Nirmala Mavila

Fibroblast Growth Factor Receptor-Mediated Activation of AKT-β-Catenin-CBP Pathway Regulates Survival and Proliferation of Murine Hepatoblasts and Hepatic Tumor Initiating Stem Cells

Fibroblast Growth Factor (FGF)-10 promotes the proliferation and survival of murine hepatoblasts during early stages of hepatogenesis through a Wnt-β-catenin dependent pathway. To determine the mechanism by which this occurs, we expanded primary culture of hepatoblasts enriched for progenitor markers CD133 and CD49f from embryonic day (E) 12.5 fetal liver and an established tumor initiating stem cell line from Mat1a−/− livers in media conditioned with recombinant (r) FGF10 or rFGF7. FGF Receptor (R) activation resulted in the downstream activation of MAPK, PI3K-AKT, and β-catenin pathways, as well as cellular proliferation. Additionally, increased levels of nuclear β-catenin phosphorylated at Serine-552 in cultured primary hepatoblasts, Mat1a−/− cells, and also in ex vivo embryonic liver explants indicate AKT-dependent activation of β-catenin downstream of FGFR activation; conversely, the addition of AKT inhibitor Ly294002 completely abrogated β-catenin activation. FGFR activation-induced cell proliferation and survival were also inhibited by the compound ICG-001, a small molecule inhibitor of β-catenin-CREB Binding Protein (CBP) in hepatoblasts, further indicating a CBP-dependent regulatory mechanism of β-catenin activity. Conclusion: FGF signaling regulates the proliferation and survival of embryonic and transformed progenitor cells in part through AKT-mediated activation of β-catenin and downstream interaction with the transcriptional co-activator CBP.

Expansion of prominin‐1‐expressing cells in association with fibrosis of biliary atresia

Biliary atresia (BA), the most common cause of end‐stage liver disease and the leading indication for pediatric liver transplantation, is associated with intrahepatic ductular reactions within regions of rapidly expanding periportal biliary fibrosis. Whereas the extent of such biliary fibrosis is a negative predictor of long‐term transplant‐free survival, the cellular phenotypes involved in the fibrosis are not well established. Using a rhesus rotavirus‐induced mouse model of BA, we demonstrate significant expansion of a cell population expressing the putative stem/progenitor cell marker, PROMININ‐1 (PROM1), adjacent to ductular reactions within regions of periportal fibrosis. PROM1positive (pos) cells express Collagen‐1α1. Subsets of PROM1pos cells coexpress progenitor cell marker CD49f, epithelial marker E‐CADHERIN, biliary marker CYTOKERATIN‐19, and mesenchymal markers VIMENTIN and alpha‐SMOOTH MUSCLE ACTIN (αSMA). Expansion of the PROM1pos cell population is associated with activation of Fibroblast Growth Factor (FGF) and Transforming Growth Factor‐beta (TGFβ) signaling. In vitro cotreatment of PROM1‐expressing Mat1a−/− hepatic progenitor cells with recombinant human FGF10 and TGFβ1 promotes morphologic transformation toward a myofibroblastic cell phenotype with increased expression of myofibroblastic genes Collagen‐1α1, Fibronectin, and α‐Sma. Infants with BA demonstrate similar expansion of periportal PROM1pos cells with activated Mothers Against Decapentaplegic Homolog 3 (SMAD3) signaling in association with increased hepatic expression of FGF10, FGFR1, and FGFR2 as well as mesenchymal genes SLUG and SNAIL. Infants with perinatal subtype of BA have higher tissue levels of PROM1 expression than those with embryonic subtype. Conclusion: Expansion of collagen‐producing PROM1pos cells within regions of periportal fibrosis is associated with activated FGF and TGFβ pathways in both experimental and human BA. PROM1pos cells may therefore play an important role in the biliary fibrosis of BA. (Hepatology 2014;60:941–953)

Time-Dependent Activation of Phox2a by the Cyclic AMP Pathway Modulates Onset and Duration of p27Kip1 Transcription

