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Sample records for mediates hepatocyte growth

  1. Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress

    PubMed Central

    Domínguez-Pérez, Mayra; Nuño-Lámbarri, Natalia; Clavijo-Cornejo, Denise; Luna-López, Armando; Souza, Verónica; Bucio, Leticia; Miranda, Roxana U.; Muñoz, Linda; Gomez-Quiroz, Luis Enrique; Uribe-Carvajal, Salvador; Gutiérrez-Ruiz, María Concepción

    2016-01-01

    Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system. PMID:27143995

  2. PXR stimulates growth factor-mediated hepatocyte proliferation by cross-talk with the FOXO transcription factor.

    PubMed

    Shizu, Ryota; Abe, Taiki; Benoki, Satoshi; Takahashi, Miki; Kodama, Susumu; Miayata, Masaaki; Matsuzawa, Atsushi; Yoshinari, Kouichi

    2016-02-01

    Growth factor-mediated hepatocyte proliferation is crucial in liver regeneration and the recovery of liver function after injury. The nuclear receptor, pregnane X receptor (PXR), is a key transcription factor for the xenobiotic-induced expression of genes associated with various liver functions. Recently, we reported that PXR activation stimulates xenobiotic-induced hepatocyte proliferation. In the present study, we investigated whether PXR activation also stimulates growth factor-mediated hepatocyte proliferation. In G0 phase-synchronized, immortalized mouse hepatocytes, serum or epidermal growth factor treatment increased cell growth and this growth was augmented by the expression of mouse PXR and co-treatment with pregnenolone 16α-carbonitrile (PCN), a PXR ligand. In a liver regeneration model using carbon tetrachloride, PCN treatment enhanced the injury-induced increase in the number of Ki-67-positive nuclei as well as Ccna2 and Ccnb1 mRNA levels in wild-type (WT) but not Pxr-null mice. Chronological analysis of this model demonstrated that PCN treatment shifted the maximum cell proliferation to an earlier time point and increased the number of M-phase cells at those time points. In WT but not Pxr-null mice, PCN treatment reduced hepatic mRNA levels of genes involved in the suppression of G0/G1- and G1/S-phase transition, e.g. Rbl2, Cdkn1a and Cdkn1b. Analysis of the Rbl2 promoter revealed that PXR activation inhibited its Forkhead box O3 (FOXO3)-mediated transcription. Finally, the PXR-mediated enhancement of hepatocyte proliferation was inhibited by the expression of dominant active FOXO3 in vitro. The results of the present study suggest that PXR activation stimulates growth factor-mediated hepatocyte proliferation in mice, at least in part, through inhibiting FOXO3 from accelerating cell-cycle progression. PMID:26574435

  3. Hepatocyte Growth Factor-mediated satellite cells niche perturbation promotes development of distinct sarcoma subtypes.

    PubMed

    Morena, Deborah; Maestro, Nicola; Bersani, Francesca; Forni, Paolo Emanuele; Lingua, Marcello Francesco; Foglizzo, Valentina; Šćepanović, Petar; Miretti, Silvia; Morotti, Alessandro; Shern, Jack F; Khan, Javed; Ala, Ugo; Provero, Paolo; Sala, Valentina; Crepaldi, Tiziana; Gasparini, Patrizia; Casanova, Michela; Ferrari, Andrea; Sozzi, Gabriella; Chiarle, Roberto; Ponzetto, Carola; Taulli, Riccardo

    2016-01-01

    Embryonal Rhabdomyosarcoma (ERMS) and Undifferentiated Pleomorphic Sarcoma (UPS) are distinct sarcoma subtypes. Here we investigate the relevance of the satellite cell (SC) niche in sarcoma development by using Hepatocyte Growth Factor (HGF) to perturb the niche microenvironment. In a Pax7 wild type background, HGF stimulation mainly causes ERMS that originate from satellite cells following a process of multistep progression. Conversely, in a Pax7 null genotype ERMS incidence drops, while UPS becomes the most frequent subtype. Murine EfRMS display genetic heterogeneity similar to their human counterpart. Altogether, our data demonstrate that selective perturbation of the SC niche results in distinct sarcoma subtypes in a Pax7 lineage-dependent manner, and define a critical role for the Met axis in sarcoma initiation. Finally, our results provide a rationale for the use of combination therapy, tailored on specific amplifications and activated signaling pathways, to minimize resistance emerging from sarcomas heterogeneity. PMID:26987019

  4. Hepatocyte Growth Factor-mediated satellite cells niche perturbation promotes development of distinct sarcoma subtypes

    PubMed Central

    Morena, Deborah; Maestro, Nicola; Bersani, Francesca; Forni, Paolo Emanuele; Lingua, Marcello Francesco; Foglizzo, Valentina; Šćepanović, Petar; Miretti, Silvia; Morotti, Alessandro; Shern, Jack F; Khan, Javed; Ala, Ugo; Provero, Paolo; Sala, Valentina; Crepaldi, Tiziana; Gasparini, Patrizia; Casanova, Michela; Ferrari, Andrea; Sozzi, Gabriella; Chiarle, Roberto; Ponzetto, Carola; Taulli, Riccardo

    2016-01-01

    Embryonal Rhabdomyosarcoma (ERMS) and Undifferentiated Pleomorphic Sarcoma (UPS) are distinct sarcoma subtypes. Here we investigate the relevance of the satellite cell (SC) niche in sarcoma development by using Hepatocyte Growth Factor (HGF) to perturb the niche microenvironment. In a Pax7 wild type background, HGF stimulation mainly causes ERMS that originate from satellite cells following a process of multistep progression. Conversely, in a Pax7 null genotype ERMS incidence drops, while UPS becomes the most frequent subtype. Murine EfRMS display genetic heterogeneity similar to their human counterpart. Altogether, our data demonstrate that selective perturbation of the SC niche results in distinct sarcoma subtypes in a Pax7 lineage-dependent manner, and define a critical role for the Met axis in sarcoma initiation. Finally, our results provide a rationale for the use of combination therapy, tailored on specific amplifications and activated signaling pathways, to minimize resistance emerging from sarcomas heterogeneity. DOI: http://dx.doi.org/10.7554/eLife.12116.001 PMID:26987019

  5. Hepatocyte growth factor as a downstream mediator of vascular endothelial growth factor-dependent preservation of growth in the developing lung.

    PubMed

    Seedorf, Gregory; Metoxen, Alexander J; Rock, Robert; Markham, Neil; Ryan, Sharon; Vu, Thiennu; Abman, Steven H

    2016-06-01

    Impaired vascular endothelial growth factor (VEGF) signaling contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). We hypothesized that the effects of VEGF on lung structure during development may be mediated through its downstream effects on both endothelial nitric oxide synthase (eNOS) and hepatocyte growth factor (HGF) activity, and that, in the absence of eNOS, trophic effects of VEGF would be mediated through HGF signaling. To test this hypothesis, we performed an integrative series of in vitro (fetal rat lung explants and isolated fetal alveolar and endothelial cells) and in vivo studies with normal rat pups and eNOS(-/-) mice. Compared with controls, fetal lung explants from eNOS(-/-) mice had decreased terminal lung bud formation, which was restored with recombinant human VEGF (rhVEGF) treatment. Neonatal eNOS(-/-) mice were more susceptible to hyperoxia-induced inhibition of lung growth than controls, which was prevented with rhVEGF treatment. Fetal alveolar type II (AT2) cell proliferation was increased with rhVEGF treatment only with mesenchymal cell (MC) coculture, and these effects were attenuated with anti-HGF antibody treatment. Unlike VEGF, HGF directly stimulated isolated AT2 cells even without MC coculture. HGF directly stimulates fetal pulmonary artery endothelial cell growth and tube formation, which is attenuated by treatment with JNJ-38877605, a c-Met inhibitor. rHGF treatment preserves alveolar and vascular growth after postnatal exposure to SU-5416, a VEGF receptor inhibitor. We conclude that the effects of VEGF on AT2 and endothelial cells during lung development are partly mediated through HGF-c-Met signaling and speculate that reciprocal VEGF-HGF signaling between epithelia and endothelia is disrupted in infants who develop BPD. PMID:27036872

  6. Effects of Adenovirus-Mediated Delivery of the Human Hepatocyte Growth Factor Gene in Experimental Radiation-Induced Heart Disease

    SciTech Connect

    Hu Shunying; Chen Yundai; Li Libing; Chen Jinlong; Wu Bin; Zhou, Xiao; Zhi Guang; Li Qingfang; Wang Rongliang; Duan Haifeng; Guo Zikuan; Yang Yuefeng; Xiao Fengjun; Wang Hua; Wang Lisheng

    2009-12-01

    Purpose: Irradiation to the heart may lead to late cardiovascular complications. The purpose of this study was to investigate whether adenovirus-mediated delivery of the human hepatocyte growth factor gene could reduce post-irradiation damage of the rat heart and improve heart function. Methods and Materials: Twenty rats received single-dose irradiation of 20 Gy gamma ray locally to the heart and were randomized into two groups. Two weeks after irradiation, these two groups of rats received Ad-HGF or mock adenovirus vector intramyocardial injection, respectively. Another 10 rats served as sham-irradiated controls. At post-irradiation Day 120, myocardial perfusion was tested by myocardial contrast echocardiography with contrast agent injected intravenously. At post-irradiation Day 180, cardiac function was assessed using the Langendorff technique with an isolated working heart model, after which heart samples were collected for histological evaluation. Results: Myocardial blood flow was significantly improved in HGF-treated animals as measured by myocardial contrast echocardiography at post-irradiation Day 120 . At post-irradiation Day 180, cardiac function was significantly improved in the HGF group compared with mock vector group, as measured by left ventricular peak systolic pressure (58.80 +- 9.01 vs. 41.94 +- 6.65 mm Hg, p < 0.05), the maximum dP/dt (5634 +- 1303 vs. 1667 +- 304 mm Hg/s, p < 0.01), and the minimum dP/dt (3477 +- 1084 vs. 1566 +- 499 mm Hg/s, p < 0.05). Picrosirius red staining analysis also revealed a significant reduction of fibrosis in the HGF group. Conclusion: Based on the study findings, hepatocyte growth factor gene transfer can attenuate radiation-induced cardiac injury and can preserve cardiac function.

  7. Requirement of kinesin-mediated membrane transport of WAVE2 along microtubules for lamellipodia formation promoted by hepatocyte growth factor.

    PubMed

    Takahashi, Kazuhide; Suzuki, Katsuo

    2008-07-01

    Lamellipodia formation necessary for epithelial cell migration and invasion is accomplished by rearrangement of the actin cytoskeleton at the leading edge through membrane transport of WAVE2. However, how WAVE2 is transported to the cell periphery where lamellipodia are formed remains to be established. We report here that hepatocyte growth factor (HGF) promoted lamellipodia formation and intracellular transport of WAVE2 to the cell periphery, depending on Rac1 activity, in MDA-MB-231 human breast cancer cells. Immunoblot analyses indicating the coimmunoprecipitation of WAVE2 with kinesin heavy chain KIF5B, one of the motor proteins, and IQGAP1 suggest that KIF5B and IQGAP1 formed a complex with WAVE2 in serum-starved cells and increased in their amount after HGF stimulation. Both downregulation of KIF5B by the small interfering RNA and depolymerization of microtubules with nocodazole abrogated the HGF-induced lamellipodia formation and WAVE2 transport. Therefore, we propose here that the promotion of lamellipodia formation by HGF in MDA-MB-231 cells is Rac1-dependent and requires KIF5B-mediated transport of WAVE2 and IQGAP1 to the cell periphery along microtubules. PMID:18514191

  8. Combined Paracrine and Endocrine AAV9 mediated Expression of Hepatocyte Growth Factor for the Treatment of Renal Fibrosis

    PubMed Central

    Schievenbusch, Stephanie; Strack, Ingo; Scheffler, Melanie; Nischt, Roswitha; Coutelle, Oliver; Hösel, Marianna; Hallek, Michael; Fries, Jochen WU; Dienes, Hans-Peter; Odenthal, Margarete; Büning, Hildegard

    2010-01-01

    In chronic renal disease, tubulointerstitial fibrosis is a leading cause of renal failure. Here, we made use of one of the most promising gene therapy vector platforms, the adeno-associated viral (AAV) vector system, and the COL4A3-deficient mice, a genetic mouse model of renal tubulointerstitial fibrosis, to develop a novel bidirectional treatment strategy to prevent renal fibrosis. By comparing different AAV serotypes in reporter studies, we identified AAV9 as the most suitable delivery vector to simultaneously target liver parenchyma for endocrine and renal tubular epithelium for paracrine therapeutic expression of the antifibrogenic cytokine human hepatocyte growth factor (hHGF). We used transcriptional targeting to drive hHGF expression from the newly developed CMV-enhancer-Ksp-cadherin-promoter (CMV-Ksp) in renal and hepatic tissue following tail vein injection of rAAV9-CMV-Ksp-hHGF into COL4A3-deficient mice. The therapeutic efficiency of our approach was demonstrated by a remarkable attenuation of tubulointerstitial fibrosis and repression of fibrotic markers such as collagen1α1 (Col1A1), platelet-derived growth factor receptor-β (PDGFR-β), and α-smooth muscle actin (SMA). Taken together, our results show the great potential of rAAV9 as an intravenously applicable vector for the combined paracrine and endocrine expression of antifibrogenic factors in the treatment of renal failure caused by tubulointerstitial fibrosis. PMID:20424598

  9. Hepatocyte Growth Factor Mediates the Antifibrogenic Action of Ocimum bacilicum Essential Oil against CCl4-Induced Liver Fibrosis in Rats.

    PubMed

    Ogaly, Hanan A; Eltablawy, Nadia A; El-Behairy, Adel M; El-Hindi, Hatim; Abd-Elsalam, Reham M

    2015-01-01

    The current investigation aimed to evaluate the antifibrogenic potential of Ocimum basilicum essential oil (OBE) and further to explore some of its underlying mechanisms. Three groups of rats were used: group I (control), group II (CCl4 model) and group III (OBE-treated) received CCl4 and OBE 2 weeks after the start of CCl4 administration. Oxidative damage was assessed by the measurement of MDA, NO, SOD, CAT, GSH and total antioxidant capacity (TAC). Liver fibrosis was assessed histopathologically by Masson's trichrome staining and α-smooth muscle actin (α-SMA) immunostaining. Expression of hepatocyte growth factor (HGF) and cytochrome P450 (CYP2EI isoform) was estimated using real-time PCR and immunohistochemistry. OBE successfully attenuated liver injury, as shown by histopathology, decreased serum transaminases and improved oxidative status of the liver. Reduced collagen deposition and α-SMA immuopositive cells indicated an abrogation of hepatic stellate cell activation by OBE. Furthermore, OBE was highly effective in stimulating HGF mRNA and protein expression and inhibiting CCl4-induced CYP2E1 down-regulation. The mechanism of antifibrogenic action of OBE is hypothesized to proceed via scavenging free radicals and activating liver regeneration by induction of HGF. These data suggest the use of OBE as a complementary treatment in liver fibrosis. PMID:26213907

  10. Mechanisms of hepatocyte growth factor-mediated signaling in differentiation of pancreatic ductal epithelial cells into insulin-producing cells

    SciTech Connect

    Li, Xin-Yu; Zhan, Xiao-Rong; Lu, Chong; Liu, Xiao-Min; Wang, Xiao-Chen

    2010-07-30

    Research highlights: {yields} A hypothesis that the differentiation of PDEC is through MAPKs or PI3K/AKT pathways. {yields} Determine if kinases (ERK1/2, p38, JNK, and AKT) are activated in these pathways. {yields} Determine signal pathway(s) that may effect on HGF-induced differentiation of PDEC. {yields} PI3K-AKT pathway is involved in the differentiation of PDECs induced by HGF. {yields} MEK-ERK pathway effect on the proliferation of PDECs but not the differentiation. -- Abstract: Pancreatic ductal epithelial cells (PDECs) were induced to differentiate into insulin-producing cells by hepatocyte growth factor (HGF) in our previous study, but the mechanism through which this induction occurs is still unknown. HGF is a ligand that activates a tyrosine kinase encoded by the c-Met proto-oncogene. This activation is followed by indirect activation of multiple downstream signal transduction pathways (including MAPKs and the PI3K/AKT signaling pathways) that initiate various biological effects. Therefore, we speculated that the differentiation of PDECs is through either the MAPK signaling pathway or the PI3K/AKT signaling pathway. To test this hypothesis, isolated PDECs from adult rats were stimulated by adding HGF to their medium for 28 days. Then, the expression levels of several protein kinases, including MAPKs (ERK1/2, p38, and JNK) and AKT, were determined by Western blotting to determine if specific protein kinases are activated in these pathways. Subsequently, re-isolated from adult rats and cultured PDECs were pre-treated with specific inhibitors of proteins shown to be activated in these signaling pathways; these cells were then induced to differentiate by the addition of HGF. The expression levels of protein kinases were determined by Western blotting, and the differentiation rate of insulin-positive cells was determined by flow cytometry. The change of PDEC differentiation rates were compared between the groups in which cells with or without inhibitors

  11. Role played by paxillin and paxillin tyrosine phosphorylation in hepatocyte growth factor/sphingosine-1-phosphate-mediated reactive oxygen species generation, lamellipodia formation, and endothelial barrier function

    PubMed Central

    Usatyuk, Peter V.; Jacobson, Jeffrey; Cress, Anne E.; Garcia, Joe G. N.; Salgia, Ravi; Natarajan, Viswanathan

    2015-01-01

    Abstract Paxillin is a multifunctional and multidomain focal adhesion adaptor protein. It serves as an important scaffolding protein at focal adhesions by recruiting and binding to structural and signaling molecules. Paxillin tyrosine phosphorylation at Y31 and Y118 is important for paxillin redistribution to focal adhesions and angiogenesis. Hepatocyte growth factor (HGF) and sphingosine-1-phosphate (S1P) are potent stimulators of lamellipodia formation, a prerequisite for endothelial cell migration. The role played by paxillin and its tyrosine phosphorylated forms in HGF- or S1P-induced lamellipodia formation and barrier function is unclear. HGF or S1P stimulated lamellipodia formation, tyrosine phosphorylation of paxillin at Y31 and Y118, and c-Abl in human lung microvascular endothelial cells (HLMVECs). Knockdown of paxillin with small interfering RNA (siRNA) or transfection with paxillin mutants (Y31F or Y118F) mitigated HGF- or S1P-induced lamellipodia formation, translocation of p47phox to lamellipodia, and reactive oxygen species (ROS) generation in HLMVECs. Furthermore, exposure of HLMVECs to HGF or S1P stimulated c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 in a time-dependent fashion, and down-regulation of c-Abl with siRNA attenuated HGF- or S1P-mediated lamellipodia formation, translocation of p47phox to lamellipodia, and endothelial barrier enhancement. In vivo, knockdown of paxillin with siRNA in mouse lungs attenuated ventilator-induced lung injury. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates HGF- or S1P-mediated lamellipodia formation, ROS generation in lamellipodia, and endothelial permeability. PMID:26697169

  12. Hypoxia-inducible Factor-dependent Production of Profibrotic Mediators by Hypoxic Hepatocytes

    PubMed Central

    Copple, Bryan L.; Bustamante, Juan J.; Welch, Timothy P.; Kim, Nam Deuk; Moon, Jeon-OK

    2011-01-01

    Background/Aims During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia-inducible factor-1α (HIF-1α), is critical for upregulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B, and plasminogen activator inhibitor-1 (PAI-1) in the liver during the development of fibrosis. What remains unknown is the cell type-specific regulation of these genes by HIF-1α in liver cell types. Accordingly, the hypothesis was tested that HIF-1α is activated in hypoxic hepatocytes and regulates production of profibrotic mediators by these cells. Methods In this study, hepatocytes were isolated from the livers of control and HIF-1α or HIF-1β-Deficient mice and exposed to hypoxia. Results Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF-1α and upregulated PAI-1, vascular endothelial cell growth factor, and the vasoactive peptides adrenomedullin-1 (ADM-1) and ADM-2. In contrast, levels of PDGF-A and PDGF-B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF-1α-Deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia-regulated transcription factors, such as HIF-2α, may also regulate these genes. In support of this, HIF-2α was activated in hypoxic hepatocytes, and exposure of HIF-1β-Deficient hepatocytes to 1% oxygen completely prevented upregulation PAI-1, VEGF, and ADM-1, suggesting that HIF-2α may also contribute to upregulation of these genes in hypoxic hepatocytes. Conclusions Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes. PMID:19302442

  13. PNPLA3 mediates hepatocyte triacylglycerol remodeling.

    PubMed

    Ruhanen, Hanna; Perttilä, Julia; Hölttä-Vuori, Maarit; Zhou, You; Yki-Järvinen, Hannele; Ikonen, Elina; Käkelä, Reijo; Olkkonen, Vesa M

    2014-04-01

    The I148M substitution in patatin-like phospholipase domain containing 3 (PNPLA3(I148M)) determines a genetic form of nonalcoholic fatty liver disease. To elucidate the mode of PNPLA3 action in human hepatocytes, we studied effects of WT PNPLA3 (PNPLA3(WT)) and PNPLA3(I148M) on HuH7 cell lipidome after [(13)C]glycerol labeling, cellular turnover of oleic acid labeled with 17 deuterium atoms ([D17]oleic acid) in triacylglycerols (TAGs), and subcellular distribution of the protein variants. PNPLA3(I148M) induced a net accumulation of unlabeled TAGs, but not newly synthesized total [(13)C]TAGs. Principal component analysis (PCA) revealed that both PNPLA3(WT) and PNPLA3(I148M) induced a relative enrichment of TAGs with saturated FAs or MUFAs, with concurrent enrichment of polyunsaturated phosphatidylcholines. PNPLA3(WT) associated in PCA with newly synthesized [(13)C]TAGs, particularly 52:1 and 50:1, while PNPLA3(I148M) associated with similar preexisting TAGs. PNPLA3(WT) overexpression resulted in increased [D17]oleic acid labeling of TAGs during 24 h, and after longer incubations their turnover was accelerated, effects not detected with PNPLA3(I148M). PNPLA3(I148M) localized more extensively to lipid droplets (LDs) than PNPLA3(WT), suggesting that the substitution alters distribution of PNPLA3 between LDs and endoplasmic reticulum/cytosol. This study reveals a function of PNPLA3 in FA-selective TAG remodeling, resulting in increased TAG saturation. A defect in TAG remodeling activity likely contributes to the TAG accumulation observed in cells expressing PNPLA3(I148M). PMID:24511104

  14. Retinoic Acid-mediated Nuclear Receptor Activation and Hepatocyte Proliferation

    PubMed Central

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

    Due to their well-known differentiation and apoptosis-inducing abilities, retinoic acid (RA) and its analogs have strong anti-cancer efficacy in human cancers. However, in vivo RA is a liver mitogen. While speculation has persisted that RA-mediated signaling is likely involved in hepatocyte proliferation during liver regeneration, direct evidence is still required. Findings in support of this proposition include observations that a release of retinyl palmitate (the precursor of RA) occurs in liver stellate cells following liver injury. Nevertheless, the biological action of this released vitamin A is virtually unknown. More likely is that the released vitamin A is converted to RA, the biological form, and then bound to a specific receptor (retinoid x receptor; RXRα), which is most abundantly expressed in the liver. Considering the mitogenic effects of RA, the RA-activated RXRα would likely then influence hepatocyte proliferation and liver tissue repair. At present, the mechanism by which RA stimulates hepatocyte proliferation is largely unknown. This review summarizes the activation of nuclear receptors (peroxisome proliferator activated receptor-α, pregnane x receptor, constitutive androstane receptor, and farnesoid x receptor) in an RXRα dependent manner to induce hepatocyte proliferation, providing a link between RA and its proliferative role.

