[119] TARGETED DISRUPTION OF THE HEPATIC TRANSFERRIN RECEPTOR 2 GENE IN MICE LEADS TO IRON OVERLOAD

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dysfunction in pe fi sed rat livers by dose response curves to cumulative doses of acetylcholine (Ach). Results: Untreated CH livers presented higher 0; levels than CT (78.3% increase; p i 0.05). Transfection of CH livers with AdECSOD produced a significant reduction in O i levels (60% 0; in relation to that observed in CHAd(iga1) and a significant increase in cGMP (969 vs 4127pmolig). This mmHg was associated to a significant decrease in PP (15f0.5 vs 17.3~t0.7 in CH transfected with Ad(3gal) without significant changes in MAP. Moreover, AdECSOD transfection significantly improved endothelial-dependent vasodilatation to Ach. AdECSOD transfection to CT rats did not produce any hemodynamic change. Conclusions: AdECSOD treatment reduces 0; levels, increases NO bioavailability, improves intrahepatic endothelial dysfunction and reduces PP in CH livers, suggesting that scavenging of 0; might be a new valuable therapeutic strategy to increase NO bioavailability and improve the hepatic circulation in cirrhosis.

11191 TARGETED DISRUPTION OF THE HEPATIC TRANSFERRIN RECEPTOR 2 GENE IN MICE LEADS TO IRON OVERLOAD D.F. Wallace, L. Summerville, VN. Subramaniam. Cancer und Cell Biology Diuision, The Queensland Institute o f Medical Research, Brishune, QLD, Australia E-mail: Daniel. [email protected] Background and Aims: Transferrin receptor 2 (TfR2) plays a key role in the regulation of iron metabolism. Mutations of TfR2 in humans cause type 3 hereditary haemochromatosis. Although highly expressed in liver, several studies have reported TtR2 expression in other cells and tissues, including the intestine, spleen, platelets and erythroid cells. To determine the contribution of liver expressed TfR2 in iron homeostasis we have generated and characterized a liver-specific TtR2-knockout mouse. Methods: To enable conditional deletion, the mouse TtR2 gene was modified by flanking exons 2 to 6 with loxP sites (floxed). Liver-specific TtR2KO mice were generated by crossing the TfR2-floxed mice with transgenic albumin-Cre mice. Tissue and serum from homozygous TfR2-floxed mice, with and without albumin-Cre were analyzed at 5 , 10 and 26 weeks of age. Serum transferrin saturation, hepatic and splenic iron concentrations were determined. The expression of iron-related mRNA transcripts was analyzed by real-time PCR. Levels of the iron-related proteins TfRl, TfR2, ferritin and prohepcidin were analyzed by immunoblotting. Results: Liver-specific TtR2-KO mice develop significant iron overload comparable to complete TtR2-KO mice. At all ages studied transferrin saturation, hepatic iron concentration and hepatic ferritin were significantly elevated. Hepatic TfK2 mRNA and protein were absent i n the livers of liver-specific TtR2-KO mice, and TtR I expression was reduced consistent with hepatic iron loading. At 5 weeks of age, hepcidinl mRNA and prohepcidin protein were decreased in liver-specific TfR2-KO compared to control mice. Conclusions: The significant iron loading and modulation of expression of iron-related genes in liver-specific TfR2-KO mice, demonstrates that the liver is the primary site for TtR2 expression and activity, and that liver-expressed TfR2 is required for the regulation of hepcidinl. This study highlights the importance of the liver in the regulation of iron homeostasis.