ABSTRACT In noradrenergic progenitors, Phox2a mediates cell cycle exit and neuronal differentiation by inducing p27Kip1 transcription in response to activation of the cyclic AMP (cAMP) pathway. The mechanism of cAMP-mediated activation of Phox2a is unknown. We identified a cluster of phosphoserine-proline sites in Phox2a by mass spectrometry. Ser206 appeared to be the most prominent phosphorylation site. A phospho-Ser206 Phox2a antibody detected dephosphorylation of Phox2a that was dependent on activation of the cAMP pathway, which occurred prior to neuronal differentiation of noradrenergic CAD cells. Employing serine-to-alanine and serine-to-aspartic acid Phox2a substitution mutants expressed in inducible CAD cell lines, we demonstrated that the transcriptional activity of Phox2a is regulated by two sequential cAMP-dependent events: first, cAMP signaling promotes dephosphorylation of Phox2a in at least one site, Ser206, thereby allowing Phox2a to bind DNA and initiate p27Kip1 transcription; second, following dephosphorylation of the phosphoserine cluster (Ser202 and Ser208), Phox2a becomes phosphorylated by protein kinase A (PKA) on Ser153, which prevents association of Phox2a with DNA and terminates p27Kip1 transcription. This represents a novel mechanism by which the same stimulus, cAMP signaling, first activates Phox2a by dephosphorylation of Ser206 and then, after a built-in delay, inactivates Phox2a via PKA-dependent phosphorylation of Ser153, thereby modulating onset and duration of p27Kip1 transcription.

Fibroblast Growth Factor signaling regulates the expansion of A6-expressing hepatocytes in association with AKT-dependent β-catenin activation

BACKGROUND & AIMS Fibroblast Growth Factors (FGFs) promote the proliferation and survival of hepatic progenitor cells (HPCs) via AKT-dependent β-catenin activation. Moreover, the emergence of hepatocytes expressing the HPC marker A6 during 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver injury is mediated partly by FGF and β-catenin signaling. Herein, we investigate the role of FGF signaling and AKT-mediated β-catenin activation in acute DDC liver injury. METHODS Transgenic mice were fed DDC chow for 14days concurrent with either Fgf10 over-expression or inhibition of FGF signaling via expression of soluble dominant-negative FGF Receptor (R)-2IIIb. RESULTS After 14days of DDC treatment, there was an increase in periportal cells expressing FGFR1, FGFR2, and AKT-activated phospho-Serine 552 (pSer552) β-Catenin in association with up-regulation of genes encoding the FGFR2IIIb ligands, Fgf7, Fgf10, and Fgf22. In response to Fgf10 over-expression, there was an increase in the number of pSer552-β-Catenin((positive)+ive) periportal cells as well as cells co-positive for A6 and hepatocyte marker, Hepatocyte Nuclear Factor-4α (HNF4α). A similar expansion of A6(+ive) cells was observed after Fgf10 over-expression with regular chow and after partial hepatectomy during ethanol toxicity. Inhibition of FGF signaling increased the periportal A6(+ive)HNF4α(+ive) cell population while reducing centrolobular A6(+ive) HNF4α(+ive) cells. AKT inhibition with Wortmannin attenuated FGF10-mediated A6(+ive)HNF4α(+ive) cell expansion. In vitro analyses using FGF10 treated HepG2 cells demonstrated AKT-mediated β-Catenin activation but not enhanced cell migration. CONCLUSIONS During acute DDC treatment, FGF signaling promotes the expansion of A6-expressing liver cells partly via AKT-dependent activation of β-Catenin expansion of A6(+ive) periportal cells and possibly by reprogramming of centrolobular hepatocytes.