  15. CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet {beta}-cells mediated by hepatocyte growth factor

    SciTech Connect

    Li, Xin-Yu; Zhan, Xiao-Rong; Liu, Xiao-Min; Wang, Xiao-Chen

    2011-01-14

    Research highlights: {yields} CREB is a regulatory target for the protein kinase Akt/PKB in pancreatic duct cells. {yields} Activation of the PI3K/AKT/CREB pathway plays a critical role in the HGF-mediated differentiation of pancreatic duct cells in vivo. {yields} CREB was causally linked to the expression of transcription factors during PDEC differentiation induced by HGF. -- Abstract: We have previously reported that the PI3K/Akt signaling pathway is involved in hepatocyte growth factor (HGF)-induced differentiation of adult rat pancreatic ductal epithelial cells (PDECs) into islet {beta}-cells in vitro. The transcription factor CREB is one of the downstream key effectors of the PI3K/Akt signaling pathway. Recent studies showing that CREB is required for the survival of certain cell types prompted us to examine whether CREB is a nuclear target for activation via the HGF-dependent Ser/Thr kinase Akt/PKB in the differentiation of pancreatic duct cell into islet {beta}-cells. In this study, we first attempted to examine whether HGF modulates the Akt-dependent activation of target gene CREB and then investigated whether CREB activity affects the differentiation of HGF-induced PDECs. Finally, we studied the role of CREB in modulating the expression of transcription factors in PDECs during the differentiation of HGF-induced PDECs. Our results demonstrated that CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet {beta}-cells mediated by HGF.

  16. Hepatocyte growth factor, hepatocyte growth factor activator and arginine in a rat fulminant colitis model

    PubMed Central

    Zwintscher, Nathan P.; Shah, Puja M.; Salgar, Shashikumar K.; Newton, Christopher R.; Maykel, Justin A.; Samy, Ahmed; Jabir, Murad; Steele, Scott R.

    2016-01-01

    Introduction Dextran sodium sulfate (DSS) is commonly used to induce a murine fulminant colitis model. Hepatocyte growth factor (HGF) has been shown to decrease the symptoms of inflammatory bowel disease (IBD) but the effect of its activator, HGFA, is not well characterized. Arginine reduces effects of oxidative stress but its effect on IBD is not well known. The primary aim is to determine whether HGF and HGFA, or arginine will decrease IBD symptoms such as pain and diarrhea in a DSS-induced fulminant colitis murine model. Methods A severe colitis was induced in young, male Fischer 344 rats with 4% (w/v) DSS oral solution for seven days; rats were sacrificed on day 10. Rats were divided into five groups of 8 animals: control, HGF (700 mcg/kg/dose), HGF and HGFA (10 mcg/dose), HGF and arginine, and high dose HGF (2800 mcg/kg/dose). Main clinical outcomes were pain, diarrhea and weight loss. Blinded pathologists scored the terminal ileum and distal colon. Results DSS reliably induced severe active colitis in 90% of animals (n = 36/40). There were no differences in injury scores between control and treatment animals. HGF led to 1.38 fewer days in pain (p = 0.036), while arginine led to 1.88 fewer days of diarrhea (P = 0.017) compared to controls. 88% of HGFA-treated rats started regaining weight (P < 0.001). Discussion/Conclusion Although treatment was unable to reverse fulminant disease, HGF and arginine were associated with decreased days of pain and diarrhea. These clinical interventions may reduce associated symptoms for severe IBD patients, even when urgent surgical intervention remains the only viable option. PMID:27144006

  17. [Hepatocyte growth factor therapy for amyotrophic lateral sclerosis].

    PubMed

    Aoki, Masashi

    2012-03-01

    Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by the death of upper and lower motor neurons. Approximately 20% of familial ALS cases are caused by mutations in the superoxide dismutase 1 (SOD1) gene. We generated rats that express a human SOD1 transgene with two different ALS-associated mutations and found that these rats develop remarkable motor neuron degeneration and paralysis. This rat model, because of the larger size of the animals as compared to ALS-affected mice, will facilitate studies involving manipulation of the cerebrospinal fluid (CSF) (e.g., implantation of intrathecal catheters for chronic therapeutic studies; CSF sampling) or spinal cord (e.g., direct administration of viral- and cell-mediated therapies). The hepatocyte growth factor (HGF) is one of the most potent survival-promoting factors for motor neurons. To examine its protective effect on motor neurons and its therapeutic potential, we administered human recombinant HGF (hrHGF) to the transgenic rats, by continuous intrathecal delivery, for 4 weeks from the onset of paralysis. Intrathecal administration of hrHGF attenuated motor neuron degeneration and prolonged the duration of the disease 62.7% compared with the contrast group. Our results indicated the therapeutic efficacy of continuous intrathecal administration of hrHGF in ALS rats. To explore the potential use of this treatment strategy in humans, we induced a contusive cervical spinal cord injury in the common marmoset, a primate, and then administered hrHGF intrathecally. The intrathecal administration of hrHGF promoted functional recovery. These projects have been supported by the "Super Special Consortium for Supporting the Development of Cutting-edge Medical Care" (tokku), a special program organized by the Cabinet Office of the Japanese government (research representative: Hideyuki Okano, M.D., Ph.D., Professor at Keio University). PMID:22402718

  18. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    SciTech Connect

    Sun, Zhichao; Yu, Xuemei; Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin; Li, Yintao; Yang, Lili; Ruan, Yuanyuan; Gu, Jianxin; Ren, Shifang; Zhang, Songwen

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

  19. Protective effects of melittin on transforming growth factor-{beta}1 injury to hepatocytes via anti-apoptotic mechanism

    SciTech Connect

    Lee, Woo-Ram; Park, Ji-Hyun; Kim, Kyung-Hyun; Park, Yoon-Yub; Han, Sang-Mi; Park, Kwan-kyu

    2011-10-15

    Melittin is a cationic, hemolytic peptide that is the main toxic component in the venom of the honey bee (Apis mellifera). Melittin has multiple effects, including anti-bacterial, anti-viral and anti-inflammatory, in various cell types. However, the anti-apoptotic mechanisms of melittin have not been fully elucidated in hepatocytes. Apoptosis contributes to liver inflammation and fibrosis. Knowledge of the apoptotic mechanisms is important to develop new and effective therapies for treatment of cirrhosis, portal hypertension, liver cancer, and other liver diseases. In the present study, we investigated the anti-apoptotic effect of melittin on transforming growth factor (TGF)-{beta}1-induced apoptosis in hepatocytes. TGF-{beta}1-treated hepatocytes were exposed to low doses (0.5 and 1 {mu}g/mL) and high dose (2 {mu}g/mL) of melittin. The low doses significantly protected these cells from DNA damage in TGF-{beta}1-induced apoptosis compared to the high dose. Also, melittin suppressed TGF-{beta}1-induced apoptotic activation of the Bcl-2 family and caspase family of proteins, which resulted in the inhibition of poly-ADP-ribose polymerase (PARP) cleavage. These results demonstrate that TGF-{beta}1 induces hepatocyte apoptosis and that an optimal dose of melittin exerts anti-apoptotic effects against TGF-{beta}1-induced injury to hepatocytes via the mitochondrial pathway. These results suggest that an optimal dose of melittin can serve to protect cells against TGF-{beta}1-mediated injury. - Highlights: > We investigated the anti-apoptotic effect of melittin on TGF-{beta}1-induced hepatocyte. > TGF-{beta}1 induces hepatocyte apoptosis. > TGF-{beta}1-treated hepatocytes were exposed to low doses and high dose of melittin. > Optimal dose of melittin exerts anti-apoptotic effects to hepatocytes.

  20. Hepatocyte-Ductal Transdifferentiation Is Mediated by Reciprocal Repression of SOX9 and C/EBPα

    PubMed Central

    O'Neill, Kathy E.; Thowfeequ, Shifaan; Li, Wan-Chun; Eberhard, Daniel; Dutton, James R.; Slack, Jonathan M.W.

    2014-01-01

    Abstract Primary hepatocytes rapidly dedifferentiate when cultured in vitro. We have studied the mechanism of hepatocyte dedifferentiation by using two culture media: one that maintains hepatocytes in a differentiated state and another that allows dedifferentiation. We show that dedifferentiation involves partial transformation of hepatocytes into cells that resemble biliary epithelial cells. Lineage labeling and time-lapse filming confirm that the dedifferentiated cells are derived from hepatocytes and not from contaminating ductal or fibroblastic cells in the original culture. Furthermore, we establish that the conversion of hepatocytes to biliary-like cells is regulated by mutual antagonism of CCAAT/enhancer binding protein alpha (C/EBPα) and SOX9, which have opposing effects on the expression of hepatocyte and ductal genes. Thus, hepatocyte dedifferentiation induces the biliary gene expression program by alleviating C/EBPα-mediated repression of Sox9. We propose that reciprocal antagonism of C/EBPα and SOX9 also operates in the formation of hepatocytes and biliary ducts from hepatoblasts during normal embryonic development. These data demonstrate that reprogramming of differentiated cells can be used to model the acquisition and maintenance of cell fate in vivo. PMID:25153359

  1. Antitumor effect of hepatocyte growth factor on hepatoblastoma.

    PubMed

    Tsunoda, Y; Shibusawa, M; Tsunoda, A; Gomi, A; Yatsuzuka, M; Okamatsu, T

    1998-01-01

    A six month-old girl presented with an abdominal mass, and high serum level of alpha-fetoprotein. She was diagnosed as having a well-differentiated hepatoblastoma by open biopsy. The biopsy specimen was transplanted on a nude mouse, and a xenograft was successfully established. Because the xenograft maintained the characteristics of the original tumor, the effect of hepatocyte growth factor (HGF) on hepatoblastoma xenograft was investigated. Recently HGF was reported to be involved in growth, invasion, and metastasis of tumor cells. Contrary to our expectations, the treatment of hepatoblastoma xenograft with recombinant 20 ng/ml HGF produced a marked inhibition of cell growth and a suppression of producing alpha-fetoprotein. PMID:9891489

  2. The emerging role of hepatocyte growth factor in renal diseases.

    PubMed

    Mao, Song; Zhang, Jianhua

    2016-06-01

    Hepatocyte growth factor (HGF), a kringle-containing polypeptide, acts on various epithelial cells to regulate cell growth, cell motility, and morphogenesis. HGF also accelerates tissue regeneration of injured organs and is regarded as a key molecule in organ regeneration. Besides the regeneration of the liver, HGF also plays a role in the renal regeneration. In addition, an adaptive alteration of HGF status in various renal diseases occurs. However, the precise role of HGF in various renal diseases remains elusive. The signaling pathways of HGF may be associated with renal diseases. In this review, we will try to provide an in-depth understanding of the underlying role of HGF and its possible interactions with other molecules in renal diseases. PMID:26460681

  3. Hepatocyte uptake and nuclear binding of epidermal growth factor (EGF)

    SciTech Connect

    Moriarity, D.M.; Underwood, T.

    1987-05-01

    The internalization of /sup 125/I-EGF and its cell-membrane receptor by target cells suggests a possible intracellular role for EGF and/or its receptor. They have examined the uptake of /sup 125/I-EGF by primary cultures of adult rat hepatocytes after 1, 24 and 48 hours of incubation in the presence of the growth factor. A significant increase in the association of radioactivity with various nuclear fractions was observed between 1 and 24 hours incubation. After 1 hour approximately 2% of the total specific binding was associated with both the nuclear sap proteins extractable with 0.14 M NaCl and with the residual nucleoplasm, while about 1% or less was associated with the nuclear membrane and the chromatin fractions. After 24 hours the percentage associated with the nuclear membrane and chromatin fractions increased 2-4 fold. Binding of /sup 125/I-EGF to isolated nuclei from intact livers of adult rats followed by fractionation of the nuclei after incubation with /sup 125/I-EGF indicated that after 60 min at 37/sup 0/C there was a substantial amount of specific binding associated with the nucleoplasm, nuclear membranes and chromatin fractions. These data indicate that specific interactions of EGF with nuclear components occur in both intact normal hepatocytes and in isolated nuclei from intact liver.

  4. Resistance to targeted cancer drugs through hepatocyte growth factor signaling

    PubMed Central

    Heynen, Guus JJE; Fonfara, Aldona; Bernards, René

    2014-01-01

    Cancer therapeutics that target a signaling pathway to which the cancer cells are addicted can deliver dramatic initial responses, but resistance is nearly always inevitable. A variety of mechanisms that cancer cells employ to escape from targeted cancer drugs have been described. We review here the role of Hepatocyte Growth Factor (HGF) and its receptor MET in drug resistance. We present data demonstrating that HGF can confer resistance to a number of kinase inhibitors in a variety of cancer cell lines and discuss our results in relation to the findings of others. Together, these data point at a major role for HGF/MET signaling in resistance to a variety of targeted cancer drugs. PMID:25426675

  5. Angiogenesis and antifibrotic action by hepatocyte growth factor in cardiomyopathy.

    PubMed

    Taniyama, Yoshiaki; Morishita, Ryuichi; Aoki, Motokuni; Hiraoka, Kazuya; Yamasaki, Keita; Hashiya, Naotaka; Matsumoto, Kunio; Nakamura, Toshikazu; Kaneda, Yasufumi; Ogihara, Toshio

    2002-07-01

    Impairment of cardiac function in cardiomyopathy has been postulated to be related to decreased blood blow and increased collagen synthesis. Therefore, a therapeutic approach to alter the blood flow or fibrosis directly by means of growth factors may open a new therapeutic concept in dilated cardiomyopathy. From this viewpoint, hepatocyte growth factor (HGF) is a unique growth factor with antifibrosis and angiogenesis effects. Using the hereditary cardiomyopathic Syrian hamster as a model of genetically determined cardiomyopathy and heart failure, the effects of overexpression of HGF on fibrosis and microvascular dysfunction were examined. HGF gene or control vector was injected by the Hemagglutinating Virus of Japan-liposome method into the anterior heart of cardiomyopathic hamsters (Bio 14.6) under echocardiography once a week, from 12 to 20 weeks of age (total, 8 times). Blood flow, as assessed by a laser Doppler imager score, and the capillary density in hearts, as assessed by alkaline phosphatase staining, were significantly increased in hamsters transfected with HGF gene compared with control-vector-transfected hamsters (P<0.01). In contrast, the fibrotic area was significantly decreased in hamsters transfected with HGF gene compared with control (P<0.01). Overall, in vivo experiments demonstrated that transfection of HGF gene into the myocardium of cardiomyopathic hamsters stimulated blood flow through the induction of angiogenesis and reduction of fibrosis. These results suggest that HGF gene transfer may be useful to protect against myocardial injury in cardiomyopathy through its cardioprotective effects such as antifibrosis and angiogenesis actions. PMID:12105137

  6. Structural basis for agonism and antagonism of hepatocyte growth factor

    SciTech Connect

    Tolbert, W. David; Daugherty-Holtrop, Jennifer; Gherardi, Ermanno; Vande Woude, George; Xu, H. Eric

    2010-11-01

    Hepatocyte growth factor (HGF) is an activating ligand of the Met receptor tyrosine kinase, whose activity is essential for normal tissue development and organ regeneration but abnormal activation of Met has been implicated in growth, invasion, and metastasis of many types of solid tumors. HGF has two natural splice variants, NK1 and NK2, which contain the N-terminal domain (N) and the first kringle (K1) or the first two kringle domains of HGF. NK1, which is a Met agonist, forms a head-to-tail dimer complex in crystal structures and mutations in the NK1 dimer interface convert NK1 to a Met antagonist. In contrast, NK2 is a Met antagonist, capable of inhibiting HGF's activity in cell proliferation without clear mechanism. Here we report the crystal structure of NK2, which forms a 'closed' monomeric conformation through interdomain interactions between the N- domain and the second kringle domain (K2). Mutations that were designed to open up the NK2 closed conformation by disrupting the N/K2 interface convert NK2 from a Met antagonist to an agonist. Remarkably, this mutated NK2 agonist can be converted back to an antagonist by a mutation that disrupts the NK1/NK1 dimer interface. These results reveal the molecular determinants that regulate the agonist/antagonist properties of HGF NK2 and provide critical insights into the dimerization mechanism that regulates the Met receptor activation by HGF.

  7. Effect of growth hormone on protein phosphorylation in isolated rat hepatocytes

    SciTech Connect

    Yamada, K.; Lipson, K.E.; Marino, M.W.; Donner, D.B.

    1987-02-10

    Hepatocytes from male rats were incubated with (/sup 32/P)P/sub i/ for 40 min at 37/sup 0/C, thereby equilibrating the cellular ATP pool with /sup 32/P. Subsequent exposure to bovine growth hormone for 10 additional min did not change the specific activity of cellular (..gamma..-/sup 32/P)ATP. Two-dimensional gel electrophoresis or chromatofocusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to fractionate phosphoproteins solubilized from control or hormone-stimulated cells. Stimulation of hepatocytes with 5 nM growth hormone for 10 min at 37/sup 0/C affected the phosphorylation of a number of proteins including an M/sub r/ 46,000 species of pI 4.7 whose phosphorylation was augmented (2.65 +/- 0.50)-fold. A significant fraction of the maximal effect of growth hormone on phosphorylation of the M/sub r/ 46,000 species was elicited by 1-5% receptor occupancy. Bovine growth hormone, which binds to somatogenic receptors with great specificity, or recombinant human growth hormone, which is not contaminated with other hormones, affected phosphorylation of hepatic proteins similarly. The M/sub r/ 46,000 phosphoprotein was isolated in a fraction enriched in cytosol after centrifugation of cellular homogenates. Phosphorylation of the M/sub r/ 46,000 phosphoprotein was also increased (1.75 +/- 0.35)-fold and (2.15 +/- 0.50)-fold by insulin and glucagon, respectively. These observations are consistent with the possibility that selective changes in the phosphorylation state of cellular proteins may mediate growth hormone actions in cells.

  8. Ichthyotoxic Cochlodinium polykrikoides Induces Mitochondrial Mediated Oxidative Stress and Apoptosis in Rat Liver Hepatocytes

    PubMed Central

    Shahraki, Jafar; Motallebi, Abbasali; Aghvami, Marjan; Pourahmad, Jalal

    2013-01-01

    In this research, we investigated the cytotoxic mechanisms of Cochlodinium polykrikoidescell lysate on isolated rat liver hepatocytes.This micro algae is responsible for a severe and widespread harmful algal bloom in the Persian Gulf and Gulf of Oman (2008-2009). Isolated hepatocytes were obtained by collagenase perfusion of Sprague-Dawley rat liver.According to our results, incubation of algal lysate with isolated rat hepatocytescaused hepatocyte membrane lysis, reactive oxygen species (ROS) formation, glutathione depletion, collapse of mitochondrial membrane potential,ATP depletion and increase in ADP/ATP ratio, cytochrome c release in to the hepatocyte cytosol,activation of caspase-3 (final mediator of apoptosis) and appearance of apoptosis phenotype. On the other hand, pre-treatment of antioxidants (α-tocopherol succinate and BHT), radical scavengers (mannitol and DMSO), mitochondrial permeability transition (MPT) pore sealing agents (cyclosporine A, carnitine and trifluoperazine), NADPH P450 reductase inhibitor (Diphenyliodonium chloride), CYP2E1 inhibitors (Phenylimidazole and 4-Methylpyrazole) and ATP generators (L-glutamine, Fructose and Xylitol)inhibitedcaspase-3 activation and cell death in algal lysate treated hepatocytes.Our data also confirmed that algal lysate activates apoptosis signaling via oxidative stress and mitochondrial pathway. Thus, ROS formation caused by the lysate exposure could directly be involved in mitochondrial MPT pore opening and activation of caspase-3 leading to C.polykrikoides lysateinduced apoptosis on rat hepatocytes. These findings contribute to a better understanding of C.polykrikoides-toxic effects on mammalian liver cells. PMID:24523763

  9. Piceatannol increases the expression of hepatocyte growth factor and IL-10 thereby protecting hepatocytes in thioacetamide-induced liver fibrosis.

    PubMed

    Abd-Elgawad, Hazem; Abu-Elsaad, Nashwa; El-Karef, Amr; Ibrahim, Tarek

    2016-07-01

    Piceatannol is a polyphenolic analog of resveratrol that selectively inhibits the non-receptor tyrosine kinase-Syk. This study investigates the potential ability of piceatannol to attenuate liver fibrosis and protect hepatocytes from injury. Thioacetamide was injected in adult male mice (100 mg/kg, i.p., 3 times/week) for 8 weeks. Piceatannol (1 or 5 mg/kg per day) was administered by oral gavage during the last 4 weeks. Liver function biomarkers, tissue malondialdehyde (MDA), cytokeratin-18 (CK18), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were measured. Necroinflammation, fibrosis, expression of transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA) were scored by histopathological examination and immunohistochemistry. Obtained results showed ability of piceatannol (1 mg/kg) to restore liver function and reduce inflammation. It significantly (p < 0.001) reduced MDA, CK18, TGF-β1, and α-SMA expression, and increased HGF and IL-10. It can be concluded that piceatannol at low dose can inhibit TGF-β1 induced hepatocytes apoptosis and exerts an anti-inflammatory effect attenuating fibrosis progression. PMID:27186801

  10. An engineered dimeric fragment of hepatocyte growth factor is a potent c-MET agonist

    PubMed Central

    Liu, Cassie J.; Jones, Douglas S.; Tsai, Ping-Chuan; Venkataramana, Abhishek; Cochran, Jennifer R.

    2015-01-01

    Hepatocyte growth factor (HGF), through activation of the c-MET receptor, mediates biological processes critical for tissue regeneration; however, its clinical application is limited by protein instability and poor recombinant expression. We previously engineered a HGF fragment (eNK1) that possesses increased stability and expression yield, and developed a c-MET agonist by coupling eNK1 through an introduced cysteine residue. Here, we further characterize this eNK1 dimer, and show it elicits significantly greater c-MET activation, cell migration, and proliferation than the eNK1 monomer. The efficacy of the eNK1 dimer was similar to HGF, suggesting its promise as a c-MET agonist. PMID:25451235

  11. Effects of hepatocyte growth factor on glutathione synthesis, growth, and apoptosis is cell density-dependent

    SciTech Connect

    Yang Heping; Magilnick, Nathaniel; Xia Meng; Lu, Shelly C.

    2008-01-15

    Hepatocyte growth factor (HGF) is a potent hepatocyte mitogen that exerts opposing effects depending on cell density. Glutathione (GSH) is the main non-protein thiol in mammalian cells that modulates growth and apoptosis. We previously showed that GSH level is inversely related to cell density of hepatocytes and is positively related to growth. Our current work examined whether HGF can modulate GSH synthesis in a cell density-dependent manner and how GSH in turn influence HGF's effects. We found HGF treatment of H4IIE cells increased cell GSH levels only under subconfluent density. The increase in cell GSH under low density was due to increased transcription of GSH synthetic enzymes. This correlated with increased protein levels and nuclear binding activities of c-Jun, c-Fos, p65, p50, Nrf1 and Nrf2 to the promoter region of these genes. HGF acts as a mitogen in H4IIE cells under low cell density and protects against tumor necrosis factor {alpha} (TNF{alpha})-induced apoptosis by limiting JNK activation. However, HGF is pro-apoptotic under high cell density and exacerbates TNF{alpha}-induced apoptosis by potentiating JNK activation. The increase in cell GSH under low cell density allows HGF to exert its full mitogenic effect but is not necessary for its anti-apoptotic effect.

