The Thyromimetic KB2115 (Eprotirome) Induces Rat Hepatocyte Proliferation.

Although the hepatomitogenic activity of T3 is well established, the wide range of harmful effects exerted by this hormone precludes its use in regenerative therapy. The aim of this study was to investigate whether an agonist of TRß, KB2115 (Eprotirome), could exert a mitogenic effect in the liver, without most of the adverse T3/TRα-dependent side effects. F-344 rats treated with KB2115 for 1 week displayed a massive increase in bromodeoxyuridine incorporation (from 20% to 40% vs. 5% of controls), which was associated with increased mitotic activity in the absence of significant signs of liver toxicity. Noteworthy, while cardiac hypertrophy typical of T3 was not observed, beneficial effects, such as lowering blood cholesterol levels, were associated to KB2115 administration. Following a single dose of KB2115, hepatocyte proliferation was evident as early as 18 h, demonstrating its direct mitogenic effect. No increase in serum transaminase levels or apoptosis was observed prior to or concomitantly with the S phase. While KB2115-induced mitogenesis was not associated to enhance expression of c-fos, c-jun, and c-myc, cyclin D1 levels rapidly increased. In conclusion, KB2115 induces hepatocyte proliferation without overt toxicity. Hence, this agent may be useful for regenerative therapies in liver transplantation or other surgical settings.

Tri-iodothyronine induces hepatocyte proliferation by protein kinase A-dependent ß-catenin activation in rodents.

UNLABELLED: Thyroid hormone (T3), like many other ligands of the steroid/thyroid hormone nuclear receptor superfamily, is a strong inducer of liver cell proliferation in rats and mice. However, the molecular basis of its mitogenic activity, which is currently unknown, must be elucidated if its use in hepatic regenerative medicine is to be considered. F-344 rats or C57BL/6 mice were fed a diet containing T3 for 2-7 days. In rats, administration of T3 led to an increased cytoplasmic stabilization and nuclear translocation of ß-catenin in pericentral hepatocytes with a concomitant increase in cyclin-D1 expression. T3 administration to wild-type (WT) mice resulted in increased hepatocyte proliferation; however, no mitogenic response in hepatocytes to T3 was evident in the hepatocyte-specific ß-catenin knockout mice (KO). In fact, T3 induced ß-catenin-TCF4 reporter activity both in vitro and in vivo. Livers from T3-treated mice demonstrated no changes in Ctnnb1 expression, activity of glycogen synthase kinase-3ß, known to phosphorylate and eventually promote ß-catenin degradation, or E-cadherin-ß-catenin association. However, T3 treatment increased ß-catenin phosphorylation at Ser675, an event downstream of protein kinase A (PKA). Administration of PKA inhibitor during T3 treatment of mice and rats as well as in cell culture abrogated Ser675-ß-catenin and simultaneously decreased cyclin-D1 expression to block hepatocyte proliferation. CONCLUSION: We have identified T3-induced hepatocyte mitogenic response to be mediated by PKA-dependent ß-catenin activation. Thus, T3 may be of therapeutic relevance to stimulate ß-catenin signaling to in turn induce regeneration in selected cases of hepatic insufficiency.

Timed regulation of P-element-induced wimpy testis-interacting RNA expression during rat liver regeneration.

UNLABELLED: Small noncoding RNAs comprise a growing family of molecules that regulate key cellular processes, including messenger RNA (mRNA) degradation, translational repression, and transcriptional gene silencing. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) represent a class of small RNAs initially identified in the germline of a variety of species, where they contribute to maintenance of genome stability, and recently found expressed also in stem and somatic cells, where their role and responsiveness to physiopathological signals remain elusive. Here, we investigated piRNA expression in rat liver and its response to the stimuli exerted by regenerative proliferation of this organ. Quantitative polymerase chain reaction analysis identify in the liver the RNAs encoding PIWIL2/HILI, PIWIL4/HIWI2, and other components of the piRNA biogenesis pathways, suggesting that this is indeed functional. RNA sequencing before, during, and after the wave of cell proliferation that follows partial hepatectomy (PH) identified ∼1,400 mammalian germline piRNAs expressed in rat liver, including 72 showing timed changes in expression 24-48 hours post-PH, a timing that corresponds to cell transition through the S phase, returning to basal levels by 168 hours, when organ regeneration is completed and hepatocytes reach quiescence. CONCLUSION: The piRNA pathway is active in somatic cells of the liver and is subject to regulation during the pathophysiological process of organ regeneration, when these molecules are available to exert their regulatory functions on the cell genome and transcriptome, as demonstrated by the identification of several liver mRNAs representing candidate targets of these regulatory RNAs.

Yes-associated protein regulation of adaptive liver enlargement and hepatocellular carcinoma development in mice.

UNLABELLED: 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. CONCLUSION: These results suggest that the Hippo pathway regulates adaptive liver enlargement and is probably inactivated in initiated cells that escape the suppressive constrain exerted on the surrounding normal tissue, thus allowing clonal expansion to HCC.

Proteomic characterization of early changes induced by triiodothyronine in rat liver.

High doses of T3 are mitogenic in liver, causing hyperplasia that has numerous differences from the compensatory regeneration induced by partial hepatectomy (PH). T3 binds to the thyroid hormone receptor (TR), which directly regulates transcription, while PH acts indirectly through signal transduction pathways. We therefore carried out a proteomic analysis to compare early effects of the two treatments. Transcriptome analysis by DNA microarray also confirmed the observed proteomic changes, demonstrating that they were caused by transcriptional regulation. Among the differentially expressed proteins, many are directly or indirectly involved in energy metabolism and response to oxidative stress. Several enzymes of lipid metabolism (e.g., Acaa2, Acads, Hadh, and Echs1) were differentially regulated by T3. In addition, altered expression levels of several mitochondrial proteins (e.g., Hspa9, Atp5b, Cps1, Glud1, Aldh2, Ak2, Acads) demonstrated the known increase of mitochondrial biogenesis mediated by T3. The present results provide insights in changes in metabolic balance occurring following T3-stimulation and define a basis for dissecting the molecular pathways of hepatocyte hyperplasia.

Expression of c-jun is not mandatory for mouse hepatocyte proliferation induced by two nuclear receptor ligands: TCPOBOP and T3.

BACKGROUND & AIMS: Mice lacking c-jun in the liver display impaired regeneration after partial hepatectomy (PH), and were reported to be more resistant to chemically-induced hepatocellular carcinoma (HCC). We investigated the role of c-jun in normal and preneoplastic hepatocyte proliferation induced by ligands of nuclear receptors, which cause liver hyperplasia in the absence of cell loss/death. METHODS: The effect of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) on hepatocyte proliferation was determined in c-jun conditional knockout (c-jun(Δli)) or in mouse liver where c-jun has been silenced. To study the role of c-jun in HCC development, c-jun(Δli) and WT mice were given diethylnitrosamine (DENA) followed by repeated injections of TCPOBOP. RESULTS: Hepatocyte proliferation induced by TCPOBOP was associated with a stronger proliferative response and earlier S phase entry in c-jun(Δli) mice, compared to WT animals. Moreover, silencing of c-jun in the liver of CD-1 mice caused increased hepatocyte proliferation. A stronger hepatocyte proliferative response of c-jun(Δli) mice was observed also following treatment with a ligand of thyroid hormone receptor. Finally, loss of c-jun did not inhibit the development of HCC induced by DENA and promoted by TCPOBOP. CONCLUSIONS: (i) c-jun may, under certain conditions, negatively regulate proliferation of normal hepatocytes, (ii) c-jun is not an absolute requirement for DENA/TCPOBOP-induced HCC formation, suggesting that the therapeutic potential of c-jun/JNK inhibition in liver tumors might be impaired by an increased stimulation of cell growth due to blockade of the c-jun pathway.

Progenitor-derived hepatocellular carcinoma model in the rat.

