STAT-induced STAT inhibitor 1 expression after partial hepatectomy in rat

I-IEPATOLOGYVol. 34, No. 4, Pt. 2, 2 0 0 1

AASLD ABSTRACTS

393A

883

884

DOES THE INCREASE IN HEPATIC MASS INDUCED BY THE PRIMARY MITOGEN TRI-IODOTHYRONINE CONFER A USEFUL INCREASE IN HEPATIC F U N C T I O N . Raza Malik, Royal Free Campus, Royal Free a n d University College Medical School, London; Rosemary Tootle, Clare Selden, H u m p h r e y Hodgson, Royal Free Campus, Royal Free a n d University College Medical School, L o n d o n Uk Background - Direct Hyperplasia - A type of liver growth in which hepatocytes are stimulated to proliferate by primary mitogens in the absence of liver injury. Direct HyperpLasia appears to be a primary event in which liver cell proliferation occurs, resulting in an increase in liver mass to above the normal value. A variety of agents can act as primary mitogens and the process by which they induce DNA synthesis and cell division is not clear. The present study characterises the effects of tri-iodothyronine (T3) as a primary mitogen for the liver. Tri-iodothyronine was a prime candidate in view of its potential exploitation as an inexpensive pharmaceuticallyavailable endogenous hormone that has been critically appraised in humans. Study la Aim - To confirm that tri-iodothyronine is a primary mitogen for the liver and to characterise the time course and magnitude of its action. Method: a) Male sprague-dawleyrats (n=7 per group) were injected with a single dose (4mg/kg) of tri-iodothyronine (T3) and sacrificed at intervals of 1,2,4,7,10 and 14 days. A control group received vehicle only twenty four hours prior to sacrifice. Cell Proliferation -Assessed by bromodeoxyuridine (BrdU) incorporation into nuclei and immunohistochemical recognition.Each animal recieved an intra peritoneal dose (50mg/kg) of BrdU one hour prior to sacrifice (flash labelling). Statistical analysis - Statistical differences were determined using the two tailed t-test and reported if p<0.05. Results: Liver mass was increased in animals treated with tri-iodothyronine as compared to controls. Maximum effect was seen on day 10 with a 20% increase in liver mass (p<0.01). A corresponding increase in total DNA (p
ACTIVATION OF SIGNAL TRANSDUCER AND ACTIVATOR TRANSCRIPTION (STAT) 3 AND SUPPRESSOR OF CYTOKINE SIGNAL (SOCS) I/STAT-INDUCED STAT I N H I B I T O R 1 EXPRESSION AFTER PARTIAL HEPATECTOMY IN RAT. Shigeru Sakuda, Sinji Tamura, Akira Yamada, Jun-Ichiro Miyagawa, Koji Yamamoto, Shinichi Kiso, Nobuyuki ho, Tetsuji Naka, Tadamitsu Kishimoto, Sumio Kawata, Yuji Matsuzawa, Grad Sch of Medicine, Osaka Univ, Suita J a p a n Aims: Recent studies have s h o w n that various transcription factors are activated shortly after partial hepatectomy (PH). The signal transducer a n d activator of transcription (STAT) 3, w h i c h is normally associated with the cytokine signal transduction pathway, is activated during the first few hours after PH a n d its activation plays a critical role in the initiation of liver growth after PH. However, the issue of where STAT3 is activated a n d h o w it is regulated is unclear. The aims of this study were to identify STAT3-activated cells a n d to clarify the expression of suppressor of cytokine signal (SOCS) 1/STAT-induced STAT inhibitor (SSI) 1, a negative feedback molecule of STAT3, after PH. Methods: Using rats, a two-thirds PH was performed, after w h i c h STAT3activated cells a n d SOCS 1-positive cells were identified by i m m u n o h i s t o c h e m istry. SOCS1 gene expression was analyzed by the reverse transcription-polymerase chain reaction. Results: Immunohistochemical analysis showed that the first STAT3-activated cells were Kupffer ceils a n d sinusoidal endothelial cells, w h i c h were detectable at 0.5 h after PH. The hepatocyte nuclei became positive for STAT3 at 1 h after PH a n d the n u m b e r of STAT3-activated hepatocytes reached a m a x i m u m at 3 h. The SOCS1 gene was expressed in regenerating livers within 0.5 h, reaching a m a x i m u m at 4.5-6 h after PH. SOCS1 immunoreactivity became to be detected predominantly in the periportal zones of hepatic lobules at 3 h after PH, a n d the localization then extended to all lobules at 12 h after PH. Conclusions: STAT3 activation was detected in Kupffer cells a n d sinusoidal endothelial cells prior to its detection in hepatocytes. Following STAT3 activation in response to PH, SOCS1 protein was expressed. SOCS1 m a y regulate STAT3 signaling negatively after PH.