Prohibitin 1 suppresses liver cancer tumorigenesis in mice and human hepatocellular and cholangiocarcinoma cells

Prohibitin 1 (PHB1) is best known as a mitochondrial chaperone, and its role in cancer is conflicting. Mice lacking methionine adenosyltransferase α1 (MATα1) have lower PHB1 expression, and we reported that c‐MYC interacts directly with both proteins. Furthermore, c‐MYC and MATα1 exert opposing effects on liver cancer growth, prompting us to examine the interplay between PHB1, MATα1, and c‐MYC and PHB1s role in liver tumorigenesis. We found that PHB1 is highly expressed in normal hepatocytes and bile duct epithelial cells and down‐regulated in most human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). In HCC and CCA cells, PHB1 expression correlates inversely with growth. PHB1 and MAT1A positively regulate each others expression, whereas PHB1 negatively regulates the expression of c‐MYC, MAFG, and c‐MAF. Both PHB1 and MATα1 heterodimerize with MAX, bind to the E‐box element, and repress E‐box promoter activity. PHB1 promoter contains a repressive E‐box element and is occupied mainly by MAX, MNT, and MATα1 in nonmalignant cholangiocytes and noncancerous tissues that switched to c‐MYC, c‐MAF, and MAFG in cancer cells and human HCC/CCA. All 8‐month‐old liver‐specific Phb1 knockout mice developed HCC, and one developed CCA. Five‐month‐old Phb1 heterozygotes, but not Phb1 flox mice, developed aberrant bile duct proliferation; and one developed CCA 3.5 months after left and median bile duct ligation. Phb1 heterozygotes had a more profound fall in the expression of glutathione synthetic enzymes and higher hepatic oxidative stress following left and median bile duct ligation. Conclusion: We have identified that PHB1, down‐regulated in most human HCC and CCA, heterodimerizes with MAX to repress the E‐box and positively regulates MAT1A while suppressing c‐MYC, MAFG, and c‐MAF expression; in mice, reduced PHB1 expression predisposes to the development of cholestasis‐induced CCA. (Hepatology 2017;65:1249‐1266).

Hepatic Prominin-1 expression is associated with biliary fibrosis

Background. Intrahepatic biliary fibrosis, as seen with cholestatic liver injuries such as biliary atresia, is mechanistically distinct from fibrosis caused by hepatocyte toxicity. We previously demonstrated the expansion of cells expressing the stem/progenitor cell marker Prominin‐1, within regions of developing fibrosis in biliary atresia. Thus, we hypothesized that Prominin‐1 expression is biliary fibrosis‐specific. Methods. Gene expression of Prominin‐1 was analyzed in adult mice undergoing either cholestatic bile duct ligation or hepatotoxic carbon tetrachloride administration by quantitative polymerase chair reaction. Lineage tracing of Prominin‐1‐expressing cells and Collagen‐1&agr;‐expressing cells was performed after bile duct ligation in Prominin‐1cre‐ert2−lacz;Gfplsl and Collagen‐1&agr;Gfp transgenic mice, respectively. Results. Prominin‐1 expression increased significantly after bile duct ligation compared with sham (6.6 ± 0.9‐fold change at 2 weeks, P < .05) but not with carbon tetrachloride (−0.7 ± 0.5‐fold change, not significant). Upregulation of Prominin‐1 was observed histologically throughout the liver as early as 5 days after bile duct ligation in Prominin‐1cre‐ert2−lacz mice by LacZ staining in nonhepatocyte cells. Lineage tracing of Prominin‐1‐expressing cells labeled prior to bile duct ligation in Prominin‐1cre‐ert2−lacz;Gfplsl mice, demonstrated increasing colocalization of GREEN FLUORESCENT PROTEIN with biliary marker CYTOKERATIN‐19 within ductular reactions up to 5 weeks after bile duct ligation consistent with biliary transdifferentiation. In contrast, rare colocalization of GREEN FLUORESCENT PROTEIN with mesenchymal marker &agr;‐SMOOTH MUSCLE ACTIN in Prominin‐1cre‐ert2−lacz;Gfplsl mice and some colocalization of GREEN FLUORESCENT PROTEIN with PROMININ‐1 in Collagen‐1&agr;Gfp mice, indicate minimal contribution of Prominin‐1 progenitor cells to the pool of collagen‐producing myofibroblasts. Conclusion. During biliary fibrosis Prominin‐1‐expressing progenitor cells transdifferentiate into cells within ductular reactions. This transdifferentiation may promote fibrosis.

Functional Human and Murine Tissue-Engineered Liver Is Generated From Adult Stem/Progenitor Cells

Liver disease affects large numbers of patients, yet there are limited treatments available to replace absent or ineffective cellular function of this crucial organ. Donor scarcity and the necessity for immunosuppression limit one effective therapy, orthotopic liver transplantation. But in some conditions such as inborn errors of metabolism or transient states of liver insufficiency, patients may be salvaged by providing partial quantities of functional liver tissue. After transplanting multicellular liver organoid units composed of a heterogeneous cellular population that includes adult stem and progenitor cells, both mouse and human tissue‐engineered liver (TELi) form in vivo. TELi contains normal liver components such as hepatocytes with albumin expression, CK19‐expressing bile ducts and vascular structures with α‐smooth muscle actin expression, desmin‐expressing stellate cells, and CD31‐expressing endothelial cells. At 4 weeks, TELi contains proliferating albumin‐expressing cells and identification of β2‐microglobulin‐expressing cells demonstrates that the majority of human TELi is composed of transplanted human cells. Human albumin is detected in the host mouse serum, indicating in vivo secretory function. Liquid chromatography/mass spectrometric analysis of mouse serum after debrisoquine administration is followed by a significant increase in the level of the human metabolite, 4‐OH‐debrisoquine, which supports the metabolic and xenobiotic capability of human TELi in vivo. Implanted TELi grew in a mouse model of inducible liver failure. Stem Cells Translational Medicine 2017;6:238–248

Prohibitin 1 Regulates the H19-Igf2 Axis and Proliferation in Hepatocytes.

Prohibitin 1 (PHB1) is a mitochondrial chaperone that regulates cell growth. Phb1 knock-out mice exhibit liver injury and hepatocellular carcinoma (HCC). Phb1 knock-out livers show induction of tumor growth-associated genes, H19 and insulin-like growth factor 2 (Igf2). These genes are controlled by the imprinting control region (ICR) containing CCCTC-binding transcription factor (CTCF)-binding sites. Because Phb1 knock-out mice exhibited induction of H19 and Igf2, we hypothesized that PHB1-mediated regulation of the H19-Igf2 axis might control cell proliferation in normal hepatocytes. H19 and Igf2 were induced (8–20-fold) in 3-week-old Phb1 knock-out livers, in Phb1 siRNA-treated AML12 hepatocytes (2-fold), and HCC cell lines when compared with control. Phb1 knockdown lowered CTCF protein in AML12 by ∼30% when compared with control. CTCF overexpression lowered basal H19 and Igf2 expression by 30% and suppressed Phb1 knockdown-mediated induction of these genes. CTCF and PHB1 co-immunoprecipitated and co-localized on the ICR element, and Phb1 knockdown lowered CTCF ICR binding activity. The results suggest that PHB1 and CTCF cooperation may control the H19-Igf2 axis. Human HCC tissues with high levels of H19 and IGF2 exhibited a 40–50% reduction in PHB1 and CTCF expression and their ICR binding activity. Silencing Phb1 or overexpressing H19 in the mouse HCC cell line, SAMe-D, induced cell growth. Blocking H19 induction prevented Phb1 knockdown-mediated growth, whereas H19 overexpression had the reverse effect. Interestingly H19 silencing induced PHB1 expression. Taken together, our results demonstrate that the H19-Igf2 axis is negatively regulated by CTCF-PHB1 cooperation and that H19 is involved in modulating the growth-suppressive effect of PHB1 in the liver.

Toll-like receptor 3 mediates PROMININ-1 expressing cell expansion in biliary atresia via Transforming Growth Factor-Beta

BACKGROUND In biliary atresia (BA), epithelial-mesenchymal hepatic progenitor cells (HPC) expressing the stem/progenitor cell marker PROMININ-1 (PROM1) undergo expansion and subsequent transdifferentiation into collagen-producing myofibroblasts within regions of evolving biliary fibrosis under the regulation of Transforming Growth Factor-β (TGFβ) signaling. We hypothesized that pro-inflammatory Toll-like Receptor-3 (TLR3) signal activation promotes the differentiation of PROM1+ HPC via TGFβ pathway activation in vitro. METHODS PROM1+ Mat1a(-/-) HPC were treated with a double-stranded RNA analog, polyionosinic-polycytidylic acid (Poly I:C), ± small molecule inhibitors nafamostat, or SB431542. RESULTS Poly I:C induced myofibroblastic-like morphologic changes, degradation of IκB-α consistent with TLR3-NFκB activation, a 15-fold increase in the expression of Vimentin, a 9-fold increase in Collagen-1a, a 4.6-fold increase in Snail at 24h (p<0.05), and an 8.2-fold increase in Prom1 at 72h (p<0.0001) by qPCR. Immunofluorescence demonstrated nuclear phosphorylated SMAD3, TLR3, and COLLAGEN-1α staining following Poly I:C treatment. Degradation of IκBα was inhibited by nafamostat. Co-treatment with either nafamostat or SB431542 blocked the morphologic change and abrogated the increased expression of Cd133, Collagen, Vimentin, and Snail1. CONCLUSIONS TLR3 activation induces myofibroblastic differentiation of PROM1+ HPC in part via TGFβ pathway activation to promote BA-associated biliary fibrosis.

Inhibition of CREB binding protein-beta-catenin signaling down regulates CD133 expression and activates PP2A-PTEN signaling in tumor initiating liver cancer cells

BackgroundThe WNT-beta-catenin pathway is known to regulate cellular homeostasis during development and tissue regeneration. Activation of WNT signaling increases the stability of cytoplasmic beta-catenin and enhances its nuclear translocation. Nuclear beta-catenin function is regulated by transcriptional co-factors such as CREB binding protein (CBP) and p300. Hyper-activated WNT-beta-catenin signaling is associated with many cancers. However, its role in inducing stemness to liver cancer cells, its autoregulation and how it regulates tumor suppressor pathways are not well understood. Here we have investigated the role of CBP-beta-catenin signaling on the expression of CD133, a known stem cell antigen and PP2A-PTEN pathway in tumor initiating liver cancer cells.MethodsHuman hepatoblastoma cell line HepG2 and clonally expanded CD133 expressing tumor initiating liver cells (TICs) from premalignant murine liver were used in this study. CBP-beta-catenin inhibitor ICG001 was used to target CBP-beta catenin signaling in liver cancer cells in vitro. Western blotting and real time PCR (qPCR) were used to quantify protein expression/phosphorylation and mRNA levels, respectively. CBP and CD133 gene silencing was performed by siRNA transfection. Fluorescence Activated Cell Sorting (FACS) was performed to quantify CD133 positive cells. Protein Phosphatase (PP2A) activity was measured after PP2AC immunoprecipitation.ResultsCBP inhibitor ICG001 and CBP silencing significantly reduced CD133 expression and anchorage independent growth in HepG2 and murine TICs. CD133 silencing in TICs decreased cell proliferation and expression levels of cell cycle regulatory genes, CyclinD1 and CyclinA2. ICG001 treatment and CBP silencing reduced the levels of phosphoSer380/Tyr382/383PTEN, phosphoSer473-AKT, Phospho-Ser552beta-catenin in TICs. ICG001 mediated de-phosphorylation of PTEN in TICs was PP2A dependent and partly prevented by co-treatment with PP2A inhibitor okadaic acid.ConclusionsCBP-beta-catenin signaling promotes stemness via CD133 induction and cell proliferation in TICs. We found a novel functional link between CBP-beta-catenin and PP2A-PTEN-AKT pathway in liver TICs. Therefore, CBP-beta-catenin-PP2A-PTEN-AKT signaling axis could be a novel therapeutic target to prevent liver tumor initiation and cancer recurrence.