Monica Pibiri

Early Increase in Cyclin-D1 Expression and Accelerated Entry of Mouse Hepatocytes into S Phase after Administration of the Mitogen 1,4-Bis(2- (3,5-Dichloropyridyloxy)) Benzene

We have previously demonstrated that hepatocyte proliferation induced by the mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) is independent of changes in cytokines, immediate early genes, and transcription factors that are considered to be necessary for regeneration of the liver after partial hepatectomy (PH) or necrosis. To further investigate the differences between mitogen-induced mouse hepatocyte proliferation and liver regeneration after PH, we have measured the expression of cyclin D1, cyclin D3, cyclin E, and cyclin A and of the cyclin-dependent kinases CDK2, CDK4, and CDK6. The involvement of the cyclin-dependent kinase inhibitors p21 and p27 and of the oncosuppressor gene p53 was also examined at different times after stimulation of hepatocyte proliferation. Results showed that a single administration of TCPOBOP caused a very rapid increase in the levels of cyclin D1, a G1 protein, when compared with two thirds PH (8 hours versus 30 hours). The early increase in cyclin D1 protein levels was associated with a faster onset of increased expression of S-phase-associated cyclin A (24 hours versus 36 hours with PH mice). Accordingly, measurement of bromodeoxyuridine (BrdU) incorporation revealed that, although approximately 8% of hepatocytes were BrdU-positive as early as 24 hours after TCPOBOP, no significant changes in BrdU incorporation were observed at the same time point after two thirds PH. The expression of other proteins involved in cell cycle control, such as cyclin-dependent kinases (CDK4, CDK2, CDK6), was also analyzed. Results showed that expression of CDK2 was induced much more rapidly in TCPOBOP-treated mice (2 hours) than in mice subjected to PH (36 hours). A different pattern of expression in the two models of hepatocyte proliferation, although less dramatic, was also observed for CDK4 and CDK6. Expression of the CDK inhibitors p21 and p27 and the oncosuppressor gene p53 variably increased after two thirds PH, whereas basically no change in protein levels was found in TCPOBOP-treated mice. The results demonstrate that profound differences in many cell cycle-regulatory proteins exist between direct hyperplasia and compensatory regeneration. Cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by the primary mitogen TCPOBOP and suggests that a direct effect of the mitogen on this cyclin may be responsible for the rapid onset of DNA synthesis observed in TCPOBOP-induced hyperplasia.

Thyroid hormone (T3) and TRβ agonist GC-1 inhibit/reverse nonalcoholic fatty liver in rats

Nonalcoholic fatty liver disease is the most common noninfectious liver disease in clinical practice, and there is an increasing need for new therapeutic approaches for the treatment of this liver disease. Here, we examined the effect of the thyroid hormone triiodothyronine (T3) and the agonist of the thyroid hormone receptor β isoform (TRβ), GC‐1, on fatty liver and steatohepatitis induced in rodents by a choline‐methionine deficient (CMD) diet. Male Fischer 344 rats fed a CMD diet for 1 wk developed a marked fatty liver and mild hepatitis. Concurrent administration of T3 resulted in a complete prevention of the fatty change associated with increased fatty acid mitochondrial and peroxisomal β‐oxidation. To investigate whether T3 could also reverse fully established fatty liver, rats were fed a CMD diet for 10 wk and then cofed T3 for 1 wk. Coadministration of T3 resulted in a complete regression of liver steatosis associated with a decrease of lipid peroxidation, cyclooxygenase‐2 expression, and activation of phospho‐STAT3 and phospho‐SAPK/JNK. Finally, additional experiments showed that GC‐1, which has no significant side effects on heart rate, prevented and reverted CMD‐induced fat accumulation, and ameliorated steatohepatitis. These results indicate that TR agonists have the potential to inhibit or reverse hepatic steatosis induced by a nutritional model.—Perra, A., Simbula, G., Simbula, M., Pibiri, M., Kowalik, M. A., Sulas, P., Cocco, M. T., Ledda‐Columbano, G. M., Columbano, A. Thyroid hormone (T3) and TRβ agonist GC‐1 inhibit/reverse nonalcoholic fatty liver in rats. FASEB J. 22, 2981–2989 (2008)

