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Hepatocyte proliferation – can we target the mechanism?
Journal of Hepatology 36 (2002) 306–309 www.elsevier.com/locate/jhep
BEYOND THE JOURNAL Associate Editors: Guadalupe Garcia-Tsao and Ronald Oude Elferink
Hepatocyte proliferation – can we target the mechanism? Cyclin D1 is an early target in hepatocyte proliferation induced by thyroid hormone (T3). Pibiri M, LeddaColombano GM, Cosso C, Simbula G, Menegazzi M, Shinozuka H, Columbano A. Department of Toxicology, Oncology and Molecular Pathology Unit, University of Cagliari, Italy. The thyroid hormone (T3) affects cell growth, differentiation, and regulates metabolic functions via its interaction with the thyroid hormone nuclear receptors (TRs). The mechanism by which TRs mediate cell growth is unknown. To investigate the mechanisms responsible for the mitogenic effect of T3, we have determined changes in activation of transcription factors, mRNA levels of immediate early genes, and levels of proteins involved in the progression from G1 to S phase of the cell cycle. We show that hepatocyte proliferation induced by a single administration of T3 to Wistar rats occurred in the absence of activation of AP-1, NF-kappa B, and STAT3 or changes in the mRNA levels of the immediate early genes c-fos, c-jun, and c-myc. These genes are considered to be essential for liver regeneration after partial hepatectomy (PH). On the other hand, T3 treatment caused an increase in cyclin D1 mRNA and protein levels that occurred much more rapidly compared to liver regeneration after 2/3 PH. The early increase in cyclin D1 expression was associated with accelerated onset of DNA synthesis, as demonstrated by a 20-fold increase of bromodeoxyuridine-positive hepatocytes at 12 h after T3 treatment and by a 20fold increase in mitotic activity at 18 h. An early increase of cyclin D1 expression was also observed after treatment with nafenopin, a ligand of a nuclear receptor (peroxisome proliferator-activated receptor alpha) of the same superfamily of steroid/thyroid receptors. T3 treatment also resulted in increased expression of cyclin E, E2F, and p107 and enhanced phosphorylation of pRb, the ultimate substrate in the pathway leading to transition from G1 to S phase. The results demonstrate that
cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by T3 and suggest that this cyclin might be a common target responsible for the mitogenic activity of ligands of nuclear receptors. FASEB J 2001;15:1006–1013 Pibiri et al. investigated the mechanisms responsible for the mitogenic effect of triiodothyronine (T3) on hepatocytes. They showed that increased expression of cyclin D1 occurs very early during rat hepatocyte proliferation induced by a single administration of T3. In liver regeneration (LR) following 2/3 partial hepatectomy, cyclin D1 is also induced before the onset of DNA synthesis (BRDU). However, following 2/3 partial hepatectomy this process occurs late in the G1 phase. Therefore, the onset of DNA synthesis and the appearance of mitoses (mitotic activity) were accelerated by T3. Moreover, as opposed to partial hepatectomy, activation of cyclin D1 after T3 was not the result of induction of immediate early genes (c-fos, c-jun, cmyc) or activation of transcription factors. A similar effect on cyclin D1 was observed by nafenopine, a peroxisome proliferator the ligand of which is of the same superfamily of steroid/thyroid receptor. They also evaluated several other players of the cell cycle and found that following the introduction of T3 the expression of cyclin E, E2F and p 107 is also increased. The phosphorylation of pRb, the ultimate substrate in the pathway leading to the transition from G1 to S phase, was enhanced by T3. They conclude that cyclin D1 might be a common target responsible for the mitogenic activity of ligands of nuclear receptors [1]. Since the description of LR in Greek mythology and up to 20 years ago, the magnificent ability of the liver to undergo complete regeneration following loss of liver mass was one of the main subjects of liver research. Therefore, this was the ‘liver regeneration era’ [2]. Although the process is very impressive, during LR quiescent hepatocytes undergo only one or two rounds of replication, then enter a nonproliferative state. Can hepatocytes undergo more than two rounds of replication? This question was
0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.