885

886

TGF~I SIGNALING AND WEE1 IN HEPATOMA CELL-CYCLE CONTROL. O s a m u Hashimoto, Takato Ueno, Rina Kimura, Hironori Koga, Ryuichiro Sakata, Toru N a k a m u r a , Riko Ogata, Masaharu Sakamoto, Takuji Torimura, Michio Sata, K u r u m e university school of medicine, K u r u m e J a p a n

MOUSE-MUSASHI-1, AN RNA-BINDING PROTEIN, IS EXPRESSED IN THE MOUSE HEPATIC PROGENITOR CELLS/EARLY HEPATOCYrES DURING LIVER DEVELOPMENT. Shinichi Kiso, Graduate School of Medicine, Osaka University, Osaka Japan; Sumio Kawata, Yamagata University School of Medicine, Yamagata Japan; Shinji Tamura, Jun-lchiro Miyagawa, Yoshiaki Doi, Takao Nanmo, Ayuko Saeki, Yoshihiro Kamada, Takako Kawasaki, Shin-lchi Sakakibara, Graduate School of Medicine, Osaka University, Osaka Japan; Hideyuki Okano, School of Medicine, Keio University, Tokyo Japan; Yuji Matsuzawa, Graduate School of Medicine, Osaka University, Osaka Japan

Aim: TGF/81 c o m m o n l y induces the g r o w t h arrest a n d apoptosis of hepatocytes. However, hepatoma cell g r o w t h u n d e r the circumstance in w h i c h TGF]31 expression is enhanced. TGF/31 was recently reported to effect at two different stages, G1 a n d G2, with or w i t h o u t Rb function (Cell 97:53-61, 1999). In h e p a t o m a cells, roles of TGF]31 in the G1 phase have been s h o w n in m a n y reports, but, the relationship between T G F b l a n d G2/M arrest is little known. In this study, we investigated that the induction of G2/M arrest b y TGF~81 treatment a n d its m e c h a n i s m in h e p a t o m a cell. Methods: W e characterized G2/M arrest i n d u c e d b y T G F ~ I (1 ng/ml) in the t u m o r suppressor p53a n d Rb-negative h e p a t o m a cell line (Hep3B), a n d compared with G1 arrest in the p 5 3 - m u t a t e d a n d Rb-positive h e p a t o m a cell line (Huh7). W e used Hep3B treated with 1ng/ml TGF/31 for 12 to 48hours(h). The expression of molecules closely related to G2/M arrest was analyzed by the following methods. (1)cell cycle profile, cell cycle a n d cyclin B; flow cytometry. (2)CDK2, cdc2, phosphorylated-cdc2 (Tyl5), CDK inhibitors (p21 a n d p27), Cdc25C, p h o s p h o r ylated-cdc2 (Tyl5) a n d Wee1; Western blot. (3) CDK2 a n d cdc2 activities; kinase assay. (4)mRNA expression of Cdc25C a n d Wee1; RT-PCR (5)Immunolocalization of cyclinB; fluorescence microscopy. (6)Stability of Wee1 by TGF]]I was analyzed with cyclohexamide treatment. Results: Hep3B arrested in the G2/M phase after TGF]81 treatment for 24h. At this point, cyclin B in G2/M phase cells were kept at high expression level in the nuclei. The expression of CDK2 a n d cdc2 was not changed. However, CDK2 a n d cdc2 kinase activities were completely down-regulated. The expression of p21, p27 a n d phosphorylated-cdc2(Ty15)was increased, whereas cdc25C was decreased after 24h. Wee1 protein in Hep3B was unstable but became substantially stabilized within 6 h of T G F b l treatment. Conclusions: These findings suggested that TGF]31 p r o m o t e d G2/M arrest in Hep3B by rapid stabilization of Wee1 a n d subsequent phosphorylation of Cdc2 o n Ty-15. It is also suggested that some TGFj81signaling pathways to hepatoma cell cycle exist in hepatocellular carcinoma.