Gadd45β is induced through a CAR‐dependent, TNF‐independent pathway in murine liver hyperplasia

We previously observed that Gadd45β/MyD118, a member of the Gadd45 family of inducible factors, showed the strongest immediate‐early induction common to two distinctive proliferation responses of the liver: (1) regeneration induced by surgical partial hepatectomy and (2) hyperplasia induced by the primary mitogen TCPOBOP, a ligand of the constitutive androstane receptor (CAR). Gadd45β is known to be stimulated by nuclear factor (NF) κB, which is activated by tumor necrosis factor alpha (TNFα) in the early response to partial hepatectomy. We therefore investigated whether TNFα and NFκB also stimulated Gadd45β as part of the response to CAR ligands, or whether activation occurred by an alternative pathway. TCPOBOP effects were characterized in three mouse genotypes: wild‐type, TNFR1−/−, and TNFR1−/−TNFR2−/−. The results showed that TCPOBOP did not activate NFκB in any of the mice, but a strong induction of Gadd45β messenger RNA was observed in all three genotypes, where TCPOBOP also induced CyP2b10, a classical target gene of activated CAR, and cyclin D1, a proliferation linked gene. Thus, the absence of TNFR signaling and induction of NFκB did not impair CAR‐mediated gene induction. Moreover, hepatocyte proliferation was strongly induced, and at significantly higher levels than wild type, in both TNFR1−/− and TNFR1−/−TNFR2−/− mice. Further studies evaluated TCPOBOP‐induced gene expression in CAR−/− mice, by microarray expression profiling and Northern blot. The induced changes in gene expression, including the stimulation of Gadd45β, were almost completely abolished—hence all were mediated via CAR activation. In conclusion, in the liver, Gadd45β can be induced by a distinctive pathway that requires CAR and is independent of TNFα‐NFκB. The greater induction of proliferation in TNFR‐null mice suggests negative cross‐talk between the CAR and TNFα‐NFκB controls that regulate proliferation. (HEPATOLOGY 2005.)

Gadd45beta is induced through a CAR-dependent, TNF-independent pathway in murine liver hyperplasia.

We previously observed that Gadd45β/MyD118, a member of the Gadd45 family of inducible factors, showed the strongest immediate‐early induction common to two distinctive proliferation responses of the liver: (1) regeneration induced by surgical partial hepatectomy and (2) hyperplasia induced by the primary mitogen TCPOBOP, a ligand of the constitutive androstane receptor (CAR). Gadd45β is known to be stimulated by nuclear factor (NF) κB, which is activated by tumor necrosis factor alpha (TNFα) in the early response to partial hepatectomy. We therefore investigated whether TNFα and NFκB also stimulated Gadd45β as part of the response to CAR ligands, or whether activation occurred by an alternative pathway. TCPOBOP effects were characterized in three mouse genotypes: wild‐type, TNFR1−/−, and TNFR1−/−TNFR2−/−. The results showed that TCPOBOP did not activate NFκB in any of the mice, but a strong induction of Gadd45β messenger RNA was observed in all three genotypes, where TCPOBOP also induced CyP2b10, a classical target gene of activated CAR, and cyclin D1, a proliferation linked gene. Thus, the absence of TNFR signaling and induction of NFκB did not impair CAR‐mediated gene induction. Moreover, hepatocyte proliferation was strongly induced, and at significantly higher levels than wild type, in both TNFR1−/− and TNFR1−/−TNFR2−/− mice. Further studies evaluated TCPOBOP‐induced gene expression in CAR−/− mice, by microarray expression profiling and Northern blot. The induced changes in gene expression, including the stimulation of Gadd45β, were almost completely abolished—hence all were mediated via CAR activation. In conclusion, in the liver, Gadd45β can be induced by a distinctive pathway that requires CAR and is independent of TNFα‐NFκB. The greater induction of proliferation in TNFR‐null mice suggests negative cross‐talk between the CAR and TNFα‐NFκB controls that regulate proliferation. (HEPATOLOGY 2005.)