Beyond the Journal
answered by two experimental models for extensive liver repopulation: the urokinase-type plasminogen activator (uPA) transgenic mouse and the fumarylacetoacetate hydrolase (Fah) null mouse [3,4]. Both models have convincingly documented that transplanted hepatocytes have enormous proliferation potential. These models signal the beginning of the ‘hepatocyte proliferation era’ and the main focus then was on repopulation of the liver by newly transplanted hepatocytes. Therefore, the proliferation era was thus followed by the ‘hepatocyte transplantation era’. Naturally, one of the most important issues was to identify efficient hepatocyte mitogens and to develop models to provide selective growth advantage to transplanted cells over endogenous hepatocytes, leading to the ‘hepatocyte mitogens era’. Indeed, a great number of studies evaluated the capacity of growth factors and cytokines involved in LR to stimulate proliferation of hepatocytes. However, none demonstrated a significant outcome [5]. Therefore, several groups worldwide evaluated the potential of hormones to stimulate hepatocytes for proliferation. The role of the thyroid hormone, mainly T3, as an attractive mitogen for hepatocytes has been extensively evaluated in recent years [6–9]. The major effect appears to be transcriptional regulation of target genes. The effect of T3 at the genomic level has been shown to be mediated by nuclear thyroid hormone receptor (TR). Thyroidectomy was found to reduce the hepatic regeneration response in rats. Moreover, injection of T3 to normal rats induced hepatocyte proliferation such as that seen after 40% hepatectomy, with a peak DNA synthesis at 24 h post-injection. Decreased serum thyroid hormone levels reduce hepatocyte turnover, whereas the introduction of thyroid hormone accelerates proliferation of injured liver cells far more than normal hepatocytes. Ohmura, Francavilla, Columbano, Shinozuka et al. have shown in numerous studies that T3 as well as 9-cis retinoic acid and some peroxisome proliferators are potent inducers of hepatocyte proliferation in rats [6,7]. Forbes et al. have reported the induction of liver hyperplasia by T3 for retroviral gene transfer [8]. In the retrorsine model for liver repopulation, T3 was shown to be a very effective stimulator for the proliferation of transplanted hepatocytes and to be as potent as 2/3 partial hepatectomy [9]. The use of T3 to stimulate hepatocyte proliferation is very attractive as its commercially available form has been known as a very safe medication for years, with an easily monitored effect, reversible if necessary, and with the possibility of repeated introduction to patients. The Columbano research team has contributed significantly to the field of hepatocyte proliferation in the last 10 years and published very interesting studies that looked into the mechanism of action of T3 on liver cells. Indeed, Pibiri et al. evaluated the mechanism of T3-induced proliferation of hepatocytes and concentrated on the effects of T3 on cyclin D1. The importance of cyclin D1 to cell proliferation has been strongly implicated in controlling the G1 phase of the cell cycle in humans. The expression of cyclin D1 mRNA
307
and protein peaks during mid-G1 in growth factor-derived cells that are re-stimulated to enter the cell cycle. Moreover, inhibition of cyclin D1 function can delay or even prevent entry of these cells into the S phase of the cell cycle. In the present article, Pibiri et al. show that increased expression of cyclin D1 is one of the earlier events during rat hepatocyte proliferation induced by T3. They also make an interesting comparison to the sequence of events that follow partial hepatectomy (PH), and suggest that the activation of cyclin D1 by T3 may be the result of the action of thyroid receptors on this gene (as they have also shown with regard to peroxisome proliferator activated receptor, PPAR·) rather than the consequence of a series of gene activation as seen in the model of PH. They suggest that during hepatocyte proliferation the same mechanism is shared by several ligands of the nuclear receptor. This study, along with recent other studies might signify the ‘mechanism era’. The question is, can we target the mechanism? R. Oren Tel Aviv Sourasky Medical Center, The Liver Unit, Tel-Aviv, Israel