  12. Renoprotective effects of hepatocyte growth factor in the stenotic kidney.

    PubMed

    Stewart, Nicholas; Chade, Alejandro R

    2013-03-15

    Renal microvascular (MV) damage and loss contribute to the progression of renal injury in renal artery stenosis (RAS). Hepatocyte growth factor (HGF) is a powerful angiogenic and antifibrotic cytokine that we showed to be decreased in the stenotic kidney. We hypothesized that renal HGF therapy will improve renal function mainly by protecting the renal microcirculation. Unilateral RAS was induced in 15 pigs. Six weeks later, single-kidney RBF and GFR were quantified in vivo using multidetector computed tomography (CT). Then, intrarenal rh-HGF or vehicle was randomly administered into the stenotic kidney (RAS, n = 8; RAS+HGF, n = 7). Pigs were observed for 4 additional weeks before CT studies were repeated. Renal MV density was quantified by 3D micro-CT ex vivo and histology, and expression of angiogenic and inflammatory factors, apoptosis, and fibrosis was determined. HGF therapy improved RBF and GFR compared with vehicle-treated pigs. This was accompanied by improved renal expression of angiogenic cytokines (VEGF, p-Akt) and tissue-healing promoters (SDF-1, CXCR4, MMP-9), reduced MV remodeling, apoptosis, and fibrosis, and attenuated renal inflammation. However, HGF therapy did not improve renal MV density, which was similarly reduced in RAS and RAS+HGF compared with controls. Using a clinically relevant animal model of RAS, we showed novel therapeutic effects of a targeted renal intervention. Our results show distinct actions on the existing renal microcirculation and promising renoprotective effects of HGF therapy in RAS. Furthermore, these effects imply plasticity of the stenotic kidney to recuperate its function and underscore the importance of MV integrity in the progression of renal injury in RAS. PMID:23269649

  13. Hepatocyte Growth Factor/cMET Pathway Activation Enhances Cancer Hallmarks in Adrenocortical Carcinoma.

    PubMed

    Phan, Liem M; Fuentes-Mattei, Enrique; Wu, Weixin; Velazquez-Torres, Guermarie; Sircar, Kanishka; Wood, Christopher G; Hai, Tao; Jimenez, Camilo; Cote, Gilbert J; Ozsari, Levent; Hofmann, Marie-Claude; Zheng, Siyuan; Verhaak, Roeland; Pagliaro, Lance; Cortez, Maria Angelica; Lee, Mong-Hong; Yeung, Sai-Ching J; Habra, Mouhammed Amir

    2015-10-01

    Adrenocortical carcinoma is a rare malignancy with poor prognosis and limited response to chemotherapy. Hepatocyte growth factor (HGF) and its receptor cMET augment cancer growth and resistance to chemotherapy, but their role in adrenocortical carcinoma has not been examined. In this study, we investigated the association between HGF/cMET expression and cancer hallmarks of adrenocortical carcinoma. Transcriptomic and immunohistochemical analyses indicated that increased HGF/cMET expression in human adrenocortical carcinoma samples was positively associated with cancer-related biologic processes, including proliferation and angiogenesis, and negatively correlated with apoptosis. Accordingly, treatment of adrenocortical carcinoma cells with exogenous HGF resulted in increased cell proliferation in vitro and in vivo while short hairpin RNA-mediated knockdown or pharmacologic inhibition of cMET suppressed cell proliferation and tumor growth. Moreover, exposure of cells to mitotane, cisplatin, or radiation rapidly induced pro-cMET expression and was associated with an enrichment of genes (e.g., CYP450 family) related to therapy resistance, further implicating cMET in the anticancer drug response. Together, these data suggest an important role for HGF/cMET signaling in adrenocortical carcinoma growth and resistance to commonly used treatments. Targeting cMET, alone or in combination with other drugs, could provide a breakthrough in the management of this aggressive cancer. PMID:26282167

  14. Growth inhibitory actions of prothrombin on normal hepatocytes: influence of matrix.

    PubMed

    Carr, Brian I; Kar, Siddhartha; Wang, Meifang; Wang, Ziqiu

    2007-09-01

    Most hepatomas have a defect in prothrombin carboxylation, and can secrete under-carboxylated prothrombin or des-gamma-carboxy-prothrombin (DCP), the function of which is unknown. We considered that the prothrombin-DCP axis might also be involved in growth control. Hepatocytes and hepatoma cells were treated with prothrombin and DNA synthesis and cytoskeletal changes were studied. Prothrombin inhibited DNA synthesis in hepatocytes on fibronectin, but not collagen matrix. Hepatoma cell lines were not inhibited. We found that hepatoma cell matrix conferred resistance to hepatocytes. Prothrombin decreased fibronectin but not collagen amounts, but only in the presence of hepatocytes and not hepatoma cells, indicating that it has a differential action on matrix proteins. It also caused changes in cell shape and actin depolymerization. In vivo, there was a decrease in plasma prothrombin activity after a partial hepatectomy (PH), concomitant with the peak of DNA synthesis in the hepatocytes at 24h after PH. Injection of warfarin at the time of PH, further inhibited PT activity and enhanced this 24h peak of DNA synthesis. Furthermore, repeated injection of prothrombin lowered the peak DNA synthesis after PH. The data support the hypothesis that prothrombin can act as a hepatocyte growth inhibitor, likely at the level of fibronectin loss and result in cytoskeletal changes. Hepatomas resist this action, possibly due to their different matrix proteins. This represents a novel mechanism for growth regulation and provides a possible biological significance for the tumor marker DCP. PMID:17490900

  15. MACROAUTOPHAGY AND CHAPERONE-MEDIATED AUTOPHAGY ARE REQUIRED FOR HEPATOCYTE RESISTANCE TO OXIDANT STRESS

    PubMed Central

    Wang, Yongjun; Singh, Rajat; Xiang, Youqing; Czaja, Mark J.

    2010-01-01

    The function of the lysosomal degradative pathway of autophagy in cellular injury is unclear as findings in nonhepatic cells have implicated autophagy as both a mediator of cell death and as a survival response. Autophagic function is impaired in steatotic and aged hepatocytes, suggesting that in these settings hepatocellular injury may be altered by the decrease in autophagy. To delineate the specific function of autophagy in the hepatocyte injury response, the effects of menadione-induced oxidative stress were examined in the RALA255-10G rat hepatocyte line when macroautophagy was inhibited by an shRNA-mediated knockdown of the autophagy gene atg5. Inhibition of macroautophagy sensitized cells to apoptotic and necrotic death from normally nontoxic concentrations of menadione. Inhibition of macroautophagy led to overactivation of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway that induced cell death. Death occurred from activation of the mitochondrial death pathway with cellular ATP depletion, mitochondrial cytochrome c release and caspase activation. Sensitization to death from menadione occurred despite up regulation of other forms of autophagy in compensation for the loss of macroautophagy. Chaperone-mediated autophagy (CMA) also mediated resistance to menadione as CMA inhibition sensitized cells to death from menadione through a mechanism different from that of a loss of macroautophagy as death occurred in the absence of JNK/c-Jun overactivation or ATP depletion. Conclusion Hepatocyte resistance to injury from menadione-induced oxidative stress is mediated by distinct functions of both macroautophagy and CMA, indicating that impaired function of either form of autophagy may promote oxidant-induced liver injury. PMID:20578144

  16. Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression

    PubMed Central

    Lamontagne, Jason; Mell, Joshua C.; Bouchard, Michael J.

    2016-01-01

    Globally, a chronic hepatitis B virus (HBV) infection remains the leading cause of primary liver cancer. The mechanisms leading to the development of HBV-associated liver cancer remain incompletely understood. In part, this is because studies have been limited by the lack of effective model systems that are both readily available and mimic the cellular environment of a normal hepatocyte. Additionally, many studies have focused on single, specific factors or pathways that may be affected by HBV, without addressing cell physiology as a whole. Here, we apply RNA-seq technology to investigate transcriptome-wide, HBV-mediated changes in gene expression to identify single factors and pathways as well as networks of genes and pathways that are affected in the context of HBV replication. Importantly, these studies were conducted in an ex vivo model of cultured primary hepatocytes, allowing for the transcriptomic characterization of this model system and an investigation of early HBV-mediated effects in a biologically relevant context. We analyzed differential gene expression within the context of time-mediated gene-expression changes and show that in the context of HBV replication a number of genes and cellular pathways are altered, including those associated with metabolism, cell cycle regulation, and lipid biosynthesis. Multiple analysis pipelines, as well as qRT-PCR and an independent, replicate RNA-seq analysis, were used to identify and confirm differentially expressed genes. HBV-mediated alterations to the transcriptome that we identified likely represent early changes to hepatocytes following an HBV infection, suggesting potential targets for early therapeutic intervention. Overall, these studies have produced a valuable resource that can be used to expand our understanding of the complex network of host-virus interactions and the impact of HBV-mediated changes to normal hepatocyte physiology on viral replication. PMID:26891448

  17. Reaction of plasma hepatocyte growth factor levels in non-small cell lung cancer patients treated with EGFR-TKIs.

    PubMed

    Tanaka, Hidenori; Kimura, Tatsuo; Kudoh, Shinzoh; Mitsuoka, Shigeki; Watanabe, Tetsuya; Suzumura, Tomohiro; Tachibana, Keisei; Noguchi, Masayuki; Yano, Seiji; Hirata, Kazuto

    2011-09-15

    Hepatocyte growth factor induces resistance to epidermal growth factor receptor tyrosine kinase inhibitors. It has been hypothesized that epidermal growth factor receptor tyrosine kinase inhibitors administration may influence the levels of plasma hepatocyte growth factor. Patients with advanced non-small cell lung cancer and relapsed after chemotherapies were eligible. Plasma hepatocyte growth factor levels were analyzed on pretreatment and post-treatment day 15 and 30. We also investigated the correlation between plasma hepatocyte growth factor levels and sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors, tissue immunoreactivity for hepatocyte growth factor and MET gene status. Thirty-one patients were enrolled. Plasma hepatocyte growth factor levels on post-treatment day 15 (630.1 ± 366.9 pg/ml) were significantly higher (p = 0.029) than the pretreatment plasma hepatocyte growth factor levels (485.9 ± 230.2 pg/ml). Plasma hepatocyte growth factor levels on the post-treatment day 30 (581.5 ± 298.1 pg/ml) tend to be higher than those before treatment (p = 0.057). Pretreatment plasma hepatocyte growth factor levels in patients with progressive disease (724.1 ± 216.4 pg/ml) were significantly higher than those in patients with stable disease (396.5 ± 148.3 pg/ml; p = 0.0008) and partial response (381.7 ± 179.0 pg/ml; p = 0.0039). The optimal pretreatment plasma hepatocyte growth factor cut-off value for diagnosis of responder was 553.5 pg/ml, and its sensitivity and specificity were 90% and 65%, respectively. Pretreatment plasma hepatocyte growth factor levels had no correlation with tissue immunoreactivities for hepatocyte growth factor, MET gene status and active EGFR mutations. Administration of epidermal growth factor receptor tyrosine kinase inhibitors significantly increased plasma hepatocyte growth factor levels. High levels of pretreatment plasma hepatocyte growth factor indicated intrinsic resistance to epidermal growth factor

  18. Human Hepatocyte Growth Factor Promotes Functional Recovery in Primates after Spinal Cord Injury

    PubMed Central

    Kitamura, Kazuya; Fujiyoshi, Kanehiro; Yamane, Jun-ichi; Toyota, Fumika; Hikishima, Keigo; Nomura, Tatsuji; Funakoshi, Hiroshi; Nakamura, Toshikazu; Aoki, Masashi; Toyama, Yoshiaki; Okano, Hideyuki; Nakamura, Masaya

    2011-01-01

    Many therapeutic interventions for spinal cord injury (SCI) using neurotrophic factors have focused on reducing the area damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF), which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF) intrathecally. Motor function was assessed using an original open field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery. In vivo magnetic resonance imaging showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-human primates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in non-human primates after contusive cervical SCI. PMID:22140459

  19. Profiling of anti-fibrotic signaling by hepatocyte growth factor in renal fibroblasts

    SciTech Connect

    Schievenbusch, Stephanie; Strack, Ingo; Scheffler, Melanie; Wennhold, Kerstin; Maurer, Julia; Nischt, Roswitha; Dienes, Hans Peter; Odenthal, Margarete

    2009-07-17

    Hepatocyte growth factor (HGF) is a multifunctional growth factor affecting cell proliferation and differentiation. Due to its mitogenic potential, HGF plays an important role in tubular repair and regeneration after acute renal injury. However, recent reports have shown that HGF also acts as an anti-inflammatory and anti-fibrotic factor, affecting various cell types such as renal fibroblasts and triggering tubulointerstitial fibrosis of the kidney. The present study provides evidence that HGF stimulation of renal fibroblasts results in the activation of both the Erk1/2 and the Akt pathways. As previously shown, Erk1/2 phosphorylation results in Smad-linker phosphorylation, thereby antagonizing cellular signals induced by TGF{beta}. By siRNA mediated silencing of the Erk1/2-Smad linkage, however, we now demonstrate that Akt signaling acts as an auxiliary pathway responsible for the anti-fibrotic effects of HGF. In order to define the anti-fibrotic function of HGF we performed comprehensive expression profiling of HGF-stimulated renal fibroblasts by microarray hybridization. Functional cluster analyses and quantitative PCR assays indicate that the HGF-stimulated pathways transfer the anti-fibrotic effects in renal interstitial fibroblasts by reducing expression of extracellular matrix proteins, various chemokines, and members of the CCN family.

  20. Emerging molecular targets in oncology: clinical potential of MET/hepatocyte growth-factor inhibitors

    PubMed Central

    Smyth, Elizabeth C; Sclafani, Francesco; Cunningham, David

    2014-01-01

    The MET/hepatocyte growth-factor (HGF) signaling pathway plays a key role in the processes of embryogenesis, wound healing, and organ regeneration. Aberrant activation of MET/HGF occurs through multiple mechanisms including gene amplification, mutation, protein overexpression, and abnormal gene splicing interrupting autocrine and paracrine regulatory feedback mechanisms. In many cancers including non-small-cell lung cancer, colorectal, gastric, renal, and hepatocellular cancer, dysregulation of MET may lead to a more aggressive cancer phenotype and may be a negative prognostic indicator. Successful therapeutic targeting of the MET/HGF pathway has been achieved using monoclonal antibodies against the MET receptor and its ligand HGF in addition to MET-specific and multitargeted small-molecule tyrosine-kinase inhibitors with several drugs in late-phase clinical trials including onartuzumab, rilotumumab, tivantinib, and cabozantinib. MET frequently interacts with other key oncogenic tyrosine kinases including epidermal growth-factor receptor (EGFR) and HER-3 and these interactions may be responsible for resistance to anti-EGFR therapies. Similarly, resistance to MET inhibition may be mediated through EGFR activation, or alternatively by increasing levels of MET amplification or acquisition of novel “gatekeeper” mutations. In order to optimize development of effective inhibitors of the MET/HGF pathway clinical trials must be enriched for patients with demonstrable MET-pathway dysregulation for which robustly standardized and validated assays are required. PMID:24959087

  1. Polyploidization delay in rat hepatocytes under liver growth inhibition by hypokinesia

    NASA Technical Reports Server (NTRS)

    Faktor, V. M.; Malyutin, V. F.; Li, S. Y.; Brodskiy, V. Y.

    1981-01-01

    A study of young rats, weighing 55 to 59 g, after being for 10 days in conditions of limited mobility, shows a retardation of body growth as well as that of liver growth. The decrease in the rate of growth is accompanied by a reduction of cell proliferation and by delay polyploidization of hepatocytes in the liver of experimental rats. The materials, methods, and results of research are discussed.

  2. Hepatocyte growth factor mimetic protects lateral line hair cells from aminoglycoside exposure

    PubMed Central

    Uribe, Phillip M.; Kawas, Leen H.; Harding, Joseph W.; Coffin, Allison B.

    2015-01-01

    Loss of sensory hair cells from exposure to certain licit drugs (e.g., aminoglycoside antibiotics, platinum-based chemotherapy agents) can result in permanent hearing loss. Here we ask if allosteric activation of the hepatocyte growth factor (HGF) cascade via Dihexa, a small molecule drug candidate, can protect hair cells from aminoglycoside toxicity. Unlike native HGF, Dihexa is chemically stable and blood-brain barrier permeable. As a synthetic HGF mimetic, it forms a functional ligand by dimerizing with endogenous HGF to activate the HGF receptor and downstream signaling cascades. To evaluate Dihexa as a potential hair cell protectant, we used the larval zebrafish lateral line, which possesses hair cells that are homologous to mammalian inner ear hair cells and show similar responses to toxins. A dose-response relationship for Dihexa protection was established using two ototoxins, neomycin and gentamicin. We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin. Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa’s protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry. Dihexa-mediated protection is attenuated by co-treatment with the HGF antagonist 6-AH, further evidence that HGF activation is a component of the observed protection. Additionally, Dihexa’s robust protection is partially attenuated by co-treatment with inhibitors of the downstream HGF targets Akt, TOR and MEK. Addition of an amino group to the N-terminal of Dihexa also attenuates the protective response, suggesting that even small substitutions greatly alter the specificity of Dihexa for its target. Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity. PMID:25674052

  3. PARTIAL PURIFICATION AND CHARACTERIZATION OF A HEPATOCYTE GROWTH FACTOR PRODUCED BY RAT HEPATOCELLULAR CARCINOMA CELLS

    EPA Science Inventory

    Serum-free medium conditioned by confluent cultures of JM1 or JM2 rat hepatocellular carcinoma cells stimulated DNA synthesis in primary cultures of adult rat hepatocytes in a dose-dependent, saturable manner and in the absence of epidermal growth factor. The hepatotrophic activi...

  4. PURIFICATION AND BIOLOGICAL CHARACTERIZATION OF HUMAN HEPATOPOETIN A: A POLYPEPTIDE GROWTH FACTOR FOR HEPATOCYTES

    EPA Science Inventory

    We have previously reported that the presence of a high molecular weight polypeptide growth factor in the plasma of normal human or rat serum which stimulates DNA synthesis in primary cultures of normal rat hepatocytes. e referred to this activity as Hepatopoietin A (HPTA) (6,7)....

  5. Knockdown of stromal interaction molecule 1 attenuates hepatocyte growth factor-induced endothelial progenitor cell proliferation.

    PubMed

    Shi, Yankun; Song, Mingbao; Guo, Ruiwei; Wang, Hong; Gao, Pan; Shi, Weibin; Huang, Lan

    2010-03-01

    Increased Ca(2+) entry through store-operated Ca(2+) channels (SOCCs) plays an essential role in the regulation of hepatocyte growth factor (HGF)-induced cell proliferation. Stromal interaction molecule 1 (STIM1) is thought to transmit endoplasmic reticulum (ER) Ca(2+) store depletion signals to the plasma membrane (PM), causing the opening of SOCCs in the PM. However, the relationship between HGF and STIM1 in endothelial progenitor cell (EPC) proliferation remains uncharacterized. The objective of this study was to evaluate the potential involvement of STIM1 in HGF-induced EPC proliferation. For this purpose, we used cultured rat bone marrow-derived EPCs and found that HGF-induced EPC proliferation at low concentrations. Store-operated Ca(2+) entry (SOCE) was elevated in HGF-treated EPCs, and the SOCC inhibitors 2-aminoethoxydiphenyl borate (2-APB) and BTP-2 inhibited the HGF-induced proliferation response. Moreover, STIM1 mRNA and protein expression levels were increased in response to HGF stimulation and knockdown of STMI1 decreased SOCE and prevented HGF-induced EPC proliferation. In conclusion, our data suggest that HGF-induced EPC proliferation is mediated partly via activation of STIM1. PMID:20404049

  6. Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract.

    PubMed Central

    Housley, R M; Morris, C F; Boyle, W; Ring, B; Biltz, R; Tarpley, J E; Aukerman, S L; Devine, P L; Whitehead, R H; Pierce, G F

    1994-01-01

    Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, was identified as a specific keratinocyte mitogen after isolation from a lung fibroblast line. Recently, recombinant (r)KGF was found to influence proliferation and differentiation patterns of multiple epithelial cell lineages within skin, lung, and the reproductive tract. In the present study, we designed experiments to identify additional target tissues, and focused on the rat gastrointestinal (GI) system, since a putative receptor, K-sam, was originally identified in a gastric carcinoma. Expression of KGF receptor and KGF mRNA was detected within the entire GI tract, suggesting the gut both synthesized and responded to KGF. Therefore, rKGF was administered to adult rats and was found to induce markedly increased proliferation of epithelial cells from the foregut to the colon, and of hepatocytes, one day after systemic treatment. Daily treatment resulted in the marked selective induction of mucin-producing cell lineages throughout the GI tract in a dose-dependent fashion. Other cell lineages were either unaffected (e.g., Paneth cells), or relatively decreased (e.g., parietal cells, enterocytes) in rKGF-treated rats. The direct effect of rKGF was confirmed by demonstrating markedly increased carcinoembryonic antigen production in a human colon carcinoma cell line, LIM1899. Serum levels of albumin were specifically and significantly elevated after daily treatment. These results demonstrate rKGF can induce epithelial cell activation throughout the GI tract and liver. Further, endogenous KGF may be a normal paracrine mediator of growth within the gut. Images PMID:7962522

  7. Hepatocyte cytoskeleton during ischemia and reperfusion - influence of ANP-mediated p38 MAPK activation

    PubMed Central

    Keller, Melanie; Gerbes, Alexander L; Kulhanek-Heinze, Stefanie; Gerwig, Tobias; Grützner, Uwe; van Rooijen, Nico; Vollmar, Angelika M; Kiemer, Alexandra K

    2005-01-01

    AIM: To determine functional consequences of this activation, whereby we focused on a potential regulation of the hepatocyte cytoskeleton during ischemia and reperfusion. METHODS: For in vivo experiments, animals received ANP (5 μg/kg) intravenously. In a different experimental setting, isolated rat livers were perfused with KH-buffer±ANP (200 nmol/L)±SB203580 (2 μmol/L). Livers were then kept under ischemic conditions for 24 h, and either transplanted or reperfused. Actin, Hsp27, and phosphorylated Hsp27 were determined by Western blotting, p38 MAPK activity by in vitro phosphorylation assay. F-actin distribution was determined by confocal microscopy. RESULTS: We first confirmed that ANP preconditioning leads to an activation of p38 MAPK and observed alterations of the cytoskeleton in hepatocytes of ANP-preconditioned organs. ANP induced an increase of hepatic F-actin after ischemia, which could be prevented by the p38 MAPK inhibitor SB203580 but had no effect on bile flow. After ischemia untreated livers showed a translocation of Hsp27 towards the cytoskeleton and an increase in total Hsp27, whereas ANP preconditioning prohibited translocation but caused an augmentation of Hsp27 phosphorylation. This effect is also mediated via p38 MAPK, since it was abrogated by the p38 MAPK inhibitor SB203580. CONCLUSION: This study reveals that ANP-mediated p38 MAPK activation leads to changes in hepatocyte cytoskeleton involving an elevation of phosphorylated Hsp27 and thereby for the first time shows functional consequences of ANP-induced hepatic p38 MAPK activation. PMID:16437711

  8. The aryl hydrocarbon receptor predisposes hepatocytes to Fas-mediated apoptosis.

    PubMed

    Park, Kyung-Tae; Mitchell, Kristen A; Huang, Gengming; Elferink, Cornelis J

    2005-03-01

    Liver homeostasis is achieved by the removal of diseased and damaged hepatocytes and their coordinated replacement to maintain a constant liver cell mass. Cirrhosis, viral hepatitis, and toxic drug effects can all trigger apoptosis in the liver as a means of removing the unwanted cells, and the Fas "death receptor" pathway comprises a major physiological mechanism by which this occurs. The susceptibility to Fas-mediated apoptosis is, in part, a function of the hepatocyte's proteome. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to influence apoptosis, conceivably by regulating the expression of genes involved in apoptotic signaling. In this article, we present evidence demonstrating that AhR expression and function promote apoptosis in liver cells in response to Fas stimulation. Reintroduction of the AhR into the AhR-negative BP8 hepatoma cells as well as into primary hepatocytes from AhR knockout mice increases the magnitude of cell death in response to Fas ligand. Enhanced apoptosis correlates with increased caspase activity and mitochondrial cytochrome c release but not with the expression of several Bcl-2 family proteins. In vivo studies showed that in contrast to wild-type mice, AhR knockout mice are protected from the lethal effects of the anti-Fas Jo2 antibody. Moreover, down-regulation of the aryl hydrocarbon receptor nuclear translocator protein in vivo by adenovirus-mediated RNA interference to suppress AhR activity provided wild-type mice partial protection from Jo2-induced lethality. PMID:15550680

  9. Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

    PubMed Central

    Volz, Yvonne; Koschut, David; Matzke-Ogi, Alexandra; Dietz, Marina S.; Karathanasis, Christos; Richert, Ludovic; Wagner, Moritz G.; Mély, Yves; Heilemann, Mike; Niemann, Hartmut H.; Orian-Rousseau, Véronique

    2015-01-01

    CD44v6, a member of the CD44 family of transmembrane glycoproteins is a co-receptor for two receptor tyrosine kinases (RTKs), Met and VEGFR-2 (vascular endothelial growth factor receptor 2). CD44v6 is not only required for the activation of these RTKs but also for signalling. In order to understand the role of CD44v6 in Met and VEGFR-2 activation and signalling we tested whether CD44v6 binds to their ligands, HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor), respectively. FACS analysis and cellular ELISA showed binding of HGF and VEGF only to cells expressing CD44v6. Direct binding of CD44v6 to HGF and VEGF was demonstrated in pull-down assays and the binding affinities were determined using MicroScale Thermophoresis, fluorescence correlation spectroscopy and fluorescence anisotropy. The binding affinity of CD44v6 to HGF is in the micromolar range in contrast with the high-affinity binding measured in the case of VEGF and CD44v6, which is in the nanomolar range. These data reveal a heparan sulfate-independent direct binding of CD44v6 to the ligands of Met and VEGFR-2 and suggest different roles of CD44v6 for these RTKs. PMID:26181364

  10. Two-signal requirement for growth-promoting function of Yap in hepatocytes

    PubMed Central

    Su, Tian; Bondar, Tanya; Zhou, Xu; Zhang, Cuiling; He, Hang; Medzhitov, Ruslan

    2015-01-01

    The transcriptional coactivator Yes-associated protein (Yap) promotes proliferation and inhibits apoptosis, suggesting that Yap functions as an oncogene. Most oncogenes, however, require a combination of at least two signals to promote proliferation. In this study, we present evidence that Yap activation is insufficient to promote growth in the otherwise normal tissue. Using a mosaic mouse model, we demonstrate that Yap overexpression in a fraction of hepatocytes does not lead to their clonal expansion, as proliferation is counterbalanced by increased apoptosis. To shift the activity of Yap towards growth, a second signal provided by tissue damage or inflammation is required. In response to liver injury, Yap drives clonal expansion, suppresses hepatocyte differentiation, and promotes a progenitor phenotype. These results suggest that Yap activation is insufficient to promote growth in the absence of a second signal thus coordinating tissue homeostasis and repair. DOI: http://dx.doi.org/10.7554/eLife.02948.001 PMID:25667983

  11. Establishment and Characterization of an In Vitro Model of Fas-Mediated Hepatocyte Cell Death.

    PubMed

    Vinken, Mathieu; Maes, Michaël; Crespo Yanguas, Sara; Willebrords, Joost; Vanhaecke, Tamara; Rogiers, Vera

    2015-01-01

    Fas-mediated apoptosis underlies a plethora of liver pathologies and toxicities. As a consequence, this process is a major research topic in the field of experimental and clinical hepatology. The present chapter describes the setup of an in vitro model of hepatocellular apoptotic cell death. In essence, this system consists of freshly isolated hepatocytes cultured in a monolayer configuration, which are exposed to a combination of Fas ligand and cycloheximide. This in vitro model has been characterized by using a set of well-acknowledged cell death markers. This experimental system allows the study of the entire course of Fas-mediated hepatocellular cell death, going from early apoptosis to secondary necrosis, and hence can serve a broad range of in vitro pharmaco-toxicological purposes. PMID:26272136

  12. Establishment and characterization of an in vitro model of Fas-mediated hepatocyte cell death

    PubMed Central

    Vinken, Mathieu; Maes, Michaël; Yanguas, Sara Crespo; Willebrords, Joost; Vanhaecke, Tamara; Rogiers, Vera

    2015-01-01

    Summary Fas-mediated apoptosis underlies a plethora of liver pathologies and toxicities. As a consequence, this process is a major research topic in the field of experimental and clinical hepatology. The present chapter describes the set-up of an in vitro model of hepatocellular apoptotic cell death. In essence, this system consists of freshly isolated hepatocytes cultured in a monolayer configuration, which are exposed to a combination of Fas ligand and cycloheximide. This in vitro model has been characterized by using a set of well-acknowledged cell death markers. This experimental system allows the study of the entire course of Fas-mediated hepatocellular cell death, going from early apoptosis to secondary necrosis, and hence can serve a broad range of in vitro pharmaco-toxicological purposes. PMID:26272136

  13. Hepatocyte exosomes mediate liver repair and regeneration via sphingosine-1-phosphate

    PubMed Central

    Nojima, Hiroyuki; Freeman, Christopher M.; Schuster, Rebecca M.; Japtok, Lukasz; Kleuser, Burkhard; Edwards, Michael J.; Gulbins, Erich; Lentsch, Alex B.