11201 HEPATITIS C E2 KINASE CONTROLS ENDOCYTOSIS AND PROLIFERATION M.M. Buck. Uniuersity of Culiftirniu und VA Medical Center; Sun Diego, Sun Diego, CA, 7JSA E-mail: [email protected] Background and Aims: The intracellular mechanisms of HCV E2 protein are unknown. We have shown that E2 is a member of the ArWPrk family of kinases. These actin regulating kinases control endocytosis through

phosphorylation of consensus sequence (L(T)XXQXTG), found on proteins involved in regulating endocytosis, including the AP50 subunit of Adaptor Protein 2, (AP2) complex. AP50, when phosphorylated, forms a bridge that connects receptors to the clathrin coated pit, facilitating endocytosis. The AP2 complex is recruited exclusively to Phosphotidylinositol 4.5biphoshate (PlP2) on the membrane where it binds through its AP50 subunit. PIP2 also controls the activation of the Phosphotidylinositol 3 Kinase (PI3K) pathway. In this study, we found that E2 is an ArWPrk kinase able to phosphorylate AP50 and control endocytosis. Therefore we studied whether it would also regulate cellular proliferation. Methods: E2 was cloned into a mammalian expression vector and transfected into normal primary mouse hepatocytes. Mutagenesis was performed using PCR to generate 10 separate single amino acid changes in 10 separate endocytic or kinase motifs. Primary Hepatocytes were isolated by perfusion with collagenase and cultured on collagen coated plates. Proliferation was measured by [3H] thymidine incorporation and nuclear staining of PCNA. Results: We found that E2 phosphorylates AP50 and increases the expression of PIP2 and clathrin. As PIP2 controls the recruitment of AP2, this is a significant regulation of endocytosis. The increased expression of clathrin enhances the formation of clathrin coated pits and leads to increased endocytosis. P13K, becomes activated, and converts PIP2 to phosphoinositol-3, 4, 5-triphopshate (PIP3). Akt and Phosphoinositol Dependent Kinase 1 (PDK1) are recruited to activated P13K, and activated PDK I is able to activate Alct through phosphorylation. Alct phosphorylates a multitude of proteins that affect cell growth, cell cycle entry, and cell survival. Conclusions: E2 is an ArWPrk kinase that controls endocytosis through its phosphorylation of the AP50 subunit of AP2 and expression of P1P2. E2 is also able to induce the powerful growth pathway of P13WAkt. Through this pathway, E2 not only blocks apoptosis, but induces proliferation in normally quiescent hepatocytes comparable to other potent mitogens, including TGF alpha and EGF.

11211 IN EXPERIMENTAL PREASClTlC LIVER CIRRHOSIS CALCIUM-DEPENDENT DIURETIC SYSTEMS ARE DOWNREGULATED, BUT MAY BE NORMALIZED BY SPECIFIC METABOLIC AND PHARMACOLOGIC STIMULI G. Sansoe”, M. Aragno2, F. Wong3, E Rosina’, A. Smedile4, M. Rizzetto4. ’Gastroentevolog~linit, Gradenigo Hospital, Torino;

2Depurtment of Experimental Medicine und Oncology, Uniuer~sityof Eirino, Torino, Ituly; ‘Depurtment of Medicine, Eironto Generul Hospital, Eironto, ON, Canuda; 4De~~urtment of Gastroenterolog~~, Uniuersity of Rivino, Torino, Italy E-mail: [email protected] Extracellular calcium stimulates cell membrane receptors called Calciumsensing Receptors (CaRs), leading to enhanced renal tubule production of PGE2, which in turn decreases both sodium reabsorption in the thick ascending limb (TAL) of Henle’s loop and free-water reabsorption in collecting ducts [ 1,2]. Parathyroid hormone (PTH) increases expression of CaRs in the kidney and reduces Na+-K+-2CI- cotransporter (BSC-I) expression in TAL cells [3]. To assess the activity of this Ca’+-dependent diuretic system in experimental preascitic cirrhosis we evaluated renal function, plasma levels of active renin, aldosterone and vasopressin, PGE2-urinary excretion, and renal tissue concentrations of BSC-I and CaRs in four groups of rats: 10 control rats receiving i.v. 5% glucose solution (vehicle), and three groups of I0 rats with CCL-induced preascitic cirrhosis receiving vehicle or 0.5mg i.v. poly-L-arginine (PLA), a CaR-selective agonist [4], or 5 subcutaneous doses o f 3 ~ g / k gPTH (one dose every 12 h before the study). The amount of vehicle infused prior to determinations was the same in each group. When compared to controls, cirrhotic rats showed reduced urine volume and absolute and fractional excretion rate of sodium (P i 0.05). Western