The primary mitogen (TCPOBOP)‐induced hepatocyte proliferation is resistant to transforming growth factor‐ β‐1 inhibition

Background: Transforming growth factor (TGF)‐β‐1 is a very efficient inhibitor of hepatocyte proliferation in various in vivo and in vitro experimental systems. However, there are no data on whether it can influence the mitogenic response induced by primary hepatocyte mitogens. Aims: In this study, we compared the proliferative response in the liver between wild‐type and transgenic mice, overexpressing active TGF‐β‐1 in their liver following the treatment by a primary hepatocyte mitogen TCPOBOP (1,4‐bis[2‐(3,5‐dichloropyridyloxy)]benzene). Methods: The proliferative response was characterized by the immunohistochemical examination of pulse and cumulative bromodeoxyuridine labelling and by quantitative real‐time polymerase chain reaction analysis of cell cycle‐related genes. Results: Neither of the applied techniques revealed significant differences between the two groups of mice; furthermore, we observed the upregulation of TGF‐β‐1 expression following the mitogenic treatment. Conclusions: TGF‐β‐1 does not inhibit the primary mitogen‐induced proliferative response of the hepatocytes. This observation may provide an explanation for the divergent consequences of hepatic proliferations induced by partial hepatectomy or primary mitogenic treatment.     

Period 2 Gene Deletion Abolishes β‐Endorphin Neuronal Response to Ethanol

Background: Ethanol exposure during early life has been shown to permanently alter the circadian expression of clock regulatory genes and the β‐endorphin precursor proopiomelanocortin (POMC) gene in the hypothalamus. Ethanol also alters the stress‐ and immune‐regulatory functions of β‐endorphin neurons in laboratory rodents. Our aim was to determine whether the circadian clock regulatory Per2 gene modulates the action of ethanol on β‐endorphin neurons in mice. Methods: Per2 mutant (mPer2^Brdml) and wild type (C57BL/6J) mice were used to determine the effect of Per2 mutation on ethanol‐regulated β‐endorphin neuronal activity during neonatal period using an in vitro mediobasal hypothalamic (MBH) cell culture model and an in vivo milk formula feeding animal model. The β‐endorphin neuronal activity following acute and chronic ethanol treatments was evaluated by measuring the peptide released from cultured cells or peptide levels in the MBH tissues, using enzyme‐linked immunosorbent assay (ELISA). Results: Per2 mutant mice showed a higher basal level of β‐endorphin release from cultured MBH cells and a moderate increase in the peptide content in the MBH in comparison with control mice. However, unlike wild type mice, Per2 mutant mice showed no stimulatory or inhibitory β‐endorphin‐secretory responses to acute and chronic ethanol challenges in vitro. Furthermore, Per2 mutant mice, but not wild type mice, failed to show the stimulatory and inhibitory responses of MBH β‐endorphin levels to acute and chronic ethanol challenges in vivo. Conclusions: These results suggest for the first time that the Per2 gene may be critically involved in regulating β‐endorphin neuronal function. Furthermore, the data revealed an involvement of the Per2 gene in regulating β‐endorphin neuronal responses to ethanol.     

Prognostic significance of β‐catenin and topoisomerase IIα in de novo acute myeloid leukemia

The Wnt/β‐catenin signaling is important for controlling self‐renewal of hematopoietic stem cells and its constitutive activation has recently been documented in a significant proportion of acute myeloid leukemia (AML) cases. Topoisomerase IIα (Topo IIα) is a marker of cell proliferation and a crucial target for anthracycline cytotoxicity, the mainstay of management employed in AML. We retrospectively investigated the prognostic roles of β‐catenin and topo IIα in a cohort of 59 patients with newly diagnosed AML by immunohistochemistry. Aberrant β‐catenin expression was demonstrated in 13 patients (22%), and it was more likely to occur in those with unfavorable karyotypes. Advanced age and poor performance status adversely influenced the achievement of complete remission, while neither aberrant β‐catenin expression nor enhanced topo IIα activity did. On multivariate survival analysis, four factors independently predicted a shortened overall survival: aberrant β‐catenin expression, high topo IIα activity, poor‐risk cytogenetics, and presence of at least one comorbidity factor. Our results suggest that both β‐catenin and topo IIα independently predicted an adverse prognosis and might serve as new markers for risk stratification in AML patients. Am. J. Hematol., 2009. © 2008 Wiley‐Liss, Inc.     