    2016-01-01

    Background & Aims Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. Methods Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. Results Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. Conclusions Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease. PMID:26254847

  14. Molecular therapy targeting Sonic hedgehog and hepatocyte growth factor signaling in a mouse model of medulloblastoma.

    PubMed

    Coon, Valerie; Laukert, Tamara; Pedone, Carolyn A; Laterra, John; Kim, K Jin; Fults, Daniel W

    2010-09-01

    The use of genetically engineered mice has provided insights into the molecular pathogenesis of the pediatric brain tumor medulloblastoma and revealed promising therapeutic targets. Ectopic expression of Sonic hedgehog (Shh) in cerebellar neural progenitor cells induces medulloblastomas in mice, and coexpression of hepatocyte growth factor (HGF) enhances Shh-induced tumor formation. To determine whether Shh + HGF-driven medulloblastomas were responsive to Shh signaling blockade and whether treatment response could be enhanced by combination therapy targeting both HGF and Shh signaling pathways, we carried out a survival study in mice. We induced medulloblastomas by retrovirus-mediated expression of Shh and HGF, after which we treated the mice systemically with (a) HGF-neutralizing monoclonal antibody L2G7, (b) Shh signaling inhibitor cyclopamine, (c) Shh-neutralizing monoclonal antibody 5E1, (d) L2G7 + cyclopamine, or (e) L2G7 + 5E1. We report that monotherapy targeting either HGF signaling or Shh signaling prolonged survival and that anti-HGF therapy had a more durable response than Shh-targeted therapy. The effect of L2G7 + 5E1 combination therapy on cumulative survival was equivalent to that of L2G7 monotherapy and that of L2G7 + cyclopamine therapy was worse. The principal mechanism by which Shh- and HGF-targeted therapies inhibited tumor growth was a potent apoptotic death response in tumor cells, supplemented by a weaker suppressive effect on proliferation. Our observation that combination therapy either failed to improve or even reduced survival in mice bearing Shh + HGF-induced medulloblastomas compared with monotherapy underscores the importance of preclinical testing of molecular-targeted therapies in animal models of tumors in which the targeted pathways are known to be active. PMID:20807782

  15. Molecular Therapy Targeting Sonic Hedgehog and Hepatocyte Growth Factor Signaling in a Mouse Model of Medulloblastoma

    PubMed Central

    Coon, Valerie; Laukert, Tamara; Pedone, Carolyn A.; Laterra, John; Kim, K. Jin; Fults, Daniel W.

    2010-01-01

    The use of genetically engineered mice has provided insights into the molecular pathogenesis of the pediatric brain tumor medulloblastoma and revealed promising therapeutic targets. Ectopic expression of Sonic Hedgehog (Shh) in cerebellar neural progenitor cells induces medulloblastomas in mice, and coexpression of hepatocyte growth factor (HGF) enhances Shh-induced tumor formation. To determine whether Shh+HGF–driven medulloblastomas were responsive to Shh signaling blockade and whether treatment response could be enhanced by combination therapy targeting both HGF and Shh signaling pathways, we carried out a survival study in mice. We induced medulloblastomas by retrovirus-mediated expression of Shh and HGF, after which we treated the mice systemically with (a) HGF-neutralizing monoclonal antibody L2G7, (b) Shh signaling inhibitor cyclopamine, (c) Shh-neutralizing monoclonal antibody 5E1, (d) L2G7+cyclopamine, or (e) L2G7+5E1. We report that monotherapy targeting either HGF signaling or Shh signaling prolonged survival and that anti-HGF therapy had a more durable response than Shh-targeted therapy. The effect of L2G7+5E1 combination therapy on cumulative survival was equivalent to that of L2G7 monotherapy and that of L2G7+cyclopamine therapy was worse. The principal mechanism by which Shh- and HGF-targeted therapies inhibited tumor growth was a potent apoptotic death response in tumor cells, supplemented by a weaker suppressive effect on proliferation. Our observation that combination therapy either failed to improve or even reduced survival in mice bearing Shh+HGF induced medulloblastomas compared with monotherapy underscores the importance of preclinical testing of molecular-targeted therapies in animal models of tumors in which the targeted pathways are known to be active. PMID:20807782

  16. NEFAs activate the oxidative stress-mediated NF-κB signaling pathway to induce inflammatory response in calf hepatocytes.

    PubMed

    Shi, Xiaoxia; Li, Dangdang; Deng, Qinghua; Li, Yu; Sun, Guoquan; Yuan, Xue; Song, Yuxiang; Wang, Zhe; Li, Xiaobing; Li, Xinwei; Liu, Guowen

    2015-01-01

    Non-esterified fatty acids (NEFAs) are important induction factors of inflammatory responses in some metabolic diseases. High plasma levels of NEFAs and oxidative stress exist in the dairy cows with ketosis. The aim of this study was to investigate whether high levels of NEFAs can induce inflammatory response and the specific molecular mechanism in the hepatocytes of dairy cow. In vitro, primary cultured bovine hepatocytes were treated with different concentrations of NEFAs, PDTC (an NF-κB inhibitor) and NAC (an antioxidant). NEFAs significantly activated NF-κB pathway. Activated NF-κB upregulated the release of pro-inflammatory cytokines, thereby inducing inflammatory response in bovine hepatocytes. When PDTC was added, activation of NF-κB-mediated inflammatory response induced by NEFAs was inhibited. NEFAs treatment results in the overproduction of the markers of oxidative stress, reactive oxygen species (ROS) and malondialdehyde (MDA), which were ameliorated by NAC treatment. These increased ROS and MDA were caused by decreasing activity of antioxidant system, including glutathione peroxidase, superoxide dismutase and catalase, in bovine hepatocytes treated with NEFAs. NAC also ameliorated NEFAs-mediated NF-κB activation and the release of pro-inflammatory cytokines. These results indicate that high concentrations of NEFAs can induce cattle hepatocytes inflammatory response through activating the oxidative stress-mediated NF-κB signaling pathway. PMID:25465477

  17. Hepatocyte growth factor-induced differentiation of bone mesenchymal stem cells toward hepatocyte-like cells occurs through nuclear factor-kappa B signaling in vitro.

    PubMed

    Yang, Tongxi; Wang, Yi; Jiang, Shasha; Liu, Xiaoping; Yu, Zhongjie

    2016-09-01

    Hepatocyte growth factor (HGF) is multifaceted cytokine that regulates proliferation, differentiation, morphology, and motility within numerous stem cells. More recently, HGF has been reported to induce the differentiation of bone mesenchymal stem cells (BMSCs) into mature hepatocytes, but the underlying biochemical and molecular signaling is largely unknown. We isolated BMSC from the bone marrow of rats, which were then cultured and exposed to HGF for 15 days. We subsequently assayed these cells for liver functionality and markers, and blocked NF-кB signaling at various stages of the pathway. The present results demonstrate that HGF induces the differentiation of BMSCs toward hepatocyte-like cells through the NF-кB signaling. More specifically, HGF upregulated the translocation of NF-кB to the nucleus. PMID:27249785

  18. NADPH Oxidase NOX4 Mediates Stellate Cell Activation and Hepatocyte Cell Death during Liver Fibrosis Development

    PubMed Central

    Sancho, Patricia; Mainez, Jèssica; Crosas-Molist, Eva; Roncero, César; Fernández-Rodriguez, Conrado M.; Pinedo, Fernando; Huber, Heidemarie; Eferl, Robert; Mikulits, Wolfgang; Fabregat, Isabel

    2012-01-01

    A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2−/−/p19ARF−/−, Stat3Δhc/Mdr2−/−) and a model of experimental induced fibrosis (CCl4) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes. PMID:23049784

  19. Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

    SciTech Connect

    Junker, L.H.; Davis, R.A. )

    1989-12-01

    The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of (14C)cholesterol from (2-14C)acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of (14C)cholesterol from (2-14C)acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis.

  20. Effects of chronic ethanol administration on receptor mediated endocytosis of asialoorosomucoid (ASOR) in isolated rat hepatocytes

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J.

    1986-05-01

    The authors have previously shown that acute and chronic ethanol administration decreases hepatic glycoprotein secretion and membrane biogenesis. The present study was undertaken to determine the effects of chronic ethanol feeding on receptor-mediated endocytosis using the endocytosis of ASOR as a model system. Rats were fed either rat chow ad lib or pair-fed with Lieber-DeCarli diet (ethanol or isocaloric glucose as 36% of total calories) for 5 to 7 weeks. Binding of /sup 125/I ASOR to isolated hepatocytes was studied at 0-4/sup 0/C. Internalization (cell-associated acid precipitable radioactivity) and degradation (acid soluble radioactivity) were determined at 37/sup 0/C for periods up to 240 min. Results were expressed as pmoles ASOR bound, degraded or internalized/10/sup 6/ cells. In ethanol-fed rats the number of pmoles ASOR bound/10/sup 6/ cells was decreased by 40-50% (p< 0.01) as compared to pair-fed and chow-fed animals. Rates of degradation and internalization of the ligand were also 50-70% lower (p< 0.01) in chronic ethanol-treated animals. No significant differences were observed for either binding or internalization of ASOR between chow-fed and pair-fed animals. These results indicate that chronic ethanol feeding decreases internalization and degradation of ASOR in rat hepatocytes.

  1. Ado-Trastuzumab Emtansine Targets Hepatocytes Via Human Epidermal Growth Factor Receptor 2 to Induce Hepatotoxicity.

    PubMed

    Yan, Haoheng; Endo, Yukinori; Shen, Yi; Rotstein, David; Dokmanovic, Milos; Mohan, Nishant; Mukhopadhyay, Partha; Gao, Bin; Pacher, Pal; Wu, Wen Jin

    2016-03-01

    Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2-positive metastatic breast cancer. It consists of trastuzumab, a humanized mAb directed against HER2, and a microtubule inhibitor, DM1, conjugated to trastuzumab via a thioether linker. Hepatotoxicity is one of the serious adverse events associated with T-DM1 therapy. Mechanisms underlying T-DM1-induced hepatotoxicity remain elusive. Here, we use hepatocytes and mouse models to investigate the mechanisms of T-DM1-induced hepatotoxicity. We show that T-DM1 is internalized upon binding to cell surface HER2 and is colocalized with LAMP1, resulting in DM1-associated cytotoxicity, including disorganized microtubules, nuclear fragmentation/multiple nuclei, and cell growth inhibition. We further demonstrate that T-DM1 treatment significantly increases the serum levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in mice and induces inflammation and necrosis in liver tissues, and that T-DM1-induced hepatotoxicity is dose dependent. Moreover, the gene expression of TNFα in liver tissues is significantly increased in mice treated with T-DM1 as compared with those treated with trastuzumab or vehicle. We propose that T-DM1-induced upregulation of TNFα enhances the liver injury that may be initially caused by DM1-mediated intracellular damage. Our proposal is underscored by the fact that T-DM1 induces the outer mitochondrial membrane rupture, a typical morphologic change in the mitochondrial-dependent apoptosis, and mitochondrial membrane potential dysfunction. Our work provides mechanistic insights into T-DM1-induced hepatotoxicity, which may yield novel strategies to manage liver injury induced by T-DM1 or other ADCs. PMID:26712117

  2. c-Jun N-terminal kinase-mediated Rubicon expression enhances hepatocyte lipoapoptosis and promotes hepatocyte ballooning

    PubMed Central

    Suzuki, Akiko; Kakisaka, Keisuke; Suzuki, Yuji; Wang, Ting; Takikawa, Yasuhiro

    2016-01-01

    AIM: To clarify the relationship between autophagy and lipotoxicity-induced apoptosis, which is termed “lipoapoptosis,” in non-alcoholic steatohepatitis. METHODS: Male C57BL/6J mice were fed a high-fat diet (HFD) for 12 wk, after which the liver histology and expression of proteins such as p62 or LC3 were evaluated. Alpha mouse liver 12 (AML12) cells treated with palmitate (PA) were used as an in vitro model. RESULTS: LC3-II, p62, and Run domain Beclin-1 interacting and cysteine-rich containing (Rubicon) proteins increased in both the HFD mice and in AML12 cells in response to PA treatment. Rubicon expression was decreased upon c-Jun N-terminal kinase (JNK) inhibition at both the mRNA and the protein level in AML12 cells. Rubicon knockdown in AML12 cells with PA decreased the protein levels of both LC3-II and p62. Rubicon expression peaked at 4 h of PA treatment in AML12, and then decreased. Treatment with caspase-9 inhibitor ameliorated the decrease in Rubicon protein expression at 10 h of PA and resulted in enlarged AML12 cells under PA treatment. The enlargement of AML12 cells by PA with caspase-9 inhibition was canceled by Rubicon knockdown. CONCLUSION: The JNK-Rubicon axis enhanced lipoapoptosis, and caspase-9 inhibition and Rubicon had effects that were cytologically similar to hepatocyte ballooning. As ballooned hepatocytes secrete fibrogenic signals and thus might promote fibrosis in the liver, the inhibition of hepatocyte ballooning might provide anti-fibrosis in the NASH liver. PMID:27605885

  3. Knockdown of triglyceride synthesis does not enhance palmitate lipotoxicity or prevent oleate-mediated rescue in rat hepatocytes.

    PubMed

    Leamy, Alexandra K; Hasenour, Clinton M; Egnatchik, Robert A; Trenary, Irina A; Yao, Cong-Hui; Patti, Gary J; Shiota, Masakazu; Young, Jamey D

    2016-09-01

    Experiments in a variety of cell types, including hepatocytes, consistently demonstrate the acutely lipotoxic effects of saturated fatty acids, such as palmitate (PA), but not unsaturated fatty acids, such as oleate (OA). PA+OA co-treatment fully prevents PA lipotoxicity through mechanisms that are not well defined but which have been previously attributed to more efficient esterification and sequestration of PA into triglycerides (TGs) when OA is abundant. However, this hypothesis has never been directly tested by experimentally modulating the relative partitioning of PA/OA between TGs and other lipid fates in hepatocytes. In this study, we found that addition of OA to PA-treated hepatocytes enhanced TG synthesis, reduced total PA uptake and PA lipid incorporation, decreased phospholipid saturation and rescued PA-induced ER stress and lipoapoptosis. Knockdown of diacylglycerol acyltransferase (DGAT), the rate-limiting step in TG synthesis, significantly reduced TG accumulation without impairing OA-mediated rescue of PA lipotoxicity. In both wild-type and DGAT-knockdown hepatocytes, OA co-treatment significantly reduced PA lipid incorporation and overall phospholipid saturation compared to PA-treated hepatocytes. These data indicate that OA's protective effects do not require increased conversion of PA into inert TGs, but instead may be due to OA's ability to compete against PA for cellular uptake and/or esterification and, thereby, normalize the composition of cellular lipids in the presence of a toxic PA load. PMID:27249207

  4. Cell lineage study in the liver using retroviral mediated gene transfer. Evidence against the streaming of hepatocytes in normal liver.

    PubMed Central

    Bralet, M. P.; Branchereau, S.; Brechot, C.; Ferry, N.

    1994-01-01

    The fate of normal hepatocytes in adult rat liver was studied after genetic labeling using the Escherichia coli beta-galactosidase gene coupled to a nuclear localization signal. The marker gene was introduced by direct in vivo retroviral-mediated gene transfer into hepatocytes 24 hours after partial hepatectomy. Analysis of beta-galactosidase expression in the liver at various time after gene transfer revealed that labeled hepatocytes were distributed throughout the entire lobule with a predominance in the periportal and mediolobular regions. Long-term experiments demonstrated that division of hepatocytes did occur as was revealed by the increasing number of beta-galactosidase-positive cells in isolated clusters. There was no evidence for the participation of stem cells in this process. Moreover, we found that after more than 1 year, the pattern of distribution of positive cells within the lobule was not modified. This suggests that hepatocytes do not migrate from the portal space to the perivenous region, as has been previously hypothesized. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8178942

  5. Improved Hepatocyte Engraftment After Portal Vein Occlusion in LDL Receptor-Deficient WHHL Rabbits and Lentiviral-Mediated Phenotypic Correction In Vitro.

    PubMed

    Goulinet-Mainot, Sylvie; Tranchart, Hadrien; Groyer-Picard, Marie-Thérèse; Lainas, Panagiotis; Saloum Diop, Papa; Holopherne, Delphine; Gonin, Patrick; Benihoud, Karim; Ba, Nathalie; Gauthier, Olivier; Franco, Dominique; Guettier, Catherine; Pariente, Danièle; Weber, Anne; Dagher, Ibrahim; Huy Nguyen, Tuan

    2012-02-01

    Innovative cell-based therapies are considered as alternatives to liver transplantation. Recent progress in lentivirus-mediated hepatocyte transduction has renewed interest in cell therapy for the treatment of inherited liver diseases. However, hepatocyte transplantation is still hampered by inefficient hepatocyte engraftment. We previously showed that partial portal vein embolization (PVE) improved hepatocyte engraftment in a nonhuman primate model. We developed here an ex vivo approach based on PVE and lentiviral-mediated transduction of hepatocytes from normal (New Zealand White, NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits: the large animal model of familial hypercholesterolemia type IIa (FH). FH is a life-threatening human inherited autosomal disease caused by a mutation in the low-density lipoprotein receptor (LDLR) gene, which leads to severe hypercholesterolemia and premature coronary heart disease. Rabbit hepatocytes were isolated from the resected left liver lobe, and the portal branches of the median lobes were embolized with Histoacryl® glue under radiologic guidance. NZW and WHHL hepatocytes were each labeled with Hoechst dye or transduced with lentivirus expressing GFP under the control of a liver-specific promoter (mTTR, a modified murine transthyretin promoter) and were then immediately transplanted back into donor animals. In our conditions, 65-70% of the NZW and WHHL hepatocytes were transduced. Liver repopulation after transplantation with the Hoechst-labeled hepatocytes was 3.5 ± 2%. It was 1.4 ± 0.6% after transplantation with either the transduced NZW hepatocytes or the transduced WHHL hepatocytes, which was close to that obtained with Hoechst-labeled cells, given the mean transduction efficacy. Transgene expression persisted for at least 8 weeks posttransplantation. Transduction of WHHL hepatocytes with an LDLR-encoding vector resulted in phenotypic correction in vitro as assessed by internalization of fluorescent LDL ligands

  6. Improved Hepatocyte Engraftment After Portal Vein Occlusion in LDL Receptor-Deficient WHHL Rabbits and Lentiviral-Mediated Phenotypic Correction In Vitro

    PubMed Central

    Goulinet-Mainot, Sylvie; Tranchart, Hadrien; Groyer-Picard, Marie-Thérèse; Lainas, Panagiotis; Saloum Diop, Papa; Holopherne, Delphine; Gonin, Patrick; Benihoud, Karim; Ba, Nathalie; Gauthier, Olivier; Franco, Dominique; Guettier, Catherine; Pariente, Danièle; Weber, Anne; Dagher, Ibrahim; Huy Nguyen, Tuan

    2012-01-01

    Innovative cell-based therapies are considered as alternatives to liver transplantation. Recent progress in lentivirus-mediated hepatocyte transduction has renewed interest in cell therapy for the treatment of inherited liver diseases. However, hepatocyte transplantation is still hampered by inefficient hepatocyte engraftment. We previously showed that partial portal vein embolization (PVE) improved hepatocyte engraftment in a nonhuman primate model. We developed here an ex vivo approach based on PVE and lentiviral-mediated transduction of hepatocytes from normal (New Zealand White, NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits: the large animal model of familial hypercholesterolemia type IIa (FH). FH is a life-threatening human inherited autosomal disease caused by a mutation in the low-density lipoprotein receptor (LDLR) gene, which leads to severe hypercholesterolemia and premature coronary heart disease. Rabbit hepatocytes were isolated from the resected left liver lobe, and the portal branches of the median lobes were embolized with Histoacryl® glue under radiologic guidance. NZW and WHHL hepatocytes were each labeled with Hoechst dye or transduced with lentivirus expressing GFP under the control of a liver-specific promoter (mTTR, a modified murine transthyretin promoter) and were then immediately transplanted back into donor animals. In our conditions, 65–70% of the NZW and WHHL hepatocytes were transduced. Liver repopulation after transplantation with the Hoechst-labeled hepatocytes was 3.5 ± 2%. It was 1.4 ± 0.6% after transplantation with either the transduced NZW hepatocytes or the transduced WHHL hepatocytes, which was close to that obtained with Hoechst-labeled cells, given the mean transduction efficacy. Transgene expression persisted for at least 8 weeks posttransplantation. Transduction of WHHL hepatocytes with an LDLR-encoding vector resulted in phenotypic correction in vitro as assessed by internalization of fluorescent LDL

  7. Overexpression of transforming growth factor-alpha causes liver enlargement and increased hepatocyte proliferation in transgenic mice.

    PubMed Central

    Webber, E. M.; Wu, J. C.; Wang, L.; Merlino, G.; Fausto, N.

