Jonathan A. Dranoff, M.D.

Associate Professor
Section of Digestive Diseases
more on this physician

Clinical interests

Liver Disease, with emphasis on cirrhosis and primary diseases of the bile ducts

Research interests

Cellular mechanisms of liver fibrosis; Regulation of bile duct growth in health and disease

Our laboratory is working on several exciting topics. The first of these is the role of extracellular nucleotides as regulators of bile ductular proliferation. It has long been known that extracellular ATP and related nucleotides can function as signaling molecules, because cells express specific G protein coupled P2Y receptors for these compounds. Several years ago, we found that bile duct epithelia express P2Y receptors with unknown function. Recently, we demonstrated that the function of these cells is to regulate bile ductular proliferation. Interestingly, these receptors are normally inactive, because neighboring portal fibroblasts express the ecto-nucleotidase NTPDase2, which inhibits activation of the P2Y receptors. However, after liver injury in obstructive cholestasis, portal fibroblasts lose NTPDase2 expression, thus allowing bile duct epithelia to grow in an unchecked fashion. We are now determining whether changes in this regulatory mechanism account for the bile ductular proliferation typical of such cholangiopathic conditions as Cystic Fibrosis, Primary Sclerosing Cholangitis, and Primary Biliary Cirrhosis.

Proposed mechanism for regulation of bile ductular proliferation by portal fibroblasts.

The cartoon above indicates checked bile ductular proliferation under normal conditions. Bile duct epithelia express P2Y receptors linked to ductular proliferation. However, these receptors remain inactive due to expression of NTPDase2, which hydrolyzes the P2Y ligands. The cartoon on the right indicates unchecked bile ductular proliferation after obstructive cholestasis. After liver injury, portal fibroblasts transdifferentiate into portal myofibroblasts and lose NTPDase2 expression. This then allows activation of bile duct epithelial P2Y receptors and subsequent ductular proliferation.

We are now seeking to understand portal fibroblasts in much greater detail. Portal fibroblasts have been noted by histologists for the past decade as a fibrogenic liver cell type distinct from hepatic stellate cells. Our laboratory has devised a method for the isolation and culture of portal fibroblasts, which has allowed us to perform molecular and functional studies on these cells. We are now attempting to determine the relative importance of portal fibroblasts and hepatic stellate cells in liver fibrogenesis. We hope that these studies will lead to advances in the understanding of the biliary fibrosis seen in cholangiopathic conditions.

NTPDase2 is expressed in the region surrounding intrahepatic bile ducts. The distribution of NTPDase2 was determined in 5 mm rat liver sections using confocal immunofluorescence. Portal structures were identified by staining filamentous actin with rhodamine phalloidin (pv = portal vein; bd = bile duct; ha = hepatic artery). NTPDase2 staining was found in the region surrounding intrahepatic bile ducts. Staining was also noted to a lesser extent in the region surrounding portal vessels. No staining was identified in hepatic vessels or in the hepatic parenchyma. Thus, unlike the vascular distribution of NTPDase1, NTPDase2 is expressed in the peri-ductular space in the portal area of the liver.

Portal fibroblasts express NTPDase2 at the plasma membrane and on intracellular structures. Expression of NTPDase2 in portal fibroblasts was determined using immunofluorescence. NTPDase2 fluorescence is seen at both the plasma membrane and on intracellular structures that may be microfilament- or microtubule-associated.

Our newest area of interest is the role of P2Y receptors in the regulation of hepatic stellate cell physiology. Hepatic stellate cells (HSC) are the primary fibrogenic cells of the liver. In the normal liver, HSC are lipid-rich and are weakly fibrogenic. After liver injury, HSC transdifferentiate into myofibroblast-like cells and are strongly fibrogenic. Recently, we found that activation of P2Y receptors stimulates cytosolic calcium signals in HSC. It seems that these calcium signals are important regulators of fibrogenesis through stimulation of transcription of collagen and other basement membrane constituents. We are now attempting to determine whether inhibition of P2Y-mediated signaling may block liver fibrosis. We are also characterizing the elements that regulate calcium signals in HSC.

HSC were loaded with the calcium-sensitive dye fluo-4/AM and then stimulated with ATP. Changes in fluorescence over time were assessed using confocal video microscopy. Images are pseudocolored according to the bar below. Stimulation with ATP rapidly induces calcium signals in HSC.

P2Y-mediated cytosolic calcium signals in HSC.

Selected Publications

• Nathanson MH, Burgstahler AD, Mennone A, Dranoff JA, Rios-Velez L, Stimulation of bile duct epithelial secretion by glybenclamide in normal and cholestatic rat liver. J. Clin. Invest. 101: 2665-2676 (1998).
• Leite MF, Dranoff JA, Gao L, Nathanson MH. Expression and subcellular localization of the ryanodine receptor in rat pancreatic acinar cells. Biochem. J. 337: 305-9 (1999).
• Dranoff JA, McClure M, Burgstahler AD, Denson LA, Crawford AR, Crawford JM, Karpen SJ, Nathanson MH. Short-term regulation of bile acid uptake by microfilament-dependent translocation of ntcp to the plasma membrane. Hepatology 30: 223-9 (1999).
• Dranoff JA, Angood PJ, Topazian M. Transnasal endoscopy for enteral feeding tube placement in critically ill patients. Am. J. Gastro. 94; 2902-4 (1999).
• Dranoff JA and Nathanson MH. Regulation of bile acid transport: beyond molecular cloning. Hepatology 29:1912-3 (1999).
• Dranoff JA, O'Neill AF, Franco AM, Cai SY, Connolly GC, Ballatori N, Boyer JL, Nathanson MH. A primitive ATP receptor from the little skate Raja erinacea. J. Biol. Chem. 275: 30701-30706 (2000).
• Dranoff JA and Nathanson MH. It's swell to have ATP in the liver. J. Hepatology 33:323-5 (2000).
• Dranoff JA, Masyuk AI, LaRusso NF, Nathanson MH. Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia. Am. J. Physiol. 281: G1059-67(2001).
• Hirata K, Pusl T, O'Neill AF, Dranoff JA, and Nathanson MH, The Type II inositol 3,4,5-triphosphate receptor can trigger Ca2+ waves in hepatocytes. Gastroenterol. 122: 1088-1100.
• Kruglov EA, Jain D, Dranoff JA. Isolation of primary rat liver fibroblasts. J. Invest. Med. 50: 179-84 (2002).
• Dranoff JA, Kruglov EA, Robson SC, Braun N, Zimmermann H, Sevigny J. The nucleoside triphosphate diphosphohydrolase NTPDase2/CD39L1 is localized to a novel compartment within the liver. Hepatology 36. 1135-44 (2002).
• Wells RG, Kruglov EA, Dranoff JA. Autocrine release of TGF-beta by portal fibroblasts regulates cell growth. FEBS Lett. 559:107-110 (2004.)
• Dranoff JA. Purinergic regulation of bile ductular secretion. In Pathobiology of bile duct epithelia, G. Alpini, D. Alvaro, M. Marzoni, E. LeSage, and N.F. LaRusso, eds. Georgetown, TX. pp. 96-100 (2004).
• Dranoff JA, Ogawa M, Kruglov EA, Gaca MD, Sevigny J, Robson SC, Wells RG. Expression of P2Y Nucleotide Receptors and Ecto-Nucleotidases in Quiescent and Activated Hepatic Stellate Cells. Am. J. Physiol. 287:G417-24 (2004).
• Dranoff JA, Kruglov EA, Toure J, Braun N, Zimmermann H, Jain D, Knowles AF, and Sevigny J, The Ectonucleotidase NTPDase2 is Selectively Down-Regulated in Biliary Fibrosis,  J. Invest. Med. 52:475-82 (2004).
• Jhandier MN, Kruglov EA, Lavoie EG, Sevigny J, and Dranoff JA. Portal Fibroblasts Regluate the Prolferation of Bile Duct Epithelia via Expression of NTPDase2. J. Biol. Chem. 280: 22986-92(2005).
• Minagawa N, Kruglov EA, Dranoff JA, Robert ME, Gores GF, and Nathanson MH. The anti-apoptotic protein Mcl-1 inhibits mitochondrial Ca2+ signals. J. Biol. Chem. 280:33637-44 (2005).
• Kruglov EA, Nathanson RA, Nguyen T, Dranoff JA. Secretion of MCP-1/CCL2 by bile duct epithelia induces myofibroblastic transdifferentiation of portal fibroblasts. Am. J. Physiol. 290:G765-71 (2006).

Contact

Campus Address
Section of Digestive Diseases
Yale University School of Medicine
333 Cedar St. LMP 1080
New Haven CT 06510

Office Address
1 Gilbert St.
TAC S-223B
New Haven CT 06510

E-mail
jonathan.dranoff@yale.edu

Office Phone
203-785-4133

Fax
203-785-7273