Yes-Associated Protein Regulation of Adaptive Liver Enlargement and Hepatocellular Carcinoma Development in Mice

The Hippo kinase cascade, a growth‐suppressive pathway that ultimately antagonizes the transcriptional coactivator Yes‐associated protein (YAP), has been shown in transgenic animals to orchestrate organ size regulation. The purpose of this study was to determine whether in non–genetically modified mice (1) the Hippo pathway is involved in the regulation of adaptive liver enlargement caused by the mitogen 1,4‐bis[2‐(3,5‐dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor and (2) a dysregulation of this pathway occurs during the development of chemically induced hepatocellular carcinoma (HCC). We show that liver enlargement caused by TCPOBOP was associated with an increase of YAP protein levels that paralleled the increase in 2‐bromodeoxyuridine incorporation. Interestingly, when a second dose of TCPOBOP was given to mice with enlarged livers, no further increases in liver mass or YAP protein levels were observed, suggesting that the Hippo pathway prevents further growth of the hyperplastic liver. Viral‐mediated exogenous expression of active YAP in mouse livers was able to partially overcome the block of hepatocyte proliferation. We also show that HCCs developed in mice given diethylnitrosamine and then subjected to repeated treatments with TCPOBOP had increased levels of YAP that were associated with down‐regulation of microRNA 375, which is known to control YAP expression, and with enhanced levels of alpha‐fetoprotein and connective tissue growth factor, two target genes of YAP.

Increased expression of c-fos, c-jun and LRF-1 is not required for in vivo priming of hepatocytes by the mitogen TCPOBOP

The notion that an increased expression of immediate early genes such as c-fos and c-jun is an absolute requirement for the G0-G1 transition of the hepatocytes has recently been challenged by the finding that rat liver cell proliferation induced by primary mitogens may occur in the absence of such changes (Columbano and Shinozuka, 1996). To further investigate the relationship between immediate early genes and hepatocyte proliferation, we have compared the hepatic levels of c-fos, c-jun and LRF-1 transcripts during mouse liver cell proliferation in two conditions: (i) direct hyperplasia induced by the non-genotoxic hepatocarcinogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, and (ii) compensatory regeneration caused by a necrogenic dose of carbon tetrachloride. The results show striking differences in the activation of early genes. In spite of a rapid stimulation of S phase by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (approximately 8% of hepatocytes were BrdU-positive as early as 24 h after mitogen treatment versus 1% of labelled hepatocytes after 2/3 partial hepatectomy), no changes in the expression of c-fos, c-jun and LRF-1 could be observed. Moreover, no change in steady state mRNA hepatic levels of IGFBP-1 (a gene highly expressed in rat liver following partial hepatectomy), and only a slight increase in c-myc and PRL-1, was found after mitogen administration. On the contrary, a rapid, massive and transient increase in the hepatic mRNA levels of all these genes was observed during carbon tetrachloride induced regeneration. The results indicate that increased expression of immediate early genes may be dependent upon the nature of the proliferative stimulus, and it may not be a prerequisite in certain in vivo conditions such as proliferation induced in the absence of liver tissue damage.

Thyroid Hormone Receptor Ligands Induce Regression of Rat Preneoplastic Liver Lesions Causing Their Reversion to a Differentiated Phenotype

Triiodothyronine (T3), through interaction with its intracellular thyroid hormone receptors (TRs), influences various physiological functions, including metabolism, development, and growth. We investigated the effect of T3 and the selective TR‐β agonist GC‐1 in two models of hepatocarcinogenesis. Preneoplastic lesions were induced in F‐344 rats via a single dose of diethylnitrosamine, followed by a choline‐deficient (CD) diet for 10 weeks. Rat subgroups were then fed the CD diet or a CD diet containing either 4 mg/kg T3 or 5 mg/kg GC‐1 for another week. Rats fed a CD diet alone showed a large number (65/cm2) of preneoplastic lesions positive for the placental form of glutathione S‐transferase (GSTP). Coadministration of T3 for the last week caused an almost complete disappearance of the foci (3/cm2). A reduction of GSTP‐positive foci was also observed in rats fed a CD + GC‐1 diet (28/cm2 versus 75/cm2 of rats fed a CD diet alone) in the absence of significant differences in labeling or apoptotic index of preneoplastic hepatocytes between the two groups. An antitumoral effect of GC‐1 was also observed with the resistant hepatocyte model of hepatocarcinogenesis. Nodule regression was associated with a return to a fully differentiated phenotype, indicated by the loss of the fetal markers GSTP and gamma glutamyl transpeptidase, and reacquisition of the activity of glucose 6‐phosphatase and adenosine triphosphatase, two enzymes expressed in normal hepatocytes. Conclusion: Our results indicate that activated TRs negatively influence the carcinogenic process through induction of a differentiation program of preneoplastic hepatocytes. The results also suggest that TRs could be a meaningful target in liver cancer therapy. (HEPATOLOGY 2009.)