BACKGROUND/AIMS: There is increasing interest in the role of RNA-binding protein during organ development. Musashi is one of the RNA-binding proteins essential for neural development and required for asymmetric cell divisions in the sensory organ development. In neurogenesis, neuron stem cell, which highly expressed mouse-Musashi-1 (m-Msi1(++)), divide a new stem cell (m-Msi-l(++)) and immature neuron cell (m-Msi-l()or(±)), asymmetrically. However, it remains unclear whether m-Msi-1 participates in liver development. In this study, to examine the role of m-Msi-1 during liver development, we investigate the expression of m-Msi-1 in the fetal hepatic cells. MATERIALS AND METHODS : (1) Analysis of m-Msi-1 gene expression. ICR mice were used throughout the experiment. Timed pregnant mice at 13.5 and 17.5 days of gestation were sacrifled and the embryos were removed from the uterus. For RT-PCR, fetal livers were isolated from the embryos under a microscopy, and were preserved in liquid nitrogen immediately after removal. Total RNAs were obtained by an acid guanidinium thiocyanate-phenol-choroform extraction procedure from fetal liver and brain. Fetal brain RNA was served as a positive control. Primers were designed to amplify portion of m-Msi-1. Direct sequencing of RT-PCR products were performed by the dideoxy method with fluorescently labeled 2', 3'-dideoxynucleoside5' -triphosphates. We also investigated the expression of m-Msi-1 in the livers of mice at 7days alter birth and adult. (2) Analysis of m-Msi-1 protein expression. For immunohistochemieal staining, the embryos were fixed in neutralized formaldehyde and embedded in paraffin. Immunohistochemical staining was performed using rat antimouse MSi-1antibody. Double-immunostaining of m-Msi-1 and hepatocyte nuclear factor 4a(HNF-4a) was performed in order to investigate whether the cells, which expressed m-Msi-1 in the fetal liver, are the hepatic cells or hematopoietic cells. RESULTS: (1) The expression of m-Msi-1 gene was observed in the fetal liver as well as brain both at 13.5 and 17.5 days of gestation. The PCR products were identified as the m-Msi-1 gene by direct sequencing. The expression levels of m-Msi-1 gene in the liver peaked at the embryonic day 13.5, when hepatogenesis is active, then decreased during the course of liver development. The liver from the mice at 7 days after birth still expressed m-Msi-1 but adult mouse liver did not any longer. (2) m-Msi-1 protein was detected in the fetal hepatic cells of the mice at 14.5 days of gestation immunohistochemically, reflecting the results of RT-PCR. An asymmetric expression of m-Msi-1 positive and m-Msi-1 negative was demonstrated in the embryonic hepatic cells during liver development, m -Msi-I is expressed in the HNF4apositive hepatic cells in the early liver at embryonic day 14.5, indicating that the mMsi-1 positive cells in the fetal liver may be hepatic progenitor cells/early hepatocytes, but not hematopietic cells. CONCLUSION m-Msi-1 protein is expressed in the hepatic pro~enitor cells/early hepatocyte in the mouse liver development, m-Msi-1 may play roles in tetal liver development though differential sorting of mRNA populations during asymmetric division of hepatic progenitor cells.