UNLABELLED: Human hepatocellular carcinoma (HCC) is a heterogeneous disease of distinct clinical subgroups. A principal source of tumor heterogeneity may be cell type of origin, which in liver includes hepatocyte or adult stem/progenitor cells. To address this issue, we investigated the molecular mechanisms underlying the fate of the enzyme-altered preneoplastic lesions in the resistant hepatocyte (RH) model. Sixty samples classified as focal lesions, adenoma, and early and advanced HCCs were microdissected after morphological and immunohistochemical evaluation and subjected to global gene expression profiling. The analysis of progression of the persistent glutathione S-transferase (GSTP)(+) focal lesions to fully developed HCC showed that approximately 50% of persistent nodules and all HCCs expressed cytokeratin 19 (CK19), whereas 14% of remodeling nodules were CK19(+). Unsupervised hierarchical clustering of the expression profiles also grouped the samples according to CK19 expression. Furthermore, supervised analysis using the differentially expressed genes in each cluster combined with gene connectivity tools identified 1308 unique genes and a predominance of the AP-1/JUN network in the CK19(+) lesions. In contrast, the CK19-negative cluster exhibited only limited molecular changes (156 differentially expressed genes versus normal liver) consistent with remodeling toward differentiated phenotype. Finally, comparative functional genomics showed a stringent clustering of CK19(+) early lesions and advanced HCCs with human HCCs characterized by poor prognosis. Furthermore, the CK19-associated gene expression signature accurately predicted patient survival (P < 0.009) and tumor recurrence (P < 0.006). CONCLUSION: Our data establish CK19 as a prognostic marker of early neoplastic lesions and strongly suggest the progenitor derivation of HCC in the rat RH model. The capacity of CK19-associated gene signatures to stratify HCC patients according to clinical prognosis indicates the usefulness of the RH model for studies of stem/progenitor-derived HCC.

TRbeta is the critical thyroid hormone receptor isoform in T3-induced proliferation of hepatocytes and pancreatic acinar cells.

BACKGROUND & AIMS: Thyroid hormones elicit many cellular and metabolic effects in various organs. Most of these actions, including mitogenesis, are mediated by the thyroid hormone 3,5,3'-triiodo-l-thyronine (T3) nuclear receptors (TRs). They are transcription factors, expressed as different isoforms encoded by the TRalpha and TRbeta genes. Here, experiments were performed to determine whether (i) T3-induces hepatocyte proliferation in mouse liver and pancreas, and, (ii) which TR isoform, is responsible for its mitogenic effect. METHODS: Cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation after T3 or the TRbeta agonist GC-1 in liver and pancreas of CD-1, C57BL, or TRalpha(0/0) mice. Cell cycle-associated proteins were measured by Western blot. RESULTS: T3 added to the diet at a concentration of 4 mg/kg caused a striking increase in BrdU incorporation in mouse hepatocytes. Increased BrdU incorporation was associated with enhanced protein levels of cyclin D1 and PCNA and decreased levels of p27. Treatment with GC-1, a selective agonist of the TRbeta isoform, also induced a strong mitogenic response of mouse hepatocytes and pancreatic acinar cells which was similar to that elicited by T3. Finally, treatment with T3 of mice TRalpha(0/0) induced a proliferative response in the liver and pancreas, similar to that of their wild type counterpart. CONCLUSIONS: These results demonstrate that T3 is a powerful inducer of cell proliferation in mouse liver and suggest that the beta-isoform is responsible for the hepatomitogenic activity of T3. The same isoform seems to also mediate the proliferation of mouse pancreatic acinar cells.

Thyroid hormone receptor ligands induce regression of rat preneoplastic liver lesions causing their reversion to a differentiated phenotype.

UNLABELLED: 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-beta 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/cm(2)) 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/cm(2)). A reduction of GSTP-positive foci was also observed in rats fed a CD + GC-1 diet (28/cm(2) versus 75/cm(2) 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.

Thyroid hormone (T3) and TRbeta 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 beta isoform (TRbeta), 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 beta-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.