Thyroid hormone induces cyclin D1 nuclear translocation and DNA synthesis in adult rat cardiomyocytes

Although mammalian cardiomyocytes lose their proliferative capacity after birth, there is evidence that postmitotic cardiomyocytes can proliferate provided that cyclin D1 accumulates in the nucleus. Here we show by Northern blot, Western analysis, and immunohistochemistry that 3,5,3′‐triiodothyronine (T3) treatment of adult rats caused an increase of cyclin D1 mRNA and protein levels. The increased cyclin D1 protein content was associated with its translocation into the nucleus of cardiomyocytes. These changes were accompanied by the re‐entry of cardiomyocytes into the cell cycle, as demonstrated by increased levels of cyclin A, PCNA, and incorporation of bromodeoxyuridine into DNA (labeling index was 30.2% in T3‐treated rats vs. 2.2% in controls). Entry into the S phase was associated with an increased mitotic activity as demonstrated by positivity of cardiomyocyte nuclei to antibodies anti‐phosphohistone‐3, a specific marker of the mitotic phase (mitotic index was 3.01/1000 cardiomyocte nuclei in hyperthyroid rats vs. 0.04 in controls). No biochemical or histological signs of tissue damage were observed in the heart of T3‐treated rats. These results demonstrated that T3 treatment is associated with a re‐entry of cardiomyocytes into the cell cycle and so may be important for the development of future therapeutic strategies aimed at inducing proliferation of cardiomyocytes.—Ledda‐Columbano, G. M., Molotzu, F., Pibiri, M., Cossu, C., Perra, A., Columbano, A. Thyroid hormone induces cyclin D1 nuclear translocation and DNA synthesis in adult rat cardiomyocytes. FASEB J. 20, 87–94 (2006)

Triiodothyronine stimulates hepatocyte proliferation in two models of impaired liver regeneration

Abstract.  Objectives: Liver regeneration is attenuated in old age and is substantially slower after 90% than after 70% partial hepatectomy (PH). We have previously demonstrated that the proliferative response to a primary mitogen is intact in aged mice, indicating that impaired liver regeneration is not due to loss of proliferative capacity. Here, we have investigated whether mitogenic effects of triiodothyronine (T3) could reverse the impaired regeneration of ageing or 90% hepatectomy, in the rat. Materials and methods: T3 (20 µg/100 g body weight) was administered to 14‐month‐old rats subjected to 70% PH or to young rats subjected to 90% PH. Cell‐proliferative capacity was determined by bromodeoxyuridine incorporation and microscopy and changes of cell cycle‐related proteins were analysed by Western blot analysis. Results: Treatment of old intact rats with T3 increased cyclin D1 expression that was followed by an enhanced proliferative response, the labelling index (LI), being 7.8% versus 1.3% of controls. T3 given before 70% PH stimulated regenerative response (LI was 10.8% versus 2.28%), and expression of cyclin D1 and proliferating cell nuclear antigen (PCNA) 24 h after PH. Pre‐treatment with T3 also improved the regenerative response of the liver after 90% hepatectomy (LI was 27.9% versus 14.2%). Conclusions: These findings show in principle that mitogen‐induced hyperplasia could be applied to human therapy in patients with reduced regenerative capacity or massive loss of hepatocytes.

Mild Synthetic Approach to Novel Indole-1-Carbinols and Preliminary Evaluation of Their Cytotoxicity in Hepatocarcinoma Cells

A mild and versatile method for the synthesis of some novel indole‐1‐carbinols has been developed via one‐pot reaction of indoles and paraformaldehyde in the presence of an excess of CaO, MgO, ZnO or TiO2. The solvent‐free reaction provided all the indole derivatives in moderate to good yields and short reaction times. Moreover, the effect of some selected indole‐1‐carbinols on cell proliferation of the hepatoma cell line FaO was evaluated.