulated in adenoma, Dra) do not exhibit properties of 2 Cl–/1 HCO3– exchange. Am. J. Physiol. 300:C276-C286, 2011.
Simpson,JE, Walker,NM, Soleimani,M and Clarke,LL. Putative anion transporter-1 (Pat-1, Slc16a6) contributes to intracellular pH regulation during H+-dipeptide transporter in the villous epithelium. Am. J. Physiol. 298: G683-G691, 2010.
Walker, NM, Simpson, JE, Brazill, JM, Gill, RK, Dudeja, PK, Schweinfest, CW, and Clarke, LL. Role of down-regulated in adenoma in HCO3– secretion across murine duodenum. Gastroenterology 136: 893-901, 2009.
Walker, NM, Simpson, JE, Yen, P-F, Gill, RK, Rigsby, EV, Brazill, JM, Dudeja, PK, Schweinfest, CW and Clarke, LL. Down-regulated in adenoma Cl–/HCO3– exchanger couples with Na+/H+ exchanger 3 for NaCl absorption in murine small intestine. Gastroenterology 136: 1645-1653, 2008
Simpson, JA, Walker, NM, Boyle, KT, Schweinfest, CW, Soleimani, M., Shull, GE and Clarke, LL. PAT-1 (Slc26a6) is the predominant apical membrane Cl–/HCO3– exchanger in the upper villous epithelium of murine duodenum. Am. J. Physiol. 292: G1079-G1082, 2007
Simpson, JE, Gawenis, LR, Walker, NM, Boyle, KT and Clarke, LL. Chloride conductance of CFTR facilitates basal Cl–/HCO3– exchange in the villous epithelium of intact murine duodenum. Am. J. Physiol. 288: G1241 – G1251, 2005.
Clarke, LL, Gawenis, LR, Bradford, EM, Judd, LM, Boyle, KT, Simpson, JE, Shull, GE, Tanabe, H, Ouellette, AJ, Franklin, CL and Walker, NM. Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of cystic fibrosis mice. Am. J. Physiol. 286: G1050-G1058, 2004.
Gawenis, LR, Franklin, CL, Simpson, JE, Palmer, BA, Walker, NM, Wiggins, TM and Clarke, LL. cAMP inhibition of murine intestinal Na+/H+ exchange requires CFTR-mediated cell shrinkage of villus epithelium. Gastroenterology 125: 1124-1148, 2003.
Zhou, Q, Clarke, LL, Nie, R, Carnes, K, Lai, L-W, Lien, Y-HH, Verkman, A, Lubahn, D, Fisher, JS, Katzenellenbogen, BS and Hess, RA. Estrogen action and male fertility: Roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function. Proc. Natl. Acad. Sci. USA 98: 14132-14137, 2001.
Clarke LL and Harline MC. Dual role of CFTR in cAMP-stimulated bicarbonate secretion across murine duodenum. Am. J. Physiol. 274: G718-G726, 1998.
Clarke LL, Grubb BR, Yankaskas JR, Cotton CU, McKenzie A and Boucher RC: Relationship of a non-CFTR chloride conductance to organ-level disease in cftr(-/-) mice. Proc. Natl. Acad. Sci. U.S.A., 91(2):479-483, 1994.
Clarke LL, Grubb BR, Gabriel SE, Smithies O, Koller BH and Boucher RC. Defective epithelial chloride transport in a gene-targeted mouse model of cystic fibrosis. Science, 257:1125-1128, 1992.
Knowles MR, Clarke LL and Boucher RC. Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis [see comments]. New Engl. J. Med., 325:533-538, 1991.