Gene profiling of maternal hepatic adaptations to pregnancy

Background: Maternal metabolic demands change dramatically during the course of gestation and must be co‐ordinated with the needs of the developing placenta and fetus. The liver is critically involved in metabolism and other important functions. However, maternal hepatic adjustments to pregnancy are poorly understood. Aim: The aim of the study was to evaluate the influences of pregnancy on the maternal liver growth and gene expression profile. Methods: Holtzman Sprague–Dawley rats were mated and sacrificed at various stages of gestation and post‐partum. The maternal livers were analysed in gravimetric response, DNA content by PicoGreen dsDNA quantitation reagent, hepatocyte ploidy by flow cytometry and hepatocyte proliferation by ki‐67 immunostaining. Gene expression profiling of non‐pregnant and gestation d18.5 maternal hepatic tissue was analysed using a DNA microarray approach and partially verified by northern blot or quantitative real‐time PCR analysis. Results: During pregnancy, the liver exhibited approximately an 80% increase in size, proportional to the increase in body weight of the pregnant animals. The pregnancy‐induced hepatomegaly was a physiological event of liver growth manifested by increases in maternal hepatic DNA content and hepatocyte proliferation. Pregnancy did not affect hepatocyte polyploidization. Pregnancy‐dependent changes in hepatic expression were noted for a number of genes, including those associated with cell proliferation, cytokine signalling, liver regeneration and metabolism. Conclusions: The metabolic demands of pregnancy cause marked adjustments in maternal liver physiology. Central to these adjustments are an expansion in hepatic capacity and changes in hepatic gene expression. Our findings provide insights into pregnancy‐dependent hepatic adaptations.     

Inhibition of nuclear factor κB and phosphatidylinositol 3‐kinase/Akt is essential for massive hepatocyte apoptosis induced by tumor necrosis factor α in mice

Background/aims: Tumor necrosis factor (TNF)‐α itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF‐α/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)‐κB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF‐α. Here, we focused on the roles of NF‐κB and phosphatidylinositol 3‐kinase (PI3K)‐regulated serine/threonine kinase Akt. Methods: TNF‐α was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form IκBα (Ad5IκB), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl‐2 family members and caspase activity were examined. Results: In the mice livers, treatment with Ad5IκB or the wortmannin suppressed the activation of NF‐κB or Akt, respectively. Suppression of either NF‐κB or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF‐α administration. However, in mice treated with both Ad5IκB and wortmannin, TNF‐α administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5IκB, wortmannin, and TNF‐α markedly increased the activation of caspase‐3 and ‐9, and activated caspase‐8 to a lesser degree, suggesting that TNF‐α‐induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF‐κB and PI3K/Akt pathways had no effect on expression of Bcl‐2 families. Conclusion: The inducible activation of NF‐κB and constitutive activation of Akt regulate hepatocyte survival against TNF‐α, which occurs independent of Bcl‐2 families.     

Frequent mutations of beta-catenin gene in sporadic secreting adrenocortical adenomas

Objective Molecular alterations remain largely unknown in most sporadic adrenocortical tumours and hyperplasias. In our previous work, we demonstrated the differential expression of several Wnt/β‐catenin signalling‐related genes implicated in ACTH‐independent macronodular adrenal hyperplasias (AIMAH). To better understand the role of Wnt/β‐catenin signalling in adrenocortical tumours, we performed mutational analysis of the β‐catenin gene. Methods We studied 53 human adrenocortical samples (33 adenomas, 4 carcinomas, 13 AIMAH, 3 ACTH‐dependent adrenal hyperplasias) and the human adrenocortical cancer cell line NCI‐H295R. All samples were screened for somatic mutations in exons 3 and 5 of the β‐catenin gene. Eleven and six samples were analysed for β‐catenin protein expression by Western blotting and immunohistochemistry, respectively. Results No mutations were detected in adrenocortical carcinomas, AIMAH and ACTH‐dependent hyperplasias. Genetic alterations were found in 5 (15%) out of 33 adenomas: three cortisol‐secreting adenomas, one aldosterone‐secreting adenoma and one nonfunctional adenoma. Two‐point mutations occurred at serine residues of codons 37 and 45 (S37C and S45F). The remaining three tumours contained deletions of 6, 55 and 271 bp. H295R cells carry an activating S45P mutation. Western blot analysis of samples with 55‐ and 271‐bp deletions showed an additional shorter and more intense band representing an accumulation of the mutated form of β‐catenin protein. In addition, cytoplasmic and/or nuclear accumulation of β‐catenin was observed in mutated adenomas by immunohistochemistry. Conclusions Activating mutations of exon 3 of the β‐catenin gene are frequent in adrenocortical adenomas, and further characterization of the Wnt/β‐catenin signalling pathway should lead to a better understanding of adrenal tumourigenesis.     

Endogenous α‐calcitonin gene‐related peptide mitigates liver fibrosis in chronic hepatitis induced by repeated administration of concanavalin A

Background: α‐Calcitonin gene‐related peptide (αCGRP) is a 37‐amino acid pleiotropic peptide that we previously showed to exert a hepatoprotective effect during concanavalin A (Con A)‐induced acute hepatitis. In the present study, we used αCGRP^?/? mice to further investigate the antifibrogenic and hepatoprotective effects of endogenous αCGRP in Con A‐induced chronic hepatitis. Methods: Chronic hepatitis was induced in αCGRP^?/? and wild‐type mice by repeated administration of Con A. Serum transaminases were measured to assess hepatic injury. The severity of fibrosis and the activation of hepatic stellate cells (HSCs) were analysed by Masson trichrome staining and immunohistochemical staining of α‐smooth muscle actin (α‐SMA) respectively. Altered expression of fibrosis‐ and inflammation‐related genes was evaluated using a quantitative real‐time polymerase chain reaction. Activation and proliferation of HSCs were analysed using both primary cultured HSCs from the mice and the LI90 HSC cell line. Results: αCGRP^?/? mice showed more severe liver fibrosis than wild‐type mice in a Con A‐induced chronic hepatitis model. In histological and gene expression analyses, αCGRP^?/? mice showed greater inflammatory and fibrotic changes, greater HSC activation and a higher incidence of apoptosis among nonparenchymal cells than wild‐type mice. Conclusions: Endogenous αCGRP mitigates liver fibrosis in chronic hepatitis induced by repeated administration of Con A. αCGRP could be a useful therapeutic target for the treatment of chronic hepatitis.     

β‐catenin regulates parathyroid hormone/parathyroid hormone–related protein receptor signals and chondrocyte hypertrophy through binding to the intracellular C‐terminal region of the receptor

Objective

To investigate the underlying mechanisms of action and functional relevance of β‐catenin in chondrocytes, by examining the role of β‐catenin as a novel protein that interacts with the intracellular C‐terminal portion of the parathyroid hormone (PTH)/PTH‐related protein (PTHrP) receptor type 1 (PTHR‐1).

Methods

The β‐catenin–PTHR‐1 binding region was determined with deletion and mutagenesis analyses of the PTHR1 C‐terminus, using a mammalian two‐hybrid assay. Physical interactions between these 2 molecules were examined with an in situ proximity ligation assay and immunostaining. To assess the effects of gain‐ and loss‐of‐function of β‐catenin, transfection experiments were performed to induce overexpression of the constitutively active form of β‐catenin (ca‐β‐catenin) and to block β‐catenin activity with small interfering RNA, in cells cotransfected with either wild‐type PTHR1 or mutant forms (lacking binding to β‐catenin). Activation of the G protein α subunits G_αs and G_αq in the cells was determined by measurement of the intracellular cAMP accumulation and intracellular Ca²⁺ concentration, while activation of canonical Wnt pathways was assessed using a TOPflash reporter assay.

Results

In differentiated chondrocytes, β‐catenin physically interacted and colocalized with the cell membrane–specific region of PTHR‐1 (584–589). Binding of β‐catenin to PTHR‐1 caused suppression of the G_αs/cAMP pathway and enhancement of the G_αq/Ca²⁺ pathway, without affecting the canonical Wnt pathway. Inhibition of Col10a1 messenger RNA (mRNA) expression by PTH was restored by overexpression of ca‐β‐catenin, even after blockade of the canonical Wnt pathway, and Col10a1 mRNA expression was further decreased by knockout of β‐catenin (via the Cre recombinase) in chondrocytes from β‐catenin–floxed mice. Mutagenesis analyses to block the binding of β‐catenin to PTHR1 caused an inhibition of chondrocyte hypertrophy markers.

Conclusion

β‐catenin binds to the PTHR‐1 C‐tail and switches the downstream signaling pathway from G_αs/cAMP to G_αq/Ca²⁺, which is a possible mechanism by which chondrocyte hypertrophy may be regulated through the PTH/PTHrP signal independent of the canonical Wnt pathway.

     

Tracing phenotypic reversibility of pancreatic β‐cells in vitro

Aims/Introduction: Studies have suggested that pancreatic β‐cells undergo dedifferentiation during proliferation in vitro. However, due to limitations of the methodologies used, the question remains whether such dedifferentiated cells can redifferentiate into β‐cells. Materials and Methods: We have established a method for cell tracing in combination with fluorescence‐activated cell sorter (FACS). Using this method, mouse pancreatic β‐cells labeled with green fluorescent protein (GFP) under the control of the insulin promoter are collected by FACS. These β‐cells can be traced and characterized throughout the culture process, even when insulin becomes undetectable, because the cells are also marked with monomeric red fluorescent protein (mRFP) driven by the CAG promoter. Results: When cultured with fetal mouse pancreatic cells, FACS sorted β‐cells lost GFP expression, but retained mRFP expression. The cells also lost expressions of genes characteristic of the β‐cell phenotype, such as Pdx1 and glucokinase, indicating dedifferentiation. More than 30% of such dedifferentiated pancreatic β‐cells were detected in S or G2/M phase. Furthermore, these dedifferentiated cells redifferentiated into insulin‐expressing cells on cultivation with a MEK1/2 inhibitor. Conclusions: Our data provide direct evidence that pre‐existing β‐cells can undergo dedifferentiation and redifferentiation in vitro, their phenotype is reversible and that dedifferentiation in β‐cells is associated with progression of the cell cycle. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00051.x, 2010)     