References [1] Pibiri M, Colombano GML, Cosso C, Simbula G, Menegazzi M, Shinozuka H, Colombano A. Delete this Cyclin D1 is an early target in hepatocyte proliferation induced by thyroid hormone (T3). Fed Am Soc Exp Biol J 2001;15:1006–1013. [2] Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997;276(5309):60–66. [3] Rhim JA, Sandgren EP, Degen JL, Palmiter RD, Brinster RL. Replacement of diseased mouse liver by hepatic cell transplantation. Science 1994;263:1149–1152. [4] Overturf K, Al-Dhalimy M, Tanguay R, Brantly M, Ou C-N, Finegold M, Grompe M. Adenovirus-mediated gene therapy in a mouse model of hereditary tyrosinemia type I. Nat Genet 1996;12:266. [5] Weber EM, Bruix J, Pierce RH, Fausto N. Tumor necrosis factor primes hepatocytes for DNA replication in the rat. Hepatology 1998;28:1226–1234. [6] Ohmura T, Katyal SL, Locker J, Ledda-Columbano G, Columbano A, Shinozuka H. Induction of cellular DNA synthesis in the pancreas and kidneys of rats by peroxisome proliferators, 9-cis retinoic acid, and 3,3 0 ,5-triiodo-l-thyronine. Cancer Res 1997;57:795–798. [7] Francavilla A, Carr BI, Azzarone A, Polimeno L, Wang Z, Van Thiel DH, et al. Hepatocyte proliferation and gene expression induced by triiodothyronine in vivo and in vitro. Hepatology 1994;20(5):1237– 1241. [8] Forbes SJ, Themis M, Alison MR, Shiota A, Kobayashi T, Coutelle C, Hodgson HJF. Triiodothyronine and deleted form of hepatocyte growth factor act synergistically to enhance liver proliferation and enable in vivo retroviral gene transfer via the peripheral venous system. Gene Ther 2000;7:784–789. [9] Oren R, Dabeva MD, Petkov PM, Moss S, Wang S, Hurston E, et al. Role of thyroid hormone in stimulating liver repopulation by transplanted hepatocytes. Hepatology 1999;30(4):903–913. PII: S0168 -8 278(01)00312 -9
BEYOND THE JOURNAL Associate Editors: Guadalupe Garcia-Tsao and Ronald Oude Elferink
Hepatocyte proliferation – can we target the mechanism? Cyclin D1 is an early target in hepatocyte proliferation induced by thyroid hormone (T3). Pibiri M, LeddaColombano GM, Cosso C, Simbula G, Menegazzi M, Shinozuka H, Columbano A. Department of Toxicology, Oncology and Molecular Pathology Unit, University of Cagliari, Italy. The thyroid hormone (T3) affects cell growth, differentiation, and regulates metabolic functions via its interaction with the thyroid hormone nuclear receptors (TRs). The mechanism by which TRs mediate cell growth is unknown. To investigate the mechanisms responsible for the mitogenic effect of T3, we have determined changes in activation of transcription factors, mRNA levels of immediate early genes, and levels of proteins involved in the progression from G1 to S phase of the cell cycle. We show that hepatocyte proliferation induced by a single administration of T3 to Wistar rats occurred in the absence of activation of AP-1, NF-kappa B, and STAT3 or changes in the mRNA levels of the immediate early genes c-fos, c-jun, and c-myc. These genes are considered to be essential for liver regeneration after partial hepatectomy (PH). On the other hand, T3 treatment caused an increase in cyclin D1 mRNA and protein levels that occurred much more rapidly compared to liver regeneration after 2/3 PH. The early increase in cyclin D1 expression was associated with accelerated onset of DNA synthesis, as demonstrated by a 20-fold increase of bromodeoxyuridine-positive hepatocytes at 12 h after T3 treatment and by a 20fold increase in mitotic activity at 18 h. An early increase of cyclin D1 expression was also observed after treatment with nafenopin, a ligand of a nuclear receptor (peroxisome proliferator-activated receptor alpha) of the same superfamily of steroid/thyroid receptors. T3 treatment also resulted in increased expression of cyclin E, E2F, and p107 and enhanced phosphorylation of pRb, the ultimate substrate in the pathway leading to transition from G1 to S phase. The results demonstrate that
cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by T3 and suggest that this cyclin might be a common target responsible for the mitogenic activity of ligands of nuclear receptors. FASEB J 2001;15:1006–1013 Pibiri et al. investigated the mechanisms responsible for the mitogenic effect of triiodothyronine (T3) on hepatocytes. They showed that increased expression of cyclin D1 occurs very early during rat hepatocyte proliferation induced by a single administration of T3. In liver regeneration (LR) following 2/3 partial hepatectomy, cyclin D1 is also induced before the onset of DNA synthesis (BRDU). However, following 2/3 partial hepatectomy this process occurs late in the G1 phase. Therefore, the onset of DNA synthesis and the appearance of mitoses (mitotic activity) were accelerated by T3. Moreover, as opposed to partial hepatectomy, activation of cyclin D1 after T3 was not the result of induction of immediate early genes (c-fos, c-jun, cmyc) or activation of transcription factors. A similar effect on cyclin D1 was observed by nafenopine, a peroxisome proliferator the ligand of which is of the same superfamily of steroid/thyroid receptor. They also evaluated several other players of the cell cycle and found that following the introduction of T3 the expression of cyclin E, E2F and p 107 is also increased. The phosphorylation of pRb, the ultimate substrate in the pathway leading to the transition from G1 to S phase, was enhanced by T3. They conclude that cyclin D1 might be a common target responsible for the mitogenic activity of ligands of nuclear receptors [1]. Since the description of LR in Greek mythology and up to 20 years ago, the magnificent ability of the liver to undergo complete regeneration following loss of liver mass was one of the main subjects of liver research. Therefore, this was the ‘liver regeneration era’ [2]. Although the process is very impressive, during LR quiescent hepatocytes undergo only one or two rounds of replication, then enter a nonproliferative state. Can hepatocytes undergo more than two rounds of replication? This question was
0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.
Beyond the Journal
answered by two experimental models for extensive liver repopulation: the urokinase-type plasminogen activator (uPA) transgenic mouse and the fumarylacetoacetate hydrolase (Fah) null mouse [3,4]. Both models have convincingly documented that transplanted hepatocytes have enormous proliferation potential. These models signal the beginning of the ‘hepatocyte proliferation era’ and the main focus then was on repopulation of the liver by newly transplanted hepatocytes. Therefore, the proliferation era was thus followed by the ‘hepatocyte transplantation era’. Naturally, one of the most important issues was to identify efficient hepatocyte mitogens and to develop models to provide selective growth advantage to transplanted cells over endogenous hepatocytes, leading to the ‘hepatocyte mitogens era’. Indeed, a great number of studies evaluated the capacity of growth factors and cytokines involved in LR to stimulate proliferation of hepatocytes. However, none demonstrated a significant outcome [5]. Therefore, several groups worldwide evaluated the potential of hormones to stimulate hepatocytes for proliferation. The role of the thyroid hormone, mainly T3, as an attractive mitogen for hepatocytes has been extensively evaluated in recent years [6–9]. The major effect appears to be transcriptional regulation of target genes. The effect of T3 at the genomic level has been shown to be mediated by nuclear thyroid hormone receptor (TR). Thyroidectomy was found to reduce the hepatic regeneration response in rats. Moreover, injection of T3 to normal rats induced hepatocyte proliferation such as that seen after 40% hepatectomy, with a peak DNA synthesis at 24 h post-injection. Decreased serum thyroid hormone levels reduce hepatocyte turnover, whereas the introduction of thyroid hormone accelerates proliferation of injured liver cells far more than normal hepatocytes. Ohmura, Francavilla, Columbano, Shinozuka et al. have shown in numerous studies that T3 as well as 9-cis