P70S6K in hepatic stellate cells: An in vivo and in vitro study

Category 3: Molecular

and cell biology (gene expression,

Conclusions: Our results suggest that HSCs could be an option to improve thie liver regeneration after a toxic injury, supporting the feasibility of BM-derived liver stem cell hypothesis.

I 265

REGULATION

AND CROSSTALK

HEPATIC STELLATE

OF ERWJNWP7OSBK

IN

CELLS: AN IN VIVO AND IN VITRO

STUDY

F. Ridolfi I, S. Saccomanno’, C. Candelaresi’, l? Sterpetti’, A. Benedetti’, F. Folli2, G. Svegliati Baroni’. ‘Department Of Gastroenterology, University OfAncona, Ancona, Italy; 2Department Of Internal Medicine, IRCCS H.S. Raffaele, Milan, Italy Liver fibrosis is characterized by hepatic stellate cell (HSC) activation. Intracellular kinases play a pivotal role in transducing extracellular signals and ERK, JNK and p70S6K regulates cell proliferation and collagen synthesis in HSC in vitro. Activation of these kinases in two models of liver injury leading to hepatic fibrosis, i.e. dimethylnitrosamine administration (DMN) and bile duct ligation (BDL), was thus evaluated. After DMN, kinases activation showed a specular behaviour increasing early at 6 h and then peaking again at 1 week. After BDL, a constant activation of ERK and p70S6k was observed, while JNK phosphorylation peaked after 7 days. Kinases activation in HSC in viva preceded cell proliferation and alpha-smooth muscle actin appearance, as a marker of HSC transformation in myofibroblast-like cells. To identify eventual relationship between these kinases in vitro, HSC were stimulated with 10% serum that induced ERKlR, JNK, and p70S6K phosphorylation, in addition to an almost 18. fold increase in HSC proliferation. By using specific kinase inhibitors, JNK and p70S6K induced ERKphosphorylation which represents a downstream component in this cascade leading to cell proliferation. HSC transiently transfected with a dominant negative form of ERKl (K71R) were incubated with a different stimulus (PDGF) and showed a reduced proliferation rate compared to control cells. This study thus indicates that intracellular kinases activation precedes HSC transformation into myofibroblast in viva. This study provides also specific targets (such as ERK) which could be inhibited by selective delivery of inhibitors or dominant negative mutant to reduce hepatic fibrosis in chronic liver diseases.

SILIBININ

(SIL) PREVENTS

17j3-D-GLUCURONIDE EXCRETORY

ESTRADIOL

(E217G)

-INDUCED

BILE SALT

FAILURE AND INTERNALISATION

CANALICULAR

BILE SALT EXPORT

ISOLATED

RAT HEPATOCYTE

POSSIBLE

INVOLVEMENT

OF THE

PUMP, BSEP, IN THE

COUPLET

(IRHC) MODEL:

OF CYCLIC AMP

EA. Crocenzi’, M.S. Portesio’, C.L. Basiglio’, R. Coleman2, M.G. Roma’. ‘Institute Of Experimental Physiology, Faculty Of Biochemical And Pharmaceutical Sciences, Univ. Of Rosario, Rosario, Argentina; 2Deparment Of Biosciences, The University Of Birmingham, Birmingham, UK Silymtin has beneficial effects in estrogen-induced cholestasis in viva (Hepatology 2001;34: 329). Since E217G induces internalisation of Bsep (.I Hepatol 2002;36 (Suppl): 6), we studied, in IRHCs, whether Sil, the active component of silymtin, prevents changes in Bsep localisation/function. E217G (50 FM) diminished the% of IRHCs accumulating apically the fluorescent bile salt, cholyl-lysylfluorescein (CLF), and Sil (2.5 FM, 30 min) fully prevented this effect. Sil beneficial effect was abolished by the intracellular-Ca2&quelating agent, BAF’TA/AM, but not by the PKA inhibitor, KT.5720 (% of IRHCs accumulating CLF, referred to controls: E217G: 43&2*; E217G+Sil: 93&l; E217G+Sil+BAF”TA/AM: .50&4*; E217G+Sil+KT5720: 92&3#; *p
signalling, jibrosis)

81

a potent inhibitor of CAMP phosphodiesterase, and CAMP protects against E217G-induced internalisation of another major canalicular pump, Mrp2 (Hepatology 2002;35: 1409), we analysed whether Sil increases CAMP in hepatocytes, and whether the CAMP analogue, DBcAMP, is able to mimic Sil effects. Sil increased hepatocellular CAMP levels by 36&6% (p
I

267

OXIDATIVE

STRESS

RELOCALISATION COUPLET

(OS) INDUCES

F-ACTIN AND BSEP

IN THE ISOLATED

(IRHC) MODEL:

AND PROTECTIVE

EFFECT

POSSIBLE

RAT HEPATOCYTE INVOLVEMENT

OF PKC

OF PKA

M.G. Roma’, l? Milkiewicz 2,3 , J Ahmed-Choudhury3, R. Coleman2. ‘IFISE (CONICET) -Fat. De Cs. Bioq. Y Farm., U.N.R., Rosario, Argentina; 2Department Of Biosciences, University Of Birmingham, Birmingham, UK; ‘Liver And Hepatobiliary UnitUniversity Of Birmingham, Birmingham, UK We showed that OS induces blebbing (a marker of actin integrity) and decreases the% of IRHCs accumulating the fluorescent bile salt, cholyllysylfluorescein (CLF), into their canalicular vacuoles (canalicular vacuolar accumulation test, cVA). We analysed here whether OS induced relocalization of the bile salt export pump, Bsep, and of the F-actin cytoskeleton. Since OS induces Ca2&dependent PKC activation, we also tested whether PKC inhibition or PKA activation, which counteracts many PKC-dependent effects, prevents and reverses OS-induced alterations. The oxidising agent, teti-butylhydroperoxide (tBOOH, 100 FM), decreased cVA of CLF by 41&2% (p
I

268

IDENTIFICATION

AND FUNCTION

HEPATIC STELLATE

OF MARCKS

IN HUMAN

CELLS (HHSC)

K. Rombouts, B. Lottini, A. Caligiuri, V. Carloni, M. Pinzani. Dipartimento Di Medicina Interna, University Of Florence, Florence, Italy Myristoylated Alanine-rich C Kinase Substrate (MARCKS) is a major Protein Kinase C substrate, responsible for regulating actin dynamics by a reversible electrostatic switch at the cell membrane. Recent evidence indicated an important role for MARCKS in cell plasticity and cell migration. In this study the presence and function of MARCKS in hHSC was evaluated. Immunofluorescence and confocal microcopy were used to identify the presence of MARCKS and phosphorylated MARCKS in hHSC, and the co-localization of phosphorylated MARCKS with F-actin filaments. Moreover, phosphorylation of MARCKS by PDGF-induced PKC activation was