Acute liver injury upregulates microRNA‐491–5p in mice,and its overexpression sensitizes Hep G2 cells for tumour necrosis factor‐α‐induced apoptosis

Background: MicroRNAs (miRNAs) have emerged as novel genetic regulators of cell functions such as proliferation, apoptosis and cancer. Aims: The aim of this study was to evaluate the role of a specific miRNA in modulating hepatic cell functions. Methods: C57Bl/6 mice were administered anti‐fas receptor antibodies to induce liver cell apoptosis. miRNAs were purified from the liver tissue and evaluated using an miRNA microarray. The role of miRNA‐491_5p, which was overexpressed in the model, in modulating hepatic cell functions was evaluated. miRNA‐491_5p was overexpressed in Hep G2 cells, followed by the addition of tumour necrosis factor (TNF)‐α, and induction of apoptosis as well as genes involved in apoptosis pathways were evaluated. The effect of miRNA‐491_5p target genes on apoptosis was also analysed by inhibiting their expression by siRNA‐induced gene silencing. Results: Upregulation of miRNA‐491_5p was found in a high‐dose anti‐fas receptor antibody group. Overexpression of microRNA‐491_5p sensitized Hep G2 cells for TNF‐α‐induced apoptosis, and also caused an inhibition of α‐fetoprotein, (AFP), heat shock protein‐90 (hsp‐90) and nuclear factor‐κB (NF‐κB). Overexpression of miRNA‐491_5p or inhibition of AFP and hsp‐90 resulted in an increased apoptosis in TNF‐α‐treated Hep G2 cells. Conclusions: One of the miRNAs that is associated with the acute liver injury mouse model, miRNA‐491_5p, sensitizes Hep G2 cells for TNF‐α‐induced apoptosis, at least in part, by inhibiting AFP, hsp‐90 and NF‐κB.     

Combination treatment of db/db mice with exendin‐4 and gastrin preserves β‐cell mass by stimulating β‐cell growth and differentiation

Aim/Introduction: Preservation of β‐cell mass is crucial for maintaining long‐term glucose homeostasis. Therapies based on incretin and its mimetics are expected to achieve this goal through various biological functions, particularly the restoration of β‐cell mass. Here we tested the effects of gastrin and exendin‐4 in type 2 diabetic animals. Materials and Methods: The effects of exendin‐4 and gastrin on β‐cell function and mass were examined in 8‐week‐old db/db mice. INS‐1 beta cells and AR42J cells were used to determine the molecular mechanism underlying the effects of the two agents. Immunohistochemistry, western blotting and RT‐PCR assays were used to assess the biological effects of the two agents. Results: Two weeks of combination administration of exendin‐4 plus gastrin resulted in a significant improvement of glucose tolerance associated with a marked preservation of β‐cell mass in db/db mice. Immunohistochemical analysis showed that such treatment resulted in the appearance of numerous irregularly‐shaped small islets and single insulin‐positive cells. While gastrin had little biological effect on INS‐1 β‐cells consistent with low expression of its intrinsic receptor on these cells, it caused differentiation of AR42J cells into insulin‐producing cells. Co‐stimulation with exendin‐4 significantly enhanced gastrin‐induced endocrine differentiation of AR42J precursor cells. These findings were further supported by enhanced expression of key genes involved in β‐cell differentiation and maturation, such as neurogenin3 (Ngn3) and MafA. Conclusions: These results suggest that combination treatment of db/db mice with exendin‐4 and gastrin preserves β‐cell mass by stimulating β‐cell growth and differentiation. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.00044.x, 2010)     

Hepatic activation of IKK/NFκB signaling induces liver fibrosis via macrophage-mediated chronic inflammation

Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-κB (NF-κB) system is activated in response to several of these stresses, we hypothesized that NF-κB activation in hepatocytes may contribute to fibrosis development. To activate the NF-κB signaling pathway in a time- and cell-type-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-κB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-κB-induced liver fibrosis in a liver-injury-independent manner. Conclusion: Our results revealed that hepatic activation of IKK/NF-κB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-κB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases. (HEPATOLOGY 2012;56:1117-1128).