retinoic acid and some peroxisome proliferators are potent inducers of hepatocyte proliferation in rats [6,7]. Forbes et al. have reported the induction of liver hyperplasia by T3 for retroviral gene transfer [8]. In the retrorsine model for liver repopulation, T3 was shown to be a very effective stimulator for the proliferation of transplanted hepatocytes and to be as potent as 2/3 partial hepatectomy [9]. The use of T3 to stimulate hepatocyte proliferation is very attractive as its commercially available form has been known as a very safe medication for years, with an easily monitored effect, reversible if necessary, and with the possibility of repeated introduction to patients. The Columbano research team has contributed significantly to the field of hepatocyte proliferation in the last 10 years and published very interesting studies that looked into the mechanism of action of T3 on liver cells. Indeed, Pibiri et al. evaluated the mechanism of T3-induced proliferation of hepatocytes and concentrated on the effects of T3 on cyclin D1. The importance of cyclin D1 to cell proliferation has been strongly implicated in controlling the G1 phase of the cell cycle in humans. The expression of cyclin D1 mRNA
307
and protein peaks during mid-G1 in growth factor-derived cells that are re-stimulated to enter the cell cycle. Moreover, inhibition of cyclin D1 function can delay or even prevent entry of these cells into the S phase of the cell cycle. In the present article, Pibiri et al. show that increased expression of cyclin D1 is one of the earlier events during rat hepatocyte proliferation induced by T3. They also make an interesting comparison to the sequence of events that follow partial hepatectomy (PH), and suggest that the activation of cyclin D1 by T3 may be the result of the action of thyroid receptors on this gene (as they have also shown with regard to peroxisome proliferator activated receptor, PPAR·) rather than the consequence of a series of gene activation as seen in the model of PH. They suggest that during hepatocyte proliferation the same mechanism is shared by several ligands of the nuclear receptor. This study, along with recent other studies might signify the ‘mechanism era’. The question is, can we target the mechanism? R. Oren Tel Aviv Sourasky Medical Center, The Liver Unit, Tel-Aviv, Israel
References [1] Pibiri M, Colombano GML, Cosso C, Simbula G, Menegazzi M, Shinozuka H, Colombano A. Delete this Cyclin D1 is an early target in hepatocyte proliferation induced by thyroid hormone (T3). Fed Am Soc Exp Biol J 2001;15:1006–1013. [2] Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997;276(5309):60–66. [3] Rhim JA, Sandgren EP, Degen JL, Palmiter RD, Brinster RL. Replacement of diseased mouse liver by hepatic cell transplantation. Science 1994;263:1149–1152. [4] Overturf K, Al-Dhalimy M, Tanguay R, Brantly M, Ou C-N, Finegold M, Grompe M. Adenovirus-mediated gene therapy in a mouse model of hereditary tyrosinemia type I. Nat Genet 1996;12:266. [5] Weber EM, Bruix J, Pierce RH, Fausto N. Tumor necrosis factor primes hepatocytes for DNA replication in the rat. Hepatology 1998;28:1226–1234. [6] Ohmura T, Katyal SL, Locker J, Ledda-Columbano G, Columbano A, Shinozuka H. Induction of cellular DNA synthesis in the pancreas and kidneys of rats by peroxisome proliferators, 9-cis retinoic acid, and 3,3 0 ,5-triiodo-l-thyronine. Cancer Res 1997;57:795–798. [7] Francavilla A, Carr BI, Azzarone A, Polimeno L, Wang Z, Van Thiel DH, et al. Hepatocyte proliferation and gene expression induced by triiodothyronine in vivo and in vitro. Hepatology 1994;20(5):1237– 1241. [8] Forbes SJ, Themis M, Alison MR, Shiota A, Kobayashi T, Coutelle C, Hodgson HJF. Triiodothyronine and deleted form of hepatocyte growth factor act synergistically to enhance liver proliferation and enable in vivo retroviral gene transfer via the peripheral venous system. Gene Ther 2000;7:784–789. [9] Oren R, Dabeva MD, Petkov PM, Moss S, Wang S, Hurston E, et al. Role of thyroid hormone in stimulating liver repopulation by transplanted hepatocytes. Hepatology 1999;30(4):903–913. PII: S0168 -8 278(01)00312 -9