    1994-01-01

    Transforming growth factor-alpha (TGF-alpha) expression is associated with hepatocyte DNA replication both in vivo and in culture. Our previous work using TGF-alpha transgenic mice showed that constitutive overexpression of this growth factor in the liver causes hepatic tumors in 75 to 80% of the animals at 12 to 15 months of age. To understand the cellular events by which TGF-alpha overexpression leads to abnormal liver growth, we examined hepatocyte proliferative activity in young and old TGF-alpha transgenic mice and hepatocyte ploidy in normal, dysplastic, and neoplastic livers of these animals. At 4 weeks of age, transgenic mice had higher liver weights and liver weight/body weight ratios than non-transgenic mice of the same age and hepatocyte proliferative activity, measured by 3H-thymidine incorporation after 3- and 7-day infusion, proliferating cell nuclear antigen staining, and mitotic index determination, was 2 to 3 times higher than in controls. In both transgenic and non-transgenic mice hepatocyte proliferation declined with age but the decrease was much more pronounced in control animals, so that at 8 months of age, hepatocyte replication was 8 to 10 times higher in transgenic animals. Surprisingly, however, transgenic and non-transgenic mice at this age had similar liver weight/body weight ratios. Labeling studies done in 3-month-old animals revealed that hepatocyte turnover was much faster in transgenic than in control animals, suggesting that a homeostatic compensatory mechanism involving cell death tended to restore normal liver weight/body weight ratios in older transgenic mice. Ploidy analyses showed that at 4 weeks of age transgenic mice had a higher proportion of diploid and tetraploid hepatocytes and that the hepatocellular tumors which developed in TGF-alpha transgenic mice at 13 months of age contained a higher fraction of diploid hepatocytes than that present in adjacent tissue or in dysplastic livers. The results demonstrate that

  8. Placental insufficiency decreases pancreatic vascularity and disrupts hepatocyte growth factor signaling in the pancreatic islet endothelial cell in fetal sheep.

    PubMed

    Rozance, Paul J; Anderson, Miranda; Martinez, Marina; Fahy, Anna; Macko, Antoni R; Kailey, Jenai; Seedorf, Gregory J; Abman, Steven H; Hay, William W; Limesand, Sean W

    2015-02-01

    Hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGFA) are paracrine hormones that mediate communication between pancreatic islet endothelial cells (ECs) and β-cells. Our objective was to determine the impact of intrauterine growth restriction (IUGR) on pancreatic vascularity and paracrine signaling between the EC and β-cell. Vessel density was less in IUGR pancreata than in controls. HGF concentrations were also lower in islet EC-conditioned media (ECCM) from IUGR, and islets incubated with control islet ECCM responded by increasing insulin content, which was absent with IUGR ECCM. The effect of ECCM on islet insulin content was blocked with an inhibitory anti-HGF antibody. The HGF receptor was not different between control and IUGR islets, but VEGFA was lower and the high-affinity VEGF receptor was higher in IUGR islets and ECs, respectively. These findings show that paracrine actions from ECs increase islet insulin content, and in IUGR ECs, secretion of HGF was diminished. Given the potential feed-forward regulation of β-cell VEGFA and islet EC HGF, these two growth factors are highly integrated in normal pancreatic islet development, and this regulation is decreased in IUGR fetuses, resulting in lower pancreatic islet insulin concentrations and insulin secretion. PMID:25249573

  9. Hepatocyte injury by activated neutrophils in vitro is mediated by proteases.

    PubMed Central

    Harbrecht, B G; Billiar, T R; Curran, R D; Stadler, J; Simmons, R L

    1993-01-01

    OBJECTIVE: This study determined the mechanism used by neutrophils (PMNs) to induce hepatocellular injury. SUMMARY BACKGROUND DATA: Neutrophils have been shown to be potent mediators of cell and tissue injury and have been hypothesized to contribute to the hepatic injury that occurs after trauma and infection. Oxygen radical scavengers protect the liver in vivo from inflammatory injury and it has been suggested that PMNs are the source of these toxic oxygen radicals. The specific mechanism used by PMNs to produce hepatocellular damage, however, has not been determined. METHODS: Neutrophils were cultured in vitro with hepatocytes (HCs) and stimulated with phorbol 12-myristate 13-acetate (PMA) to induce HC injury in the presence of oxygen radical scavengers and protease inhibitors. RESULTS: PMA induced a PMN-mediated HC injury that was dependent on the number of PMNs present and the concentration of PMA. Protease inhibitors reduced the extent of HC injury, while oxygen radical scavengers had no effect. Hydrogen peroxide, directly applied, was able to injure HCs, but only at concentrations greater than those that could be produced by PMA-stimulated PMNs. CONCLUSIONS: PMNs are cytotoxic to cultured HCs, predominantly due to the release of proteolytic enzymes, while HCs appear relatively resistant to oxidative injury. Involvement of neutrophil toxic oxygen radicals in hepatic damage in vivo may require impairment of HC antioxidant defenses or may involve injury to nonparenchymal liver cells with secondary effects on HCs. PMID:8342991

  10. Protective Role of Morin, a Flavonoid, against High Glucose Induced Oxidative Stress Mediated Apoptosis in Primary Rat Hepatocytes

    PubMed Central

    Kapoor, Radhika; Kakkar, Poonam

    2012-01-01

    Apoptosis is an early event of liver damage in diabetes and oxidative stress has been linked to accelerate the apoptosis in hepatocytes. Therefore, the compounds that can scavenge ROS may confer regulatory effects on high-glucose induced apoptosis. In the present study, primary rat hepatocytes were exposed to high concentration (40 mM) of glucose. At this concentration decreased cell viability and enhanced ROS generation was observed. Depleted antioxidant status of hepatocytes under high glucose stress was also observed as evident from transcriptional level and activities of antioxidant enzymes. Further, mitochondrial depolarisation was accompanied by the loss of mitochondrial integrity and altered expression of Bax and Bcl-2. Increased translocation of apoptotic proteins like AIF (Apoptosis inducing factor) & Endo-G (endonuclease-G) from its resident place mitochondria to nucleus was also observed. Cyt-c residing in the inter-membrane space of mitochondria also translocated to cytoplasm. These apoptotic proteins initiated caspase activation, DNA fragmentation, chromatin condensation, increased apoptotic DNA content in glucose treated hepatocytes, suggesting mitochondria mediated apoptotic mode of cell death. Morin, a dietary flavonoid from Psidium guajava was effective in increasing the cell viability and decreasing the ROS level. It maintained mitochondrial integrity, inhibited release of apoptotic proteins from mitochondria, prevented DNA fragmentation, chromatin condensation and hypodiploid DNA upon exposure to high glucose. This study confirms the capacity of dietary flavonoid Morin in regulating apoptosis induced by high glucose via mitochondrial mediated pathway through intervention of oxidative stress. PMID:22899998

  11. Growth Hormone-Regulated mRNAs and miRNAs in Chicken Hepatocytes

    PubMed Central

    Wang, Huijuan; Shao, Fang; Yu, JianFeng; Jiang, Honglin; Han, Yaoping; Gong, Daoqing; Gu, Zhiliang

    2014-01-01

    Growth hormone (GH) is a key regulatory factor in animal growth, development and metabolism. Based on the expression level of the GH receptor, the chicken liver is a major target organ of GH, but the biological effects of GH on the chicken liver are not fully understood. In this work we identified mRNAs and miRNAs that are regulated by GH in primary hepatocytes from female chickens through RNA-seq, and analyzed the functional relevance of these mRNAs and miRNAs through GO enrichment analysis and miRNA target prediction. A total of 164 mRNAs were found to be differentially expressed between GH-treated and control chicken hepatocytes, of which 112 were up-regulated and 52 were down-regulated by GH. A total of 225 chicken miRNAs were identified by the RNA-Seq analysis. Among these miRNAs 16 were up-regulated and 1 miRNA was down-regulated by GH. The GH-regulated mRNAs were mainly involved in growth and metabolism. Most of the GH-upregulated or GH-downregulated miRNAs were predicted to target the GH-downregulated or GH-upregulated mRNAs, respectively, involved in lipid metabolism. This study reveals that GH regulates the expression of many mRNAs involved in metabolism in female chicken hepatocytes, which suggests that GH plays an important role in regulating liver metabolism in female chickens. The results of this study also support the hypothesis that GH regulates lipid metabolism in chicken liver in part by regulating the expression of miRNAs that target the mRNAs involved in lipid metabolism. PMID:25386791

  12. Characteristics of inositol trisphosphate-mediated Ca/sup 2 +/ release from permeabilized hepatocytes

    SciTech Connect

    Joseph, S.K.; Williamson, J.R.

    1986-11-05

    Ca/sup 2 +/ release triggered by inositol trisphosphate (Ins(1,4,5)P/sub 3/) has been measured in saponin-permeabilized hepatocytes with /sup 45/Ca/sup 2 +/ or Quin 2. The initial rate of Ca/sup 2 +/ release was not greatly affected by the incubation temperature. The amount of Ca/sup 2 +/ released by Ins(1,4,5)P/sub 3/ was not affected by pH (6.5-8.0). La/sup 3 +/ (100 ..mu..M) markedly inhibited the effect of 1 ..mu..M Ins(1,4,5)P/sub 3/. The possibility that La/sup 3 +/ chelates Ins(1,4,5)P/sub 3/ cannot be excluded since the effect of La/sup 3 +/ could be overcome by increasing the Ins(1,4,5)P/sub 3/ concentration. Ins(1,4,5)P/sub 3/-mediated Ca/sup 2 +/ release showed a requirement for permeant cations in the incubation medium. Optimal release was observed with potassium gluconate. Other monovalent cations, with the exception of Li/sup +/, can substitute for K/sup +/. Permeant anions, at concentrations above 40 mM, inhibited Ca/sup 2 +/ release produced by Ins(1,4,5)P/sub 3/. Cl/sup -/, Br/sup -/, I/sup -/, and SO/sup 2 -//sub 4/ were equally effective as inhibitors. Ins(1,4,5)P/sub 3/ also caused the release of /sup 54/Mn/sup 2 +/ and /sup 85/Sr/sup 2 +/ accumulated by the permeabilized hepatocytes. The results are consistent with Ins(1,4,5)P/sub 3/ promoting the membrane translocation of divalent cations through an ion channel rather than an ion carrier. The translocation of positive charge through this channel is balanced by ancillary movements of monovalent cations and anions across the reticular membranes. The transport systems responsible for these compensatory ion movements may represent a potential site for the regulation of the hormone-mediated Ca/sup 2 +/ signal.

  13. Keratinocyte growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium.

    PubMed Central

    Panos, R J; Rubin, J S; Csaky, K G; Aaronson, S A; Mason, R J

    1993-01-01

    Epithelial-mesenchymal interactions mediate aspects of normal lung growth and development and are important in the restoration of normal alveolar architecture after lung injury. To determine if fibroblasts are a source of soluble growth factors for alveolar type II cells, we investigated the effect of fibroblast-conditioned medium (CM) on alveolar type II cell DNA synthesis. Serum-free CM from confluent adult human lung fibroblasts was concentrated fivefold by lyophilization. Type II cells were isolated from adult rats by elastase dissociation and incubated with [3H]thymidine and varying dilutions of concentrated CM and serum from day 1 to 3 of culture. Stimulation of type II cell DNA synthesis by fibroblast-CM was maximal after 48 h of conditioning and required the presence of serum. The activity of the CM was eliminated by boiling and by treatment with trypsin, pepsin, or dithiothreitol and was additive with saturating concentrations of acidic fibroblast growth factor, epidermal growth factor, and insulin. The growth factor activity bound to heparin-Sepharose and was eluted with 0.6 and 1.0 M NaCl. Neutralizing antibody studies demonstrated that the primary mitogens isolated in the 0.6 and 1.0 M NaCl fractions were keratinocyte growth factor (KGF, fibroblast growth factor 7) and hepatocyte growth factor/scatter factor (HGF/SF), respectively. HGF/SF was demonstrated in the crude CM and KGF was detected in the 0.6 M NaCl eluent by immunoblotting. Northern blot analysis confirmed that the lung fibroblasts expressed both KGF and HGF/SF transcripts. Human recombinant KGF and HGF/SF induced a concentration- and serum-dependent increase in rat alveolar type II cell DNA synthesis. We conclude that adult human lung fibroblasts produce at least two soluble heparin-binding growth factors, KGF and HGF/SF, which promote DNA synthesis and proliferation of rat alveolar type II cells in primary culture. KGF and HGF/SF may be important stimuli for alveolar type II cell

  14. IGF-I mediated inhibition of leptin receptor expression in porcine hepatocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A study was conducted to elucidate hormonal control of leptin receptor gene expression in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (52 kg) and seeded into collagen-coated T-25 flasks. Monolayer cultures were established in medium containing fetal bovine serum fo...

  15. HCV-Mediated Apoptosis of Hepatocytes in Culture and Viral Pathogenesis

    PubMed Central

    Silberstein, Erica; Ulitzky, Laura; Lima, Livia Alves; Cehan, Nicoleta; Teixeira-Carvalho, Andréa; Roingeard, Philippe; Taylor, Deborah R.

    2016-01-01

    Chronic Hepatitis C Virus (HCV) infection is associated with progressive liver injury and subsequent development of fibrosis and cirrhosis. The death of hepatocytes results in the release of cytokines that induce inflammatory and fibrotic responses. The mechanism of liver damage is still under investigation but both apoptosis and immune-mediated processes may play roles. By observing the changes in gene expression patterns in HCV-infected cells, both markers and the causes of HCV-associated liver injury may be elucidated. HCV genotype 1b virus from persistently infected VeroE6 cells induced a strong cytopathic effect when used to infect Huh7.5 hepatoma cells. To determine if this cytopathic effect was a result of apoptosis, ultrastructural changes were observed by electron microscopy and markers of programmed cell death were surveyed. Screening of a human PCR array demonstrated a gene expression profile that contained upregulated markers of apoptosis, including tumor necrosis factor, caspases and caspase activators, Fas, Bcl2-interacting killer (BIK) and tumor suppressor protein, p53, as a result of HCV genotype 1b infection. The genes identified in this study should provide new insights into understanding viral pathogenesis in liver cells and may possibly help to identify novel antiviral and antifibrotic targets. PMID:27280444

  16. Ischemic Preconditioning protects hepatocytes from ischemia-reperfusion injury via TGR5-mediated anti-apoptosis.

    PubMed

    Zhuang, Lin; Fan, Ye; Lu, Ling; Ding, Wenbin; Ni, Chuangye; Wang, Xuehao; Zhang, Feng; Rao, Jianhua

    2016-05-13

    Ischemic preconditioning (IP) has been shown to protect hepatic tissue from liver ischemia-reperfusion injury (IRI). TGR5, as a new-type bile acid receptor, has been shown protective roles in several liver diseases. However, the relationship between TGR5 and IP is still unknown. This study investigated effects of IP on TGR5 as well as the roles of TGR5 on hepatic tissue lesions and apoptosis in liver IRI. We showed that TGR5 was significantly upregulated in liver tissues after IP. To further analyzed effects of the TGR5 on liver IRI, wild type and TGR5 knockout mice were used to establish the liver IRI model. IP effectively alleviated liver IRI, but TGR5 deficiency significantly neutralized IP-related liver protection, as evidenced by serum alanine aminotransferase levels, histological liver damage, hepatocellular apoptosis and cytokines expressions. In addition, molecules related to apoptosis were detected by Western Blot, which showed that activation of TGR5 by IP increased expression of Bcl-2, and inhibited expressions of IRAK4 and cleaved caspase-3, but TGR5 deficiency abolished IP-induced expressions of anti-apoptosis molecule. In vitro, effects of TGR5 on hepatocytes were further analyzed by TGR5 agonist (INT-777) and hypoxia/reoxygenation (H/R), which displayed that INT-777 markedly attenuated H/R-induced hepatocellular apoptosis. In conclusion, our study indicates that IP alleviates hepatocellular apoptosis, and reduces liver IRI through TGR5-mediated anti-apoptosis functions. PMID:27045083

  17. MET inhibitor PHA-665752 suppresses the hepatocyte growth factor-induced cell proliferation and radioresistance in nasopharyngeal carcinoma cells

    SciTech Connect

    Liu, Tongxin; Li, Qi; Sun, Quanquan; Zhang, Yuqin; Yang, Hua; Wang, Rong; Chen, Longhua; Wang, Wei

    2014-06-20

    Highlights: • We demonstrated that irradiation induced MET overexpression and activation. • The aberrant MET signal mediated by HGF induced proliferation and radioresistance of NPC cells. • MET inhibitor PHA-665752 effectively suppressed HGF induced cell proliferation and radioresistance in NPC cells. • PHA-665752 suppressed the three downstream pathway of HGF/MET signal in a dose-dependent manner. - Abstract: Although ionizing radiation (IR) has provided considerable improvements in nasopharyngeal carcinoma (NPC), in subsets of patients, radioresistance is still a major problem in the treatment. In this study, we demonstrated that irradiation induced MET overexpression and activation, and the aberrant MET signal mediated by hepatocyte growth factor (HGF) induced radioresistance. We also found that MET inhibitor PHA-665752 effectively suppressed HGF induced cell proliferation and radioresistance in NPC cells. Further investigation indicated that PHA-665752 suppressed the phosphorylation of the Akt, ERK1/2, and STAT3 proteins in a dose-dependent manner. Our data indicated that the combination of IR with a MET inhibitor, such as PHA-665752, might be a promising therapeutic strategy for NPC.

  18. Hepatocyte growth factor/hepatopoietin A is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cells.

    PubMed

    Schirmacher, P; Geerts, A; Pietrangelo, A; Dienes, H P; Rogler, C E

    1992-01-01

    Hepatocyte growth factor/hepatopoietin A is a complete mitogen for parenchymal liver cells, and its expression is increased as an early response to acute liver injury. To identify the liver cell population responsible for hepatocyte growth factor gene expression, we investigated tissue sections and isolated and purified cell fractions from normal rat liver by in situ and Northern blot hybridization. Hepatocyte growth factor transcripts were present in sinusoidal liver cells, which were preferentially located in the periportal parenchyma. Northern hybridization analysis of RNA isolated from purified liver cell fractions demonstrated that HGF messenger RNA is present only in fat-storing cells. No specific hepatocyte growth factor gene expression was detected in parenchymal cells, endothelial cells and Kupffer cells. Myofibroblast-like transition of fat-storing cells, which is linked to fibrogenesis in chronic liver disease, results in the loss of hepatocyte growth factor expression. Hepatocyte growth factor gene expression in the normal liver, a new function of fat-storing cells, suggests that this growth factor may play a role in the physiological balance between cell death and replacement in the liver and that hepatocyte growth factor may also act in a paracrine manner. Furthermore, loss of hepatocyte growth factor expression in myofibroblast-like cells derived from fat-storing cells may be responsible for reduced parenchymal cell regeneration in chronic liver disease. PMID:1530788

  19. Inhibition of neoplastic development in the liver by hepatocyte growth factor in a transgenic mouse model.

    PubMed Central

    Santoni-Rugiu, E; Preisegger, K H; Kiss, A; Audolfsson, T; Shiota, G; Schmidt, E V; Thorgeirsson, S S

    1996-01-01

    Overexpression of the c-myc oncogene is associated with a variety of both human and experimental tumors, and cooperation of other oncogenes and growth factors with the myc family are critical in the evolution of the malignant phenotype. The interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in a transgenic mouse model has been analyzed. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Furthermore, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice, whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine. Images Fig. 2 Fig. 3 PMID:8790372

  20. Suppression of CYP2B Induction by Alendronate-Mediated Farnesyl Diphosphate Synthase Inhibition in Primary Cultured Rat Hepatocytes

    PubMed Central

    Jackson, Nancy M.; Kocarek, Thomas A.

    2008-01-01

    We previously reported that squalestatin 1-mediated induction of CYP2B expression is attributable to squalene synthase inhibition and accumulation of an endogenous isoprenoid(s) that is capable of activating the constitutive androstane receptor. To determine whether squalestatin 1-mediated CYP2B induction is strictly dependent upon the biosynthesis of farnesyl pyrophosphate (FPP), the substrate for squalene synthase, the effects of alendronate, a nitrogen-containing bisphosphonate inhibitor of farnesyl diphosphate synthase, were determined on basal, squalestatin 1-inducible, and phenobarbital-inducible CYP2B expression in primary cultured rat hepatocytes. Alendronate treatment alone had no effect on CYP2B or CYP3A mRNA expression in the hepatocyte cultures, but alendronate co-treatment completely suppressed squalestatin 1-mediated CYP2B mRNA induction at concentrations (60 and 100 μM) that effectively inhibited cellular farnesyl diphosphate synthase activity, as assessed by reductions of squalestatin 1-mediated FPP accumulation, and that were not toxic to the cells, as indicated by a lack of effect on MTT activity. Alendronate co-treatment also partially suppressed phenobarbital-inducible CYP2B expression, and this suppressive effect was attenuated by additional co-treatment with the upstream pathway inhibitor, pravastatin. These findings demonstrate that squalestatin 1-mediated CYP2B induction cannot occur in the absence of FPP biosynthesis, but also indicate that one or more upstream isoprenoids, possibly isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate, function to antagonize the CYP2B induction process. PMID:18617600

  1. Asialoglycoprotein receptor mediates the toxic effects of an asialofetuin-diphtheria toxin fragment A conjugate on cultured rat hepatocytes

    SciTech Connect

    Cawley, D.B.; Simpson, D.L.; Herschman, H.R.

    1981-06-01

    We have constructed a toxic hybrid protein that is recognized by asialoglycoprotein (ASGP) receptors of cultured rat hepatocytes. The conjugate consists of fragment A of diphtheria toxin (DTA) linked by a disulfide bond to asialofetuin (ASF). This conjugate is highly toxic, inhibiting protein synthesis in primary rat hepatocytes at concentrations as low as 10 pM. The ASF-DTA conjugate was 600 and 1800 times as toxic as diphtheria toxin and DTA, respectively, on primary rat hepatocytes. The ASGP receptor recognizes galactose-terminated proteins. We tested a series of glycoproteins for their ability to block the action of the ASF-DTA conjugate. Fetuin and orosomucoid, two glycoproteins with terminal sialic acid on their oligosaccharide chains, did not block the action of the conjugate. Their galactose-terminated asialo derivatives, ASF and asialoorosomucoid, as expected, did block the action of the conjugate. The N-acetylglucosaminyl-terminated derivative (asialoagalactoorosomucoid) had no appreciable effect on the activity of the conjugate. We tested the ASF-DTA conjugate on six cell types; except for primary rat hepatocytes, none of them were affected by a high concentration (10 nM) of ASF-DTA conjugate. A fetuin-DTA conjugate was less toxic by a factor of 300 than the ASF-DTA conjugate and exerted its effects primarily through non-receptor-mediated mechanisms. The highly toxic ASF-DTA conjugate is cell-type specific, and its action is mediated by a well-characterized receptor, whose mechanism of receptor-ligand internalization has been extensively investigated.

  2. Purification and characterization of a growth factor from rat platelets for mature parenchymal hepatocytes in primary cultures.

    PubMed Central

    Nakamura, T; Teramoto, H; Ichihara, A

    1986-01-01

    A growth factor (HGF) stimulating DNA synthesis of adult rat hepatocytes in primary culture was found in rat platelets. HGF was purified from rat platelets to homogeneity by a three-step procedure: stimulation of its release from platelets by thrombin, cation-exchanger fast protein liquid chromatography on a Mono S column, and heparin-Sepharose chromatography. HGF was clearly distinguishable from the platelet-derived growth factor (PDGF) by fast protein liquid chromatography. HGF was a heat- and acid-labile cationic protein that was inactivated by reduction with dithiothreitol. Its molecular mass was estimated to be 27 kDa by NaDodSO4/PAGE and its amino acid composition was very different from that of PDGF. The purified HGF stimulated DNA synthesis in adult rat hepatocytes at 2 ng/ml and was maximally effective at 20 ng/ml; its effect was additive or synergistic with those of insulin and EGF, depending on their combinations. HGF did not stimulate DNA synthesis of Swiss 3T3 cells, while PDGF did not stimulate that of hepatocytes. Thus, HGF showed clearly different cell specificity from PDGF in its growth-promoting activities. These findings indicate that HGF is a growth factor in platelets for mature hepatocytes. Images PMID:3529086

  3. PROLINE IS REQUIRED FOR THE STIMULATION OF DNA SYNTHESIS IN HEPATOCYTE CULTURES BY EGF (EPIDERMAL GROWTH FACTOR)

    EPA Science Inventory

    Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis in rat parenchymal hepatocytes both in vivo and in vitro (4,9). The authors report here that this response in vitro is dependent on the amino acids present in the media. Of all the amino acids, proline has th...

  4. Total synthesis of biotinylated N domain of human hepatocyte growth factor.

    PubMed

    Raibaut, Laurent; Vicogne, Jérome; Leclercq, Bérénice; Drobecq, Hervé; Desmet, Rémi; Melnyk, Oleg

    2013-06-15

    Hepatocyte growth factor/scatter factor (HGF/SF) is the high affinity ligand of MET tyrosine kinase receptor. We report here the total synthesis of a biotinylated analogue of human HGF/SF N domain. Functionally, N domain is part of the HGF/SF high affinity binding site for MET and also the main HGF/SF binding site for heparin. The 97 Aa linear chain featuring a C-terminal biotin group was assembled in high yield using an N-to-C one-pot three segments assembly strategy relying on a sequential Native Chemical Ligation (NCL)/bis(2-sulfanylethyl)amido (SEA) native peptide ligation process. The folded protein displayed the native disulfide bond pattern and showed the ability to bind heparin. PMID:23523386

  5. Directed random walks and constraint programming reveal active pathways in hepatocyte growth factor signaling.

    PubMed

    Kittas, Aristotelis; Delobelle, Aurélien; Schmitt, Sabrina; Breuhahn, Kai; Guziolowski, Carito; Grabe, Niels

    2016-01-01

    An effective means to analyze mRNA expression data is to take advantage of established knowledge from pathway databases, using methods such as pathway-enrichment analyses. However, pathway databases are not case-specific and expression data could be used to infer gene-regulation patterns in the context of specific pathways. In addition, canonical pathways may not always describe the signaling mechanisms properly, because interactions can frequently occur between genes in different pathways. Relatively few methods have been proposed to date for generating and analyzing such networks, preserving the causality between gene interactions and reasoning over the qualitative logic of regulatory effects. We present an algorithm (MCWalk) integrated with a logic programming approach, to discover subgraphs in large-scale signaling networks by random walks in a fully automated pipeline. As an exemplary application, we uncover the signal transduction mechanisms in a gene interaction network describing hepatocyte growth factor-stimulated cell migration and proliferation from gene-expression measured with microarray and RT-qPCR using in-house perturbation experiments in a keratinocyte-fibroblast co-culture. The resulting subgraphs illustrate possible associations of hepatocyte growth factor receptor c-Met nodes, differentially expressed genes and cellular states. Using perturbation experiments and Answer Set programming, we are able to select those which are more consistent with the experimental data. We discover key regulator nodes by measuring the frequency with which they are traversed when connecting signaling between receptors and significantly regulated genes and predict their expression-shift consistently with the measured data. The Java implementation of MCWalk is publicly available under the MIT license at: https://bitbucket.org/akittas/biosubg. PMID:26518250

  6. Heparin-binding growth factor type 1 (acidic fibroblast growth factor): a potential biphasic autocrine and paracrine regulator of hepatocyte regeneration.

    PubMed Central

    Kan, M; Huang, J S; Mansson, P E; Yasumitsu, H; Carr, B; McKeehan, W L

    1989-01-01

    Heparin-binding growth factor type 1 (HBGF-1; sometimes termed acidic fibroblast growth factor) is potentially an important factor in liver regeneration. HBGF-1 alone (half-maximal effect at 60 pM) stimulated hepatocyte DNA synthesis and bound to a high-affinity receptor (Kd = 62 pM; 5000 per cell). Epidermal growth factor (EGF) neutralized or masked the mitogenic effect of HBGF-1 concurrent with appearance of low-affinity HBGF-1 binding sites. HBGF-1 reduced the inhibitory effect of transforming growth factor type beta (TGF-beta) on the EGF stimulus. Nanomolar levels of HBGF-1 decreased the EGF stimulus. An increase in hepatic HBGF-1 gene expression after partial hepatectomy precedes increases in expression of the EGF homolog, TGF-alpha, and nonparenchymal-cell-derived TGF-beta in the regenerating liver. Expression of HBGF-1 mRNA occurs in both hepatocytes and nonparenchymal cells and persists for 7 days in liver tissue after partial hepatectomy. HBGF-1 acting through a high-affinity receptor is a candidate for the early autocrine stimulus that drives hepatocyte DNA synthesis prior to or concurrent with the EGF/TGF-alpha stimulus. It may allow hepatocyte proliferation to proceed in the presence of low levels of TGF-beta. An EGF/TGF-alpha-dependent change in HBGF-1 receptor phenotype and increasing levels of nonparenchymal-cell-derived HBGF-1 and TGF-beta may serve to limit hepatocyte proliferation. Images PMID:2477840

  7. Towards liver-directed gene therapy: retrovirus-mediated gene transfer into human hepatocytes.

    PubMed

    Grossman, M; Raper, S E; Wilson, J M

    1991-11-01

    Liver-directed gene therapy is being considered in the treatment of inherited metabolic diseases. One approach we are considering is the transplantation of autologous hepatocytes that have been genetically modified with recombinant retroviruses ex vivo. We describe, in this report, techniques for isolating human hepatocytes and efficiently transducing recombinant genes into primary cultures. Hepatocytes were isolated from tissue of four different donors, plated in primary culture, and exposed to recombinant retroviruses expressing either the LacZ reporter gene or the cDNA for rabbit LDL receptor. The efficiency of gene transfer under optimal conditions, as determined by Southern blot analysis, varied from a maximum of one proviral copy per cell to a minimum of 0.1 proviral copy per cell. Cytochemical assays were used to detect expression of the recombinant derived proteins, E. coli beta-galactosidase and rabbit LDL receptor. Hepatocytes transduced with the LDL receptor gene expressed levels of receptor protein that exceeded the normal endogenous levels. The ability to isolate and genetically modify human hepatocytes, as described in this report, is an important step towards the development of liver-directed gene therapies in humans. PMID:1767337

  8. Epinephrine effects on mitochondrial Krebs cycle are not mediated by typical adrenergic receptors in isolated rat hepatocytes

    SciTech Connect

    Mohan, C.; Memon, R.A.; Bessman, S.P. )

    1990-02-26

    Oxidation of 2,3-{sup 14}C succinate (suc) carbons in the intra-mitochondrial Krebs cycle was used as a probe to investigate the effects of epinephrine (epi) on isolated rat hepatocytes. Hepatocytes were incubated at 30{degrees}C in Krebs-Henseleit bicarbonate buffer, pH 7.4, with 0.5 mM concentration of each of the 20 natural amino acids, 0.5 mm concentration of each of the 20 natural amino acids, 2,3-{sup 14}C suc and epi (10 uM), phenylephrine (pheni) (10uM) or isoproterenol (10 uM). Epi and phepi caused a significant increase in {sup 14}CO{sub 2} formation from 2,3-{sup 14}C suc, however, phentolamine, an {infinity}-antagonist, failed to inhibit this increased oxidation of suc carbons. Isoproterenol had no effect on hepatocyte metabolism and propranolol, a {beta}-antagonist, failed to cause any reduction in basal or epi stimulated oxidation of 2,3-{sup 14}C carbons. Unlike insulin, neither epi nor phepi had any significant effect on the anabolic utilization of suc carbons for protein or lipid synthesis. Anabolic channeling of Krebs cycle intermediates into amino acids was reduced by epi treatment of hepatocytes. Although epi treatment can enhance the oxidation of substrate through the Krebs cycle reactions, only insulin is capable of channeling these substrates into anabolic reactions. Data presented also suggest that epi effects on mitochondrial Krebs cycle oxidation are mediated through an atypical {infinity}-adrenergic receptor which is unresponsive to inhibition by non-selective {infinity}-antagonists.

  9. Pulmonary and systemic hepatocyte and keratinocyte growth factors in patients with chronic obstructive pulmonary disease

    PubMed Central

    Sauleda, Jaume; Noguera, Aina; Blanquer, David; Pons, Jaume; López, Meritxell; Villena, Cristina; Agustí, Alvar GN

    2008-01-01

    Background The potential role of growth factors in chronic obstructive pulmonary disease (COPD) has begun to be addressed only recently and is still poorly understood. For this study, we investigated potential abnormalities of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in patients with COPD. Methods To this end, we compared the levels of HGF and KGF, measured by enzyme-linked immunosorbent assay (ELISA), in bronchoalveolar lavage (BAL) fluid and in serum in 18 patients with COPD (62 ± 9 yrs, forced expiratory volume in one second [FEV1] 57 ± 12% ref, X ± standard deviation of mean), 18 smokers with normal lung function (58 ± 8 yrs, FEV1 90 ± 6% ref) and 8 never smokers (67 ± 9 yrs, 94 ± 14% ref). Results We found that in BAL, HGF levels were higher in patients with COPD than in the other two groups whereas, in serum, HGF concentration was highest in smokers with normal lung function (p < 0.01). KGF levels were not significantly different between groups, neither in blood nor in BAL (most values were below the detection limit). Conclusions These results highlight a different response of HGF in BAL and serum in smokers with and without COPD that may be relevant for tissue repair in COPD. PMID:19281086

  10. Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoposis

    SciTech Connect

    Zhou Yijun . E-mail: zhou-yijun@hotmail.com; Wang Jiahe; Zhang Jin

    2006-06-02

    Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-{kappa}B, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-{kappa}B, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease.

  11. Hepatocyte growth factor and its receptor (c-MET) in prostatic carcinoma.

    PubMed Central

    Humphrey, P. A.; Zhu, X.; Zarnegar, R.; Swanson, P. E.; Ratliff, T. L.; Vollmer, R. T.; Day, M. L.

    1995-01-01

    Hepatocyte growth factor (scatter factor) and its receptor, the c-met proto-oncogene product (c-MET), have been implicated in embryogenesis, tissue reorganization, and tumor progression. Little is known, however, of the expression and functional significance of these molecules in prostatic cells and tissue. In this investigation, we assessed the expression of hepatocyte growth factor (HGF) and c-MET in prostatic tissues and cell lines and also determined the effect of purified recombinant HGF on cell proliferation and scattering of prostatic carcinoma cell lines. HGF was expressed by human prostatic stromal myofibroblasts in primary culture but not by three human prostatic carcinoma cell lines (LNCaP, DU 145, and PC-3) as assessed by Northern blot analysis. HGF was also detected by reverse transcriptase-polymerase chain reaction in both benign and malignant tissues from radical prostatectomy specimens. c-MET transcripts were identified by Northern blot in two androgen-insensitive human prostatic carcinoma cell lines (DU 145 and PC-3) but not the androgen-sensitive LNCaP cell line. Additional evidence of linkage of androgen responsiveness and c-MET was provided by experiments in which androgen deprivation of normal rat prostates via castration produced a marked up-regulation of c-MET expression as determined by Northern blot and immunohistochemistry. c-MET protein was detected by immunohistochemical analysis in a substantial percentage (58 of 128 or 45%) of prostatic carcinomas and was found more often in metastatic growths of human prostatic carcinoma (15 of 20 patients) compared with primary tumors (43 of 108 patients; P < 0.005). Moreover, in Dunning R-3327 rat prostatic carcinoma cell lines, c-MET expression was highest in the androgen-insensitive subline with the highest metastatic capacity. Purified recombinant human HGF induced dose-dependent cellular proliferation and scattering in the DU 145 carcinoma cell line. These data indicate that HGF may function in

  12. Adenoviral delivery of truncated MMP-8 fused with the hepatocyte growth factor mutant 1K1 ameliorates liver cirrhosis and promotes hepatocyte proliferation

    PubMed Central

    Liu, Jinghua; Li, Jianbo; Fu, Weiwei; Tang, Jiacheng; Feng, Xu; Chen, Jiang; Liang, Yuelong; Jin, Ren’an; Xie, Anyong; Cai, Xiujun

    2015-01-01

    Liver cirrhosis is a chronic liver disease caused by chronic liver injury, which activates hepatic stellate cells (HSCs) and the secretion of extracellular matrix (ECM). Cirrhosis accounts for an extensive level of morbidity and mortality worldwide, largely due to lack of effective treatment options. In this study, we have constructed a fusion protein containing matrix metal-loproteinase 8 (MMP-8) and the human growth factor mutant 1K1 (designated cMMP8-1K1) and delivered it into hepatocytes and in vivo and in cell culture via intravenous injection of fusion protein-harboring adenovirus. In doing so, we found that the cMMP8-1K1 fusion protein promotes the proliferation of hepatocytes, likely resulting from the combined inhibition of type I collagen secretion and the degradation of the ECM in the HSCs. This fusion protein was also observed to ameliorate liver cirrhosis in our mouse model. These changes appear to be linked to changes in downstream gene expression. Taken together, these results suggest a possible strategy for the treatment of liver cirrhosis and additional work is warranted. PMID:26527860

  13. Characteristics of inositol trisphosphate mediated Ca/sup 2 +/ release from permeabilized hepatocytes

    SciTech Connect

    Joseph, S.K.; Williamson, J.R.

    1986-05-01

    Ca/sup 2 +/ release triggered by inositol trisphosphate (IP/sub 3/) has been measured in saponin-permeabilized hepatocytes with /sup 45/Ca/sup 2 +/ or Quin 2. The initial rate of Ca/sup 2 +/ release was not markedly affected by the incubation temperature (175 +/- 40 pmol/s/mg at 30/sup 0/C versus 133 +/- 24 pmol/s/mg at 4/sup 0/C). This result is consistent with the membrane translocation of Ca/sup 2 +/ occurring through an ion-channel rather than an ion-carrier. The amount of Ca/sup 2 +/ released by IP/sub 3/ was not affected by pH (6.5-8.0) or by compounds that inhibit voltage-gated Ca/sup 2 +/ channels. La/sup 3 +/ (100 ..mu..M) markedly inhibits the effect of 1 ..mu..M IP/sub 3/. The possibility that La/sup 3 +/ chelates IP/sub 3/ cannot be excluded since the effect of La/sup 3 +/ can be overcome by increasing the IP/sub 3/ concentration. IP/sub 3/-mediated Ca/sup 2 +/ release displays a requirement for a permeant cation in the incubation medium. Optimal release is observed with K/sup +/ gluconate. Other monovalent cations, with the exception of Li/sup +/, can substitute for K/sup +/. Permeant anions, at concentrations above 40 mM, inhibit Ca/sup 2 +/ release produced by IP/sub 3/. Cl/sup -/, Br/sup -/, I/sup -/, and SO/sub 4//sup 2 -/ were equally effective. Ca/sup 2 +/ release was not inhibited by DIDS or Furosemide. /sup 85/Sr/sup 2 +/ and /sup 54/Mn/sup 2 +/ fluxes were also stimulated by IP/sub 3/. These results suggest that IP/sub 3/ acts to gate a divalent cation channel. The translocation of positive charge through this channel is balanced by ancillary movements of monovalent cations and anions across the reticular membrane.

  14. Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition.

    PubMed

    Ordovás, Laura; Boon, Ruben; Pistoni, Mariaelena; Chen, Yemiao; Wolfs, Esther; Guo, Wenting; Sambathkumar, Rangarajan; Bobis-Wozowicz, Sylwia; Helsen, Nicky; Vanhove, Jolien; Berckmans, Pieter; Cai, Qing; Vanuytsel, Kim; Eggermont, Kristel; Vanslembrouck, Veerle; Schmidt, Béla Z; Raitano, Susanna; Van Den Bosch, Ludo; Nahmias, Yaakov; Cathomen, Toni; Struys, Tom; Verfaillie, Catherine M

    2015-11-10

    Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes. PMID:26455413

  15. Cytochrome P450 peroxidase/peroxygenase mediated xenobiotic metabolic activation and cytotoxicity in isolated hepatocytes.

    PubMed

    Anari, M R; Khan, S; Liu, Z C; O'Brien, P J

    1995-12-01

    Cytochrome P450 (P450) can utilize organic hydroperoxides and peracids to support hydroxylation and dealkylation of various P450 substrates. However, the biological significance of this P450 peroxygenase/peroxidase activity in the bioactivation of xenobiotics in intact cells has not been demonstrated. We have shown that tert-butyl hydroperoxide (tBHP) markedly enhances 3-20-fold the cytotoxicity of various aromatic hydrocarbons and their phenolic metabolites. The tBHP-enhanced hepatocyte cytotoxicity of 4-nitroanisole (4-NA) and 4-hydroxyanisole (4-HA) was also accompanied by an increase in the hepatocyte O-demethylation of 4-NA and 4-HA up to 7.5- and 21-fold, respectively. Hepatocyte GSH conjugation by 4-HA was also markedly increased by tBHP. An LC/MS analysis of the GSH conjugates identified hydroquinone-GSH and 4-methoxy-catechol:GSH conjugates as the predominant adducts. Pretreatment of hepatocytes with P450 inhibitors, e.g., phenylimidazole, prevented tBHP-enhanced 4-HA metabolism, GSH depletion, and cytotoxicity. In conclusion, hydroperoxides can therefore be used by intact cells to support the bioactivation of xenobiotics through the P450 peroxidase/peroxygenase system. PMID:8605292

  16. NOREPINEPHRINE AND EPIDERMAL GROWTH FACTOR: DYNAMICS OF THEIR INTERACTION IN THE STIMULATION OF HEPATOCYTE DNA SYNTHESIS

    EPA Science Inventory

    Primary cultures of adult rat hepatocytes are stimulated to enter DNA synthesis by norepinephrine (NE). This stimulation is maximal if the hepatocytes are incubated with NE for more than 12 hr, beginning no later than 2-4 hr after the cells are first plated. After 24 hr in cultur...

  17. Role of TLR4-Mediated PI3K/AKT/GSK-3β Signaling Pathway in Apoptosis of Rat Hepatocytes

    PubMed Central

    Zhang, Xian; Jiang, Daorong; Jiang, Wei; Zhao, Min; Gan, Jianhe

    2015-01-01

    We investigated the mechanism of the Toll-like receptor 4- (TLR4-) mediated PI3K/AKT/GSK-3β signaling pathway in rat hepatocytes apoptosis induced by LPS. The cultured rat hepatocytes were treated with LPS alone or first pretreated with TLR4 inhibitor, AKT inhibitor, and GSK-3β inhibitor, respectively, and then stimulated with the same dose of LPS. Cell viability, cell apoptotic rate, and apoptosis morphology were assessed; the level of P-AKTSer473, P-GSK-3βSer9, and active Caspase-3 and the ratio of Bax/Bcl-2 were evaluated. The results indicated that cell viability decreased, while cell apoptotic rate increased with time after LPS stimulation. The expression of P-AKTSer473 and P-GSK-3βSer9 in the LPS group decreased compared with the control, while the level of active Caspase-3 and the ratio of Bax/Bcl-2 were significantly increased. These effects were attenuated by pretreatment with CLI-095. In addition, the apoptotic ratio decreased after pretreatment with LiCl but increased following pretreatment with LY294002. The expression of P-AKTSer473 further decreased following pretreatment with LY294002 and the expression of P-GSK-3βSer9 increased following pretreatment with LiCl. Moreover, pretreatment with CLI-095 weakened LPS-induced nuclear translocation of GSK-3β. Our findings suggest that the TLR4-mediated PI3K/AKT/GSK-3β signaling pathway is present in rat hepatocytes and participates in apoptosis of BRL-3A cells. PMID:26770978

  18. Hepatocyte Growth Factor Levels in the Saliva and Gingival Crevicular Fluid in Smokers with Periodontitis

    PubMed Central

    Anil, Sukumaran; Vellappally, Sajith; Preethanath, R. S.; Mokeem, Sameer A.; AlMoharib, Hani S.; Chalisserry, Elna P.; Al Kheraif, Abdulaziz A.

    2014-01-01

    Hepatocyte growth factor (HGF) production by oral fibroblasts is enhanced by various molecules that are induced during inflammatory conditions including periodontitis. HGF plays an important role in the progression of periodontitis, by stimulating intense growth of epithelial cells and preventing regeneration of connective tissue attachments. Smokers have a greater risk factor in the pathogenesis and progression of periodontal disease. The objective of the study was to estimate the level of HGF in saliva and gingival crevicular fluid (GCF) in smokers with periodontitis and to compare these levels with that of nonsmokers with periodontitis and healthy controls. The HGF levels were found to be significantly high in the saliva and GCF of smokers with periodontitis compared to both never-smokers with periodontitis and the healthy control group. The elevated levels of HGF in the saliva and GCF in the study population could explain the intrinsic mechanism triggering the severity of the periodontitis in smokers. Further studies are necessary to validate the current observations and to establish a sensitive marker to predict periodontal disease activity. PMID:25389376

  19. Hepatocyte Growth Factor Inhibits Epithelial to Myofibroblast Transition in Lung Cells via Smad7

    PubMed Central

    Shukla, Manasi N.; Rose, Jane L.; Ray, Rabindranath; Lathrop, Kira L.; Ray, Anuradha; Ray, Prabir

    2009-01-01

    Idiopathic pulmonary fibrosis is a lethal parenchymal lung disease characterized by denudation of the lung epithelium, fibroblast proliferation, and collagen deposition. Cellular changes underlying disease progression involve injury to alveolar epithelial cells, epithelial to mesenchymal transition, proliferation of α-smooth muscle actin (α-SMA)–expressing myofibroblasts and of fibroblasts resulting in enhanced deposition of extracellular matrix proteins. Hepatocyte growth factor (HGF) inhibits progression of bleomycin-induced pulmonary fibrosis in mice. The mechanism underlying the inhibitory effect of HGF was investigated in an in vitro model. We show that HGF markedly antagonizes basal and transforming growth factor (TGF)-β–induced expression of myofibroblast markers such as α-SMA, collagen type 1, and fibronectin in rat alveolar epithelial cells. HGF also inhibited TGF-β–induced α-SMA expression in primary murine alveolar epithelial cells. Since TGF-β is known to regulate α-SMA expression, the effect of HGF on components of TGF-β signaling was investigated. HGF induced expression of Smad7, an inhibitor of TGF-β signaling, in a mitogen-activated protein kinase–dependent manner. HGF also induced the nuclear export of Smad7 and Smad ubiquitin regulatory factor 1 (Smurf1) to the cytoplasm. HGF-dependent decrease in α-SMA was abolished with specific siRNAs targeted to Smad7. Thus, induction of Smad7 by HGF serves to limit acquisition of the myofibroblast phenotype in alveolar epithelial cells. PMID:18988920

  20. Reinke's edema: investigations on the role of MIB-1 and hepatocyte growth factor.

    PubMed

    Artico, M; Bronzetti, E; Ionta, B; Bruno, M; Greco, A; Ruoppolo, G; De Virgilio, A; Longo, L; De Vincentiis, M

    2010-01-01

    Reinke's edema is a benign disease of the human vocal fold, which mainly affects the sub-epithelial layer of the vocal fold. Microscopic observations show a strongly oedematous epithelium with loosened intercellular junctions, a disruption of the extracellular connections between mucosal epithelium and connective tissue, closely adherent to the thyroarytenoid muscle. Thickening of the basal layer of epithelium, known as Reinke's space, high deposition of fibronectin and chronic inflammatory infiltration it is also visible. We analyzed, together with the hepatocyte growth factor (HGF), the expression level of MIB-1 in samples harvested from patients affected by Reinke's edema, in order to define its biological role and consider it as a possible prognostic factor in the follow-up after surgical treatment. We observed a moderate expression of HGF in the lamina propria of the human vocal fold and in the basal membrane of the mucosal epithelium. Our finding suggests that this growth factor acts as an antifibrotic agent in Reinke's space and affects the fibronectin deposition in the lamina propria. MIB-1, on the contrary, showed a weak expression in the basement membrane of the mucosal epithelium and a total absence in the lamina propria deep layer, thus suggesting that only the superficial layer is actively involved in the reparatory process with a high regenerative capacity, together with a high deposition of fibronectin. The latter is necessary for the cellular connections reconstruction, after the inflammatory infiltration. PMID:20819770

  1. Reinke's Edema: investigations on the role of MIB-1 and hepatocyte growth factor

    PubMed Central

    Artico, M.; Bronzetti, E.; Ionta, B.; Bruno, M.; Greco, A.; Ruoppolo, G.; De Virgilio, A.; Longo, L.; De Vincentiis, M.

    2010-01-01

    Reinke's edema is a benign disease of the human vocal fold, which mainly affects the sub-epithelial layer of the vocal fold. Microscopic observations show a strongly oedematous epithelium with loosened intercellular junctions, a disruption of the extracellular connections between mucosal epithelium and connective tissue, closely adherent to the thyroarytenoid muscle. Thickening of the basal layer of epithelium, known as Reinke's space, high deposition of fibronectin and chronic inflammatory infiltration it is also visible. We analyzed, together with the hepatocyte growth factor (HGF), the expression level of MIB-1 in samples harvested from patients affected by Reinke's edema, in order to define its biological role and consider it as a possible prognostic factor in the follow-up after surgical treatment. We observed a moderate expression of HGF in the lamina propria of the human vocal fold and in the basal membrane of the mucosal epithelium. Our finding suggests that this growth factor acts as an anti - fibrotic agent in Reinke's space and affects the fibronectin deposition in the lamina propria. MIB-1, on the contrary, showed a weak expression in the basement membrane of the mucosal epithelium and a total absence in the lamina propria deep layer, thus suggesting that only the superficial layer is actively involved in the reparatory process with a high regenerative capacity, together with a high deposition of fibronectin. The latter is necessary for the cellular connections reconstruction, after the inflammatory infiltration. PMID:20819770

  2. Combined Stimulation with the Tumor Necrosis Factor α and the Epidermal Growth Factor Promotes the Proliferation of Hepatocytes in Rat Liver Cultured Slices.

    PubMed

    Finot, Francis; Masson, Régis; Desmots, Fabienne; Ribault, Catherine; Bichet, Nicole; Vericat, Joan A; Lafouge, Patricia; Guguen-Guillouzo, Christiane; Loyer, Pascal

    2012-01-01

    The culture liver slices are mainly used to investigate drug metabolism and xenobiotic-mediated liver injuries while apoptosis and proliferation remain unexplored in this culture model. Here, we show a transient increase in LDH release and caspase activities indicating an ischemic injury during the slicing procedure. Then, caspase activities decrease and remain low in cultured slices demonstrating a low level of apoptosis. The slicing procedure is also associated with the G0/G1 transition of hepatocytes demonstrated by the activation of stress and proliferation signalling pathways including the ERK1/2 and JNK1/2/3 MAPKinases and the transient upregulation of c-fos. The cells further progress up to mid-G1 phase as indicated by the sequential induction of c-myc and p53 mRNA levels after the slicing procedure and at 24 h of culture, respectively. The stimulation by epidermal growth factor induces the ERK1/2 phosphorylation but fails to activate expression of late G1 and S phase markers such as cyclin D1 and Cdk1 indicating that hepatocytes are arrested in mid-G1 phase of the cell cycle. However, we found that combined stimulation by the proinflammatory cytokine tumor necrosis factor α and the epidermal growth factor promotes the commitment to DNA replication as observed in vivo during the liver regeneration. PMID:23119170

  3. Combined Stimulation with the Tumor Necrosis Factor α and the Epidermal Growth Factor Promotes the Proliferation of Hepatocytes in Rat Liver Cultured Slices

    PubMed Central

    Finot, Francis; Masson, Régis; Desmots, Fabienne; Ribault, Catherine; Bichet, Nicole; Vericat, Joan A.; Lafouge, Patricia; Guguen-Guillouzo, Christiane; Loyer, Pascal

    2012-01-01

    The culture liver slices are mainly used to investigate drug metabolism and xenobiotic-mediated liver injuries while apoptosis and proliferation remain unexplored in this culture model. Here, we show a transient increase in LDH release and caspase activities indicating an ischemic injury during the slicing procedure. Then, caspase activities decrease and remain low in cultured slices demonstrating a low level of apoptosis. The slicing procedure is also associated with the G0/G1 transition of hepatocytes demonstrated by the activation of stress and proliferation signalling pathways including the ERK1/2 and JNK1/2/3 MAPKinases and the transient upregulation of c-fos. The cells further progress up to mid-G1 phase as indicated by the sequential induction of c-myc and p53 mRNA levels after the slicing procedure and at 24 h of culture, respectively. The stimulation by epidermal growth factor induces the ERK1/2 phosphorylation but fails to activate expression of late G1 and S phase markers such as cyclin D1 and Cdk1 indicating that hepatocytes are arrested in mid-G1 phase of the cell cycle. However, we found that combined stimulation by the proinflammatory cytokine tumor necrosis factor α and the epidermal growth factor promotes the commitment to DNA replication as observed in vivo during the liver regeneration. PMID:23119170

  4. Hepatocyte-targeting gene transfer mediated by galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative

    PubMed Central

    Wang, Yuqiang; Su, Jing; Cai, Wenwei; Lu, Ping; Yuan, Lifen; Jin, Tuo; Chen, Shuyan; Sheng, Jing

    2013-01-01

    Biscarbamate cross-linked polyethylenimine derivative (PEI-Et) has been reported as a novel nonviral vector for efficient and safe gene transfer in our previous work. However, it had no cell-specificity. To achieve specific delivery of genes to hepatocytes, galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative (GPE) was prepared through modification of PEI-Et with poly(ethylene glycol) and lactobionic acid, bearing a galactose group as a hepatocyte-targeting moiety. The composition of GPE was characterized by proton nuclear magnetic resonance. The weight-average molecular weight of GPE measured with a gel permeation chromatography instrument was 9489 Da, with a polydispersity of 1.44. GPE could effectively condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed that GPE/pDNA complexes were completely formed at weigh ratios (w/w) over 3. The particle size of GPE/pDNA complexes was 79–100 nm and zeta potential was 6–15 mV, values which were appropriate for cellular uptake. The morphology of GPE/pDNA complexes under atomic force microscopy appeared spherical and uniform in size, with diameters of 53–65 nm. GPE displayed much higher transfection efficiency than commercially available PEI 25 kDa in BRL-3A cell lines. Importantly, GPE showed good hepatocyte specificity. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration or weight ratio in BRL-3A cell lines. To sum up, our results indicated that GPE might carry great potential in safe and efficient hepatocyte-targeting gene delivery. PMID:23576866

  5. Hepatocyte-targeting gene transfer mediated by galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative.

    PubMed

    Wang, Yuqiang; Su, Jing; Cai, Wenwei; Lu, Ping; Yuan, Lifen; Jin, Tuo; Chen, Shuyan; Sheng, Jing

    2013-01-01

    Biscarbamate cross-linked polyethylenimine derivative (PEI-Et) has been reported as a novel nonviral vector for efficient and safe gene transfer in our previous work. However, it had no cell-specificity. To achieve specific delivery of genes to hepatocytes, galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative (GPE) was prepared through modification of PEI-Et with poly(ethylene glycol) and lactobionic acid, bearing a galactose group as a hepatocyte-targeting moiety. The composition of GPE was characterized by proton nuclear magnetic resonance. The weight-average molecular weight of GPE measured with a gel permeation chromatography instrument was 9489 Da, with a polydispersity of 1.44. GPE could effectively condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed that GPE/pDNA complexes were completely formed at weigh ratios (w/w) over 3. The particle size of GPE/pDNA complexes was 79-100 nm and zeta potential was 6-15 mV, values which were appropriate for cellular uptake. The morphology of GPE/pDNA complexes under atomic force microscopy appeared spherical and uniform in size, with diameters of 53-65 nm. GPE displayed much higher transfection efficiency than commercially available PEI 25 kDa in BRL-3A cell lines. Importantly, GPE showed good hepatocyte specificity. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration or weight ratio in BRL-3A cell lines. To sum up, our results indicated that GPE might carry great potential in safe and efficient hepatocyte-targeting gene delivery. PMID:23576866

  6. Association of serum hepatocyte growth factor with pericardial fat volume in patients with coronary artery disease

    PubMed Central

    Liu, Jingning; Liu, Zhengxia; Cai, Shikun; Lu, Peng; Lu, Xiang; Peng, Gang

    2015-01-01

    Hepatocyte growth factor (HGF), as a metabolic regulator, was shown to be secreted by adipose tissue and associated with metabolic syndrome (MS) and coronary artery disease (CAD). Pericardial fat, as a visceral fat, was found to be a significant predictor of CAD. We investigated the relationship between serum HGF levels and pericardial fat volume (PFV) in individuals aged between 40-65 years without liver or renal diseases, and also without medicine consumption. Serum HGF levels were found to be significantly higher in participants with CAD than those without CAD (P<0.001). In addition, the serum HGF levels had a significant positive correlation with the PFV in all the participants (r=0.485, P<0.001). Multivariate linear regression demonstrated that the serum HGF levels were significantly associated with PFV (β value=0.454, P<0.001) after adjustment for the metabolic parameters. Further regression assessment found that the serum HGF levels were significantly associated with PFV in participants with CAD (β value=0.586, P<0.001). The serum HGF levels were significant and independent predictors for determining the presence of CAD (OR=1.002, 95% CI: 1.000-1.004, P=0.011). This study therefore demonstrated that the serum HGF levels positively correlated with PFV in participants with CAD and can therefore be a significant predictor for the presence of CAD. PMID:26221348

  7. Hepatocyte Growth Factor Prevents Acute Renal Failure of Accelerates Renal Regeneration in mice

    NASA Astrophysics Data System (ADS)

    Kawaida, Kouichi; Matsumoto, Kunio; Shimazu, Hisaaki; Nakamura, Toshikazu

    1994-05-01

    Although acute renal failure is encountered with administration of nephrotoxic drugs, ischemia, or unilateral nephrectomy, there has been no effective drug which can be used in case of acute renal failure. Hepatocyte growth factor (HGF) is a potent hepatotropic factor for liver regeneration and is known to have mitogenic, motogenic, and morphogenic activities for various epithelial cells, including renal tubular cells. Intravenous injection of recombinant human HGF into mice remarkably suppressed increases in blood urea nitrogen and serum creatinine caused by administration of cisplatin, a widely used antitumor drug, or HgCl_2, thereby indicating that HGF strongly prevented the onset of acute renal dysfunction. Moreover, exogenous HGF stimulated DNA synthesis of renal tubular cells after renal injuries caused by HgCl_2 administration and unilateral nephrectomy and induced reconstruction of the normal renal tissue structure in vivo. Taken together with our previous finding that expression of HGF was rapidly induced after renal injuries, these results allow us to conclude that HGF may be the long-sought renotropic factor for renal regeneration and may prove to be effective treatment for patients with renal dysfunction, especially that caused by cisplatin.

  8. Naked gene therapy of hepatocyte growth factor for dextran sulfate sodium-induced colitis in mice

    SciTech Connect

    Kanbe, Takamasa |; Murai, Rie; Mukoyama, Tomoyuki; Murawaki, Yoshiyuki |; Hashiguchi, Ko-ichi; Yoshida, Yoko; Tsuchiya, Hiroyuki; Kurimasa, Akihiro; Harada, Ken-ichi; Yashima, Kazuo; Nishimuki, Eiji; Shabana, Noriko; Kishimoto, Yukihiro; Kojyo, Haruhiko; Miura, Kunihiko; Kawasaki, Hironaka; Murawaki, Yoshikazu; Shiota, Goshi . E-mail: gshiota@grape.med.tottori-u.ac.jp

    2006-07-14

    Ulcerative colitis (UC) is progressive and relapsing disease. To explore the therapeutic effects of naked gene therapy of hepatocyte growth factor (HGF) on UC, the SR{alpha} promoter driving HGF gene was intrarectally administered to the mice in which colitis was induced by dextran sulfate sodium (DSS). Expression of the transgene was seen in surface epithelium, lamina propria, and muscularis mucosae. The HGF-treated mice showed reduced colonic mucosal damage and increased body weights, compared with control mice (P < 0.01 and P < 0.05, respectively). The HGF-treated mice displayed increased number of PCNA-positive cells and decreased number of apoptotic cells than in control mice (P < 0.01, each). Phosphorylated AKT was dramatically increased after HGF gene administration, however, phosphorylated ERK1/2 was not altered. Microarray analysis revealed that HGF induced expression of proliferation- and apoptosis-associated genes. These data suggest that naked HGF gene delivery causes therapeutic effects through regulation of many downstream genes.

  9. Hepatocyte growth factor can substitute for M-CSF to support osteoclastogenesis

    SciTech Connect

    Adamopoulos, Iannis E. . E-mail: iadamopoulos@path.wustl.edu; Xia Zhidao; Lau, Y.S.; Athanasou, Nicholas A.

    2006-11-17

    Osteopetrotic mice lacking functional macrophage-colony stimulating factor (M-CSF) recover with ageing, suggesting that alternative osteoclastogenesis pathways exist. Hepatocyte growth factor (HGF) and M-CSF signal through tyrosine kinase receptors and phosphorylate common transducers and effectors such as Src, Grb2, and PI3-Kinase. HGF is known to play a role in osteoclast formation, and in this study we have determined whether HGF could replace M-CSF to support human osteoclastogenesis. We found that the HGF receptor, c-Met, is expressed by the CD14{sup +} monocyte fraction of human peripheral blood mononuclear cells (PBMC). HGF was able to support monocyte-osteoclast differentiation in the presence of receptor activator for nuclear factor {kappa}B ligand as evidenced by the formation of numerous multinucleated tartrate-resistant acid phosphatase and vitronectin receptor positive cells which formed F-actin rings and were capable of lacunar resorption. The addition of a neutralising antibody to M-CSF did not inhibit osteoclast differentiation. HGF is a well-established survival factor and viability assays and live/dead staining showed that it promoted the survival and proliferation of monocytes and osteoclasts in a manner similar to M-CSF. Our findings indicate that HGF can substitute for M-CSF to support human osteoclast formation.

  10. Intramyocardial transfer of hepatocyte growth factor as an adjunct to CABG: phase I clinical study.

    PubMed

    Kim, J S; Hwang, H Y; Cho, K R; Park, E-A; Lee, W; Paeng, J C; Lee, D S; Kim, H-K; Sohn, D-W; Kim, K-B

    2013-07-01

    The purpose of this phase I clinical trial was to evaluate the safety, tolerability and potential efficacy of VM202, naked DNA expressing two isoforms of hepatocyte growth factor, as an adjunct therapy to coronary artery bypass grafting (CABG) in patients with ischemic heart disease (IHD). Nine patients were assigned to receive increasing doses (0.5 to 2.0 mg) of VM202 injected into the right coronary artery (RCA) territory following completion of CABG for the left coronary artery territory. Patients were evaluated for safety and tolerability, and changes in myocardial functions were monitored via echocardiography, cardiac magnetic resonance imaging and myocardial single photon emission computed tomography throughout 6-month follow-up period. No serious complication related to VM202 was observed throughout the 6-month follow-up period. Global myocardial functions (wall motion score index, P=0.0084; stress perfusion, P=0.0002) improved during the follow-up period. In the RCA region, there was an increase in the stress perfusion (baseline vs 3-month, P=0.024; baseline vs 6-month, P=0.024) and also in the wall thickness of the diastolic and systolic phases. Intramyocardial injection of VM202 can be safely used in IHD patients with the tolerable dose of 2.0 mg. In addition, VM202 might appear to have improved regional myocardial perfusion and wall thickness in the injected region. PMID:23151518

  11. Live cell imaging shows hepatocyte growth factor-induced Met dimerization.

    PubMed

    Koschut, David; Richert, Ludovic; Pace, Giuseppina; Niemann, Hartmut H; Mély, Yves; Orian-Rousseau, Véronique

    2016-07-01

    The canonical model of receptor tyrosine kinase (RTK) activation assumes that ligand-induced dimerization of inactive receptor monomers is a prerequisite for autophosphorylation. For several RTK families, recent results of fluorescence microscopy provided evidence for preformed receptor dimers that may or may not require ligand binding for kinase activity. Here we report, for the first time, the application of advanced quantitative fluorescence microscopy techniques to study changes in the oligomerization state of the RTK Met in response to stimulation by its endogenous ligand hepatocyte growth factor (HGF). We used inducible C-terminal fusions between Met and enhanced green fluorescent protein (EGFP) or red fluorescent protein (RFP) in combination with fluorescence resonance energy transfer (FRET)-based fluorescence-lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS). A small fraction of HGF-independent Met dimers appeared to be present in cells even at low receptor density. At high receptor density, both the fraction of Met dimers and the level of Met autophosphorylation increased in the absence of HGF. Stimulation with HGF at low receptor density significantly increased the fraction of Met dimers on live cells. We found no indications of Met oligomers larger than dimers. Our findings thus confirm a model of Met activation through HGF-induced dimerization and at the same time they support previous reports of Met dimers in unstimulated cells. The tools established in this work will be useful to further characterize the mechanism of Met activation and to define the contribution of co-receptors. PMID:27094128

  12. Hepatocyte growth factor enhances the barrier function in primary cultures of rat brain microvascular endothelial cells.

    PubMed

    Yamada, Narumi; Nakagawa, Shinsuke; Horai, Shoji; Tanaka, Kunihiko; Deli, Maria A; Yatsuhashi, Hiroshi; Niwa, Masami

    2014-03-01

    The effects of hepatocyte growth factor (HGF) on barrier functions were investigated by a blood-brain barrier (BBB) in vitro model comprising a primary culture of rat brain capillary endothelial cells (RBEC). In order to examine the response of the peripheral endothelial cells to HGF, human umbilical vascular endothelial cells (HUVEC) and human dermal microvascular endothelial cells (HMVEC) were also treated with HGF. HGF decreased the permeability of RBEC to sodium fluorescein and Evans blue albumin, and dose-dependently increased transendothelial electrical resistance (TEER) in RBEC. HGF altered the immunochemical staining pattern of F-actin bands and made ZO-1 staining more distinct on the linear cell borders in RBEC. In contrast, HGF increased sodium fluorescein and Evans blue albumin permeability in HMVEC and HUVEC, and decreased TEER in HMVEC. In HMVEC, HGF reduced cortical actin bands and increased stress fiber density, and increased the zipper-like appearance of ZO-1 staining. Western blot analysis showed that HGF significantly increased the amount of ZO-1 and VE-cadherin. HGF seems to act on the BBB to strengthen BBB integrity. These findings indicated that cytoskeletal rearrangement and cell-cell adhesion, such as through VE-cadherin and ZO-1, are candidate mechanisms for the influence of HGF on the BBB. The possibility that HGF has therapeutic significance in protecting the BBB from damage needs to be considered. PMID:24370951

  13. Hepatocyte growth factor is crucial for development of the carapace in turtles.

    PubMed

    Kawashima-Ohya, Yoshie; Narita, Yuichi; Nagashima, Hiroshi; Usuda, Ryo; Kuratani, Shigeru

    2011-01-01

    Turtles are characterized by their shell, composed of a dorsal carapace and a ventral plastron. The carapace first appears as the turtle-specific carapacial ridge (CR) on the lateral aspect of the embryonic flank. Accompanying the acquisition of the shell, unlike in other amniotes, hypaxial muscles in turtle embryos appear as thin threads of fibrous tissue. To understand carapacial evolution from the perspective of muscle development, we compared the development of the muscle plate, the anlage of hypaxial muscles, between the Chinese soft-shelled turtle, Pelodiscus sinensis, and chicken embryos. We found that the ventrolateral lip (VLL) of the thoracic dermomyotome of P. sinensis delaminates early and produces sparse muscle plate in the lateral body wall. Expression patterns of the regulatory genes for myotome differentiation, such as Myf5, myogenin, Pax3, and Pax7 have been conserved among amniotes, including turtles. However, in P. sinensis embryos, the gene hepatocyte growth factor (HGF), encoding a regulatory factor for delamination of the dermomyotomal VLL, was uniquely expressed in sclerotome and the lateral body wall at the interlimb level. Implantation of COS-7 cells expressing a HGF antagonist into the turtle embryo inhibited CR formation. We conclude that the de novo expression of HGF in the turtle mesoderm would have played an innovative role resulting in the acquisition of the turtle-specific body plan. PMID:21535464

  14. Effect of hepatocyte growth factor encapsulated in targeted liposomes on liver cirrhosis.

    PubMed

    Li, Feng; Sun, Jian-Yong; Wang, Ji-Yao; Du, Shi-Lin; Lu, Wei-Yue; Liu, Min; Xie, Chao; Shi, Jian-Ying

    2008-10-01

    Hepatocyte growth factor (HGF) was encapsulated into sterically stabilized liposomes (SSL) in order to protect it from in vivo degradation. Cyclic Arg-Gly-Asp (RGD) peptides were combined with maleimide-[poly (ethylene glycol)]-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (MAL-PEG-DOPE) incorporated into SSL. The average percentage of HGF encapsulated into liposomes was 32.38%, the size of liposomes was 91.56 nm and the polydispersity index was 0.164. In vivo, histological observation of the rat livers revealed that injection of RGD-SSL-HGF induced more significant remission of liver cirrhosis than injection of SSL-HGF, HGF alone, HGF plus RGD-SSL and saline. When the histological score, the collagen surface density, the hydroxyproline content and the expression of procollagen alpha1 (I) and alpha1 (III) mRNA in the liver were evaluated, all values were smallest in the RGD-SSL-HGF group. In contrast, an increase in apoptotic alpha-SMA-positive cells was noted in the RGD-SSL-HGF group. Together, this data suggests that targeted liposomes encapsulating HGF is a promising therapeutic modality in terms of promoting the remission of liver cirrhosis by promoting collagen fiber digestion, inhibiting collagen production, and promoting apoptosis of alpha-SMA-positive cells in rats with cirrhosis. PMID:18692530

  15. Effects of macroporous hydroxyapatite carriers on the growth and function of human hepatoblasts derived from fetal hepatocytes.

    PubMed

    Ishii, Takaaki; Saito, Hiroshi; Komizu, Yuji; Tomoshige, Ryuichi; Matsushita, Taku

    2016-08-01

    Improvement of three-dimensional (3D) culture conditions, including substrates for cell growth, is needed for various cell-based applications. In this study, we developed hydroxyapatite (HAp) macroporous carriers having several pore size distributions and tried to obtain the findings about the effective pore sizes for the growth and function of hepatoblasts derived from human fetal hepatocytes. Cellular CYP3A4 activity was significantly enhanced when 20% HAp macroporous carrier was used, reaching 1.49±0.28 pmol/10(6) cells/min of benzyloxyresorufin-O-dealkylation activity, which is comparable to that of primary human hepatocytes from livers of adult donors. Analysis of the pore size (the radius of curvature) distribution of each HAp carrier using a 3D-electron beam surface roughness analyzer revealed two peaks of pore size distribution at 30-40 μm and 70-80 μm, respectively. Thirty-five percent of the pores in the 20% carrier had a size distribution within 50-80 μm. Especially, pores of 70-80 μm were more abundant in the 20% HAp carrier than in the 10% and 30% HAp carriers. These results suggested that a HAp carrier with the pore size distribution of 50-80 μm might be effective for cell growth and function in human hepatoblasts derived from fetal hepatocytes. PMID:26968126

  16. Hepatocyte growth factor-modified adipose tissue-derived stem cells improve erectile function in streptozotocin-induced diabetic rats.

    PubMed

    Liu, Tao; Peng, Yifeng; Jia, Chao; Fang, Xiang; Li, Jing; Zhong, Wan

    2015-01-01

    TGFβ1-Smad signaling pathway is closely related to various tissues fibrosis. Hepatocyte growth factor (HGF) has been shown to antagonize TGFβ1-Smad signaling and may improve kidney tissue fibrosis in diabetic models. Penile fibrosis is a pathological condition which occurs during diabetic erectile dysfunction (ED). The aim of this study was to examine the effect of the treatment of ED in diabetic rats with a combination of HGF and adipose tissue-derived stem cells (ADSC). In this diabetes model, rats were injected intraperitoneally with 60 mg streptozotocin (STZ) to induce diabetes. Three months later, the diabetic rats were divided into a negative control(NC) group, an ADSC-treated group and an ADSC + HGF-treated group while normal rats were assigned into a sham group. Rats in the sham and NC groups were injected in the corpus cavernosum with phosphate-buffered saline, while rats in the other groups were injected with either ADSC or ADSC + HGF. One month later, erectile function was examined in each group and penile tissues were collected for experiments. The expression of smooth muscle actin (SMA) and platelet-endothelial cell adhesion molecule-1 (PECAM-1) was analyzed by Western blotting. The smooth muscle and collagen deposition in corpus cavernosum was evaluated by Masson staining, while endothelial changes were assessed immunohistochemically. Cell apoptosis was detected by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. The results revealed that ADSC alone can significantly improve erectile function in diabetic rats, but in combination with HGF the improvement was more prominent, showing higher content of smooth muscle and endothelial cells and lower cell apoptotic index in corpus cavernosum. Treatment with HGF can significantly enhance the beneficial effect of ADSC on erectile function in diabetic rats, and this effect might be closely related to the down-regulation of TGFβ1-Smad signaling. PMID:26339935

  17. Hepatocyte Growth Factor Modulates MET Receptor Tyrosine Kinase and β-Catenin Functional Interactions to Enhance Synapse Formation.

    PubMed

    Xie, Zhihui; Eagleson, Kathie L; Wu, Hsiao-Huei; Levitt, Pat

    2016-01-01

    MET, a pleiotropic receptor tyrosine kinase implicated in autism risk, influences multiple neurodevelopmental processes. There is a knowledge gap, however, in the molecular mechanism through which MET mediates developmental events related to disorder risk. In the neocortex, MET is expressed transiently during periods of peak dendritic outgrowth and synaptogenesis, with expression enriched at developing synapses, consistent with demonstrated roles in dendritic morphogenesis, modulation of spine volume, and excitatory synapse development. In a recent coimmunoprecipitation/mass spectrometry screen, β-catenin was identified as part of the MET interactome in developing neocortical synaptosomes. Here, we investigated the influence of the MET/β-catenin complex in mouse neocortical synaptogenesis. Western blot analysis confirms that MET and β-catenin coimmunoprecipitate, but N-cadherin is not associated with the MET complex. Following stimulation with hepatocyte growth factor (HGF), β-catenin is phosphorylated at tyrosine(142) (Y142) and dissociates from MET, accompanied by an increase in β-catenin/N-cadherin and MET/synapsin 1 protein complexes. In neocortical neurons in vitro, proximity ligation assays confirmed the close proximity of these proteins. Moreover, in neurons transfected with synaptophysin-GFP, HGF stimulation increases the density of synaptophysin/bassoon (a presynaptic marker) and synaptophysin/PSD-95 (a postsynaptic marker) clusters. Mutation of β-catenin at Y142 disrupts the dissociation of the MET/β-catenin complex and prevents the increase in clusters in response to HGF. The data demonstrate a new mechanism for the modulation of synapse formation, whereby MET activation induces an alignment of presynaptic and postsynaptic elements that are necessary for assembly and formation of functional synapses by subsets of neocortical neurons that express MET/β-catenin complex. PMID:27595133

  18. Hepatocyte Growth Factor Modulates MET Receptor Tyrosine Kinase and β-Catenin Functional Interactions to Enhance Synapse Formation

    PubMed Central

    Xie, Zhihui; Eagleson, Kathie L.

    2016-01-01

    MET, a pleiotropic receptor tyrosine kinase implicated in autism risk, influences multiple neurodevelopmental processes. There is a knowledge gap, however, in the molecular mechanism through which MET mediates developmental events related to disorder risk. In the neocortex, MET is expressed transiently during periods of peak dendritic outgrowth and synaptogenesis, with expression enriched at developing synapses, consistent with demonstrated roles in dendritic morphogenesis, modulation of spine volume, and excitatory synapse development. In a recent coimmunoprecipitation/mass spectrometry screen, β-catenin was identified as part of the MET interactome in developing neocortical synaptosomes. Here, we investigated the influence of the MET/β-catenin complex in mouse neocortical synaptogenesis. Western blot analysis confirms that MET and β-catenin coimmunoprecipitate, but N-cadherin is not associated with the MET complex. Following stimulation with hepatocyte growth factor (HGF), β-catenin is phosphorylated at tyrosine142 (Y142) and dissociates from MET, accompanied by an increase in β-catenin/N-cadherin and MET/synapsin 1 protein complexes. In neocortical neurons in vitro, proximity ligation assays confirmed the close proximity of these proteins. Moreover, in neurons transfected with synaptophysin-GFP, HGF stimulation increases the density of synaptophysin/bassoon (a presynaptic marker) and synaptophysin/PSD-95 (a postsynaptic marker) clusters. Mutation of β-catenin at Y142 disrupts the dissociation of the MET/β-catenin complex and prevents the increase in clusters in response to HGF. The data demonstrate a new mechanism for the modulation of synapse formation, whereby MET activation induces an alignment of presynaptic and postsynaptic elements that are necessary for assembly and formation of functional synapses by subsets of neocortical neurons that express MET/β-catenin complex. PMID:27595133

  19. Zonal differences in ethanol-induced impairments in receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J. )

    1991-02-01

    We have shown previously that ethanol-induced defects in receptor-mediated endocytosis of asialoorosomucoid occurred as early as 1 wk after ethanol feeding. This study was undertaken as an initial attempt to establish a possible role of defective receptor-mediated endocytosis in liver injury by investigating whether differences exist in the effects of ethanol on receptor-mediated endocytosis in hepatocytes isolated from different regions of the liver. Perivenule cells, present in the distal half of the liver, are thought to be more susceptible to ethanol-induced liver injury than are the periportal cells located in the proximal half of the liver acini. For these studies, we fed male Sprague-Dawley rats for 7 days with liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate. Perivenule and periportal hepatocytes were then isolated using a digitonin-collagenase perfusion method. In control animals, cells isolated from the perivenule region bound significantly more ligand than did cells from the periportal region. Amounts of ligand internalized and degraded were also greater in perivenule than in periportal cells in these animals. After ethanol feeding, cells isolated from both the perivenule and periportal regions bound significantly less ligand than their respective controls. This impairment in surface and total binding was more pronounced in perivenule than in periportal cells. Internalization and degradation of the ligand were also more adversely affected in the centrilobular region as shown by decreases of greater than 60% in perivenule cells and by only 20% to 30% in periportal cells of ethanol-fed animals compared with controls.

  20. The Acute Transcriptomic and Proteomic Response of HC-04 Hepatoma Cells to Hepatocyte Growth Factor and its Implications for Plasmodium falciparum Sporozoite Invasion*

    PubMed Central

    Tao, Dingyin; King, Jonas G.; Tweedell, Rebecca E.; Jost, Philipp J.; Boddey, Justin A.; Dinglasan, Rhoel R.

    2014-01-01

    The routine study of human malaria liver-stage biology in vitro is hampered by low infection efficiency of human hepatocellular carcinoma (HCC) lines (<0.1%), poor understanding of steady-state HCC biology, and lack of appropriate tools for trace sample analysis. HC-04 is the only HCC that supports complete development of human malaria parasites. We hypothesized that HCCs are in various intermediate stages of the epithelial-mesenchymal transition (EMT) and HC-04s retain epithelial characteristics that permit infection. We developed a facile analytical approach to test this hypothesis viz. the HC-04 response to hepatocyte growth factor (HGF). We used online two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) to quantify protein expression profiles in HC-04 pre-/post-HGF treatment and validated these results by RT-qPCR and microscopy. We successfully increased protein identification efficiency over offline-2D methods by 12-fold, using less sample material, allowing robust protein quantification. We observed expected up-regulation and down-regulation of EMT protein markers in response to HGF, but also unexpected cellular responses. We also observed that HC-04 is generally more susceptible to HGF-mediated signaling than what was observed for HepG2, a widely used, but poor malaria liver stage-HCC model. Our analytical approach to understanding the basic biology of HC-04 helps us understand the factors that may influence its utility as a model for malaria liver-stage development. We observed that HC-04 treatment with HGF prior to the addition of Plasmodium falciparum sporozoites did not facilitate cell invasion, which suggests unlinking the effect of HGF on malaria liver stage development from hepatocyte invasion. Finally, our 2D-LC-MS/MS approach and broadly applicable experimental strategy should prove useful in the analysis of various hepatocyte-pathogen interactions, tumor progression, and early disease events. PMID:24532842

  1. Involvement of hepatocyte growth factor-induced epithelial-mesenchymal transition in human adenomyosis.

    PubMed

    Khan, Khaleque Newaz; Kitajima, Michio; Hiraki, Koichi; Fujishita, Akira; Nakashima, Masahiro; Masuzaki, Hideaki

    2015-02-01

    Adenomyosis is commonly believed to arise from the basalis endometrium. As an estromedin growth factor, hepatocyte growth factor (HGF) exhibits multiple functions in endometriosis, a disease commonly believed to arise from the functionalis endometrium. Here, we investigated the role of HGF in the occurrence of epithelial-mesenchymal transition (EMT) in adenomyosis. Full-thickness-biopsy specimens from endometrium to myometrium were collected after hysterectomy from women with and without adenomyosis. The relationship between HGF and E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell markers) was examined at the gene and protein levels using endometrial epithelial cells (EECs) in culture and tissues by quantitative RT-PCR and immunohistochemistry. The gene and protein expressions of two transcriptional repressors of E-cadherin, SLUG and SNAIL, were examined using Ishikawa cells and in response to HGF and estrogen (E2). HGF down-regulated E-cadherin and up-regulated N-cadherin mRNA expression in EECs, and an inverse relationship in protein expression between HGF and E-cadherin was observed in basalis endometria derived from women with diffuse and focal adenomyosis. HGF induced morphological changes of EECs from a cobblestone-like appearance to spindle-shaped cells and promoted migration of EECs. Ishikawa cells exhibited up-regulation of SLUG/SNAIL gene expression in response to both HGF and E2 with an additive effect between them. HGF- and E2-promoted SLUG/SNAIL gene expression was significantly abrogated after pretreatment of cells with anti-HGF antibody or ICI 182720, an estrogen receptor antagonist. HGF may be involved in gland invagination deep into the myometrium by inducing EMT at the endo-myometrial junction in women with adenomyosis. PMID:25505196

  2. Differential effects of vasopressin and phenylephrine on protein kinase C-mediated protein phosphorylations in isolated hepatocytes

    SciTech Connect

    Cooper, R.H.; Johanson, R.A.; Wiliamson, J.R.

    1986-05-01

    Receptor-mediated breakdown of inositol lipids produces two intracellular signals, diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which causes release of intracellular vesicular Ca/sup 2 +/. This study examined the effects of Ca/sup 2 +/-ionophores, vasopressin, phenylephrine, and phorbol ester (PMA) on hepatocyte protein phosphorylations. (/sup 32/P) Phosphoproteins from hepatocytes prelabeled with /sup 32/P were resolved by 2-dimensional SDS-PAGE and corresponding autoradiographs were quantitated by densitometric analysis. The phosphorylation of five proteins, a plasma membrane bound 16 kDa protein with pI 6.4, a cytosolic 16 kDa protein with pI 5.8, and proteins with Mr's of 36 kDa, 52 kDa, and 68 kDa, could be attributed to phosphorylation by protein kinase C since the phosphorylation was stimulated by PMA. When the vasopressin concentration was varied, low vasopressin stimulated the phosphorylation of only the membrane bound 16 kDa protein of the above set of proteins, while higher vasopressin concentrations were required to stimulate the phosphorylation of all five proteins. Phenylephrine, even at supramaximal concentrations, stimulated the phosphorylation of only the membrane bound 16 kDa protein. These results suggest that phenylephrine is a less potent activator of protein kinase C than vasopressin by virtue of limited or localized diacylglycerol production.

  3. Xanthine oxidase-derived reactive oxygen species mediate 4-oxo-2-nonenal-induced hepatocyte cell death

    SciTech Connect

    Sakuma, Satoru Negoro, Miki; Kitamura, Takahiro; Fujimoto, Yohko

    2010-12-01

    Among the aldehydes derived from lipid peroxidation, there have been several reports concerning the toxicity of 4-hydroxy-2-nonenal (4-HNE), whereas little information is available about 4-oxo-2-nonenal (4-ONE). In the present study, we examined the effects of 4-HNE and 4-ONE on the cell viability of primary rat hepatocyte cultures. At concentrations of 5, 10, and 20 {mu}M, 4-HNE had no significant effect on the cell viability of primary rat hepatocytes cultures, whereas 4-ONE potently decreased the cell viability in a dose-dependent manner (5-20 {mu}M, 23-69% inhibition). The TUNEL assay showed that 4-ONE causes apoptosis in the cells. 4-ONE also increased 2',7'-dichlorofluorescein-fluorescence intensity from 2',7'-dichlorodihydrofluorescein, an indicator of reactive oxygen species (ROS) generation. Allopurinol, a xanthine oxidase (XO) inhibitor, diminished the 4-ONE-induced increase in the 2',7'-dichlorofluorescein-fluorescence intensity and the decrease in viability, indicating the role of XO in mediating 4-ONE-induced cell death. These observations suggest that 4-ONE has the potential to induce liver cell death via XO-derived ROS generation.

  4. Hepatocyte FRS2α is essential for the endocrine fibroblast growth factor to limit the amplitude of bile acid production induced by prandial activity.

    PubMed

    Wang, C; Yang, C; Chang, J Y F; You, P; Li, Y; Jin, C; Luo, Y; Li, X; McKeehan, W L; Wang, F

    2014-01-01

    In addition to being positively regulated by prandial activity, bile acid production is also negatively controlled by the endocrine fibroblast growth factor 19 (FGF19) or the mouse ortholog FGF15 from the ileum that represses hepatic cholesterol 7 α-hydroxylase (Cyp7a1) expression through activating FGF receptor four (FGFR4). However, how these two regulatory mechanisms interplay to control bile acid homeostasis in the body and the downstream pathways by which FGFR4 regulates Cyp7a1 expression are not fully understood. Here we report that hepatocyte FGFR substrate 2α (FRS2α), a scaffold protein essential for canonical FGFRs to activate the ERK and AKT pathways, was required for the regulation of bile acid production by the FGF15/19-FGFR4 signaling axis. This occurred through limiting the extent of increases in Cyp7a1 expression induced by prandial activity. Excess FGFR4 kinase activity reduced the amplitude of the increase whereas a lack of FGFR4 augmented the increase of Cyp7a1 expression in the liver. Ablation of Frs2α alleles in hepatocytes abrogated the regulation of Cyp7a1 expression by FGFR4. Together, the results demonstrate that FRS2α-mediated pathways are essential for the FGF15/FGF19-FGFR4 signaling axis to control bile acid homeostasis. PMID:25056539

  5. Serum levels of hepatocyte growth factor as a potential tumor marker in patients with malignant melanoma.

    PubMed

    Hügel, Rainer; Muendlein, Axel; Volbeding, Lennart; Drexel, Heinz; Richtig, Erika; Wehkamp, Ulrike; Painsi, Clemes; Lange-Asschenfeldt, Bernhard; Hauschild, Axel; Egberts, Friederike

    2016-08-01

    Serum markers can be important tools for prognostic classification and treatment monitoring in cancer patients. The MAP-kinase pathway, which is upregulated in the majority of melanoma patients, can be activated by hepatocyte-growth factor (HGF) through the proto-oncogene c-MET. The aim of this study was to evaluate the predictive and prognostic value of circulating HGF in terms of treatment outcome and survival compared with a widely established serum marker, protein S-100B, in patients with advanced metastatic melanoma. HGF and S-100B were measured in serum samples of 101 patients with metastatic melanoma (American Joint Committee on Cancer stage IV) before and after treatment and 50 patients with stage I/II melanoma. HGF and S-100B correlated significantly with the stage of disease (P=0.032 and P<0.001, respectively). In stage IV melanoma patients, baseline serum levels of HGF and S-100B were significantly associated with treatment response (P=0.012 and 0.006, respectively). Furthermore, the Cox regression analysis confirmed that serum levels of HGF and S-100B proved to have a significant prognostic impact on progression-free survival (hazard ratio=1.39 and 1.29, respectively) and overall survival (hazard ratio=1.27 and 1.29, respectively) in advanced metastatic melanoma patients. In melanoma patients, serum levels of HGF and S-100B correlate significantly with the stage of disease. In stage IV melanoma, both markers are prognostic factors and correlate significantly with progression-free survival and overall survival. Measurement of serum HGF levels might be a useful additional tool in the management of melanoma patients. PMID:27206057

  6. Hepatocyte growth factor sensitizes brain tumors to c-MET kinase inhibition

    PubMed Central

    Zhang, Ying; Farenholtz, Kaitlyn E.; Yang, Yanzhi; Guessous, Fadila; diPierro, Charles G.; Calvert, Valerie S.; Deng, Jianghong; Schiff, David; Xin, Wenjun; Lee, Jae K.; Purow, Benjamin; Christensen, James; Petricoin, Emanuel; Abounader, Roger

    2013-01-01

    Purpose The receptor tyrosine kinase (RTK) c-MET and its ligand hepatocyte growth factor (HGF) are deregulated and promote malignancy in cancer and brain tumors. Consequently, clinically applicable c-MET inhibitors have been developed. The purpose of this study was to investigate the not well known molecular determinants that predict responsiveness to c-MET inhibitors, and to explore new strategies for improving inhibitor efficacy in brain tumors. Experimental design We investigated the molecular factors and pathway activation signatures that determine sensitivity to c-MET inhibitors in a panel of glioblastoma and medulloblastoma cells, glioblastoma stem cells (GSCs), and established cell line-derived xenografts using functional assays, reverse protein microarrays, and in vivo tumor volume measurements, but validation with animal survival analyses remains to be done. We also explored new approaches for improving the efficacy of the inhibitors in vitro and in vivo. Results We found that HGF co-expression is a key predictor of response to c-MET inhibition among the examined factors, and identified an ERK/JAK/p53 pathway activation signature that differentiates c-MET inhibition in responsive and non-responsive cells. Surprisingly, we also found that short pre-treatment of cells and tumors with exogenous HGF moderately but statistically significantly enhanced the anti-tumor effects of c-MET inhibition. We observed a similar ligand-induced sensitization effect to an EGFR small molecule kinase inhibitor. Conclusions These findings allow the identification of a subset of patients that will be responsive to c-MET inhibition, and propose ligand pre-treatment as a potential new strategy for improving the anti-cancer efficacy of RTK inhibitors. PMID:23386689

  7. Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes.

    PubMed Central

    Crljen, Vladiana; Volinia, Stefano; Banfic, Hrvoje

    2002-01-01

    Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH(3) and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2 beta activity, which is sensitive to wortmannin (10 nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices with HGF and could be mimicked by the Ca(2+) ionophore A23187 and blocked by the cell-penetrant Ca(2+) chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2 beta revealed a single immunoreactive band of 180 kDa in BLM and BBM, while after stimulation with HGF a gel shift of 18 kDa was noticed only in BBM, suggesting that the observed enzyme activation is achieved by proteolysis. When BBM were subjected to short-term (15 min) exposure to mu-calpain, a similar gel shift together with an increase in PI3K-C2 beta activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2 beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that in renal cells there is a spatial separation of the inositol lipid signalling system between BLM and BBM, and that HGF causes activation of PLC and

  8. Burkholderia pseudomallei rpoS mediates iNOS suppression in human hepatocyte (HC04) cells

    PubMed Central

    Sanongkiet, Sucharat; Ponnikorn, Saranyoo; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2016-01-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen and the causative agent of melioidosis, a widespread disease in Southeast Asia. Reactive nitrogen, in an intermediate form of nitric oxide (NO), is one of the first lines of defense used by host cells to eliminate intracellular pathogens, through the stimulation of inducible nitric oxide synthase (iNOS). Studies in phagocytotic cells have shown that the iNOS response is muted in B. pseudomallei infection, and implicated the rpoS sigma factor as a key regulatory factor mediating suppression. The liver is a main visceral organ affected by B. pseudomallei, and there is little knowledge about the interaction of liver cells and B. pseudomallei. This study investigated the induction of iNOS, as well as autophagic flux and light-chain 3 (LC3) localization in human liver (HC04) cells in response to infection with B. pseudomallei and its rpoS deficient mutant. Results showed that the rpoS mutant was unable to suppress iNOS induction and that the mutant showed less induction of autophagy and lower co-localization with LC3, and this was coupled with a lower intracellular growth rate. Combining these results suggest that B. pseudomallei rpoS is an important factor in establishing infection in liver cells. PMID:27324398

  9. Burkholderia pseudomallei rpoS mediates iNOS suppression in human hepatocyte (HC04) cells.

    PubMed

    Sanongkiet, Sucharat; Ponnikorn, Saranyoo; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2016-08-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen and the causative agent of melioidosis, a widespread disease in Southeast Asia. Reactive nitrogen, in an intermediate form of nitric oxide (NO), is one of the first lines of defense used by host cells to eliminate intracellular pathogens, through the stimulation of inducible nitric oxide synthase (iNOS). Studies in phagocytotic cells have shown that the iNOS response is muted in B. pseudomallei infection, and implicated the rpoS sigma factor as a key regulatory factor mediating suppression. The liver is a main visceral organ affected by B. pseudomallei, and there is little knowledge about the interaction of liver cells and B. pseudomallei This study investigated the induction of iNOS, as well as autophagic flux and light-chain 3 (LC3) localization in human liver (HC04) cells in response to infection with B. pseudomallei and its rpoS deficient mutant. Results showed that the rpoS mutant was unable to suppress iNOS induction and that the mutant showed less induction of autophagy and lower co-localization with LC3, and this was coupled with a lower intracellular growth rate. Combining these results suggest that B. pseudomallei rpoS is an important factor in establishing infection in liver cells. PMID:27324398

  10. Cytotoxicity of luteolin in primary rat hepatocytes: the role of CYP3A-mediated ortho-benzoquinone metabolite formation and glutathione depletion.

    PubMed

    Shi, Fuguo; Zhao, Peng; Li, Xiaobing; Pan, Hong; Ma, Shiping; Ding, Li

    2015-11-01

    Luteolin (LUT), an active ingredient in traditional Chinese medicines and an integral part of the human diet, has shown promising pharmacological activities with a great potential for clinical use. The purpose of this study was to evaluate the role of cytochrome P450 (CYP450)-mediated reactive ortho-benzoquinone metabolites formation and glutathione (GSH) depletion in LUT-induced cytotoxicity in primary rat hepatocytes. A reactive ortho-benzoquinone metabolite was identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in rat liver microsomes (RLMs) and rat hepatocytes. Using a specific chemical inhibitor method, the CYP3A subfamily was found to be responsible for the reactive metabolite formation in RLMs. Induction of CYP3A by dexamethasone enhanced LUT-induced cytotoxicity, whereas inhibition of CYP3A by ketoconazole (Keto) decreased the cytotoxicity. The cytotoxicity and cell apoptosis induced by LUT were related to the amount of reactive metabolite formation. Furthermore, Keto inhibited the LUT-induced GSH exhaustion. The cytotoxicity was significantly enhanced by pretreatment with L-buthionine sulfoximine to deplete the intracellular GSH. A time course experiment showed that GSH depletion by LUT was not via oxidation of GSH and occurred prior to the increase in 2', 7'-dichlorofluorescein in hepatocytes. Collectively, these data suggest that CYP3A-mediated reactive metabolite formation plays a critical role in LUT-induced hepatotoxicity, and the direct GSH depletion is an initiating event in LUT-mediated cytotoxicity in primary rat hepatocytes. PMID:25612170

  11. Hepatocyte growth factor in vitreous and serum from patients with proliferative diabetic retinopathy

    PubMed Central

    Canton, A.; Burgos, R.; Hernandez, C.; Mateo, C.; Segura, R.; Mesa, J.; Simo, R.

    2000-01-01

    BACKGROUND—Hepatocyte growth factor (HGF) is an endothelium specific growth factor that has been implicated in angiogenesis, a crucial event for the development of proliferative diabetic retinopathy (PDR). The aim of the study is to determine the intravitreous concentrations of HGF in diabetic patients with PDR, and to investigate whether its serum levels could contribute to its intravitreous concentration.
METHODS—17 diabetic patients and seven non-diabetic patients in whom a vitrectomy was performed were studied. Both groups were matched by serum levels of HGF. Venous blood and vitreous samples were collected simultaneously at the time of vitreoretinal surgery. Vitreous and serum HGF were determined by ELISA.
RESULTS—Intravitreous concentrations of HGF (median and range) were higher in diabetic patients (17.04 ng/ml (9.98-80)) in comparison with non-diabetic patients (5.88 ng/ml (2.57-14.20); p=0.003). Intravitreous HGF concentrations were strikingly higher than serum HGF concentrations both in diabetic patients (17.04 ng/ml (9.98-80) v 0.66 ng/ml (0.26-1.26); p<0.001) and in the control group (5.88 ng/ml (2.57-14.20) v 0.68 ng/ml (0.49-0.96); p=0.003). No correlation was found between serum and vitreous levels of HGF in both groups (diabetic patients, r= −0.31; p=0.5 and control subjects r= −0.15; p=0.5).
CONCLUSION—The high vitreous levels of HGF observed in diabetic patients with PDR cannot be attributed to serum diffusion across the blood-retinal barrier. Therefore, intraocular synthesis appears to be the main contributing factor for the high vitreous HGF concentrations in diabetic patients, a cytokine that seems to be directly involved in the pathogenesis of PDR.

 PMID:10873984

  12. Mechanism of a transcriptional cross talk between transforming growth factor-beta-regulated Smad3 and Smad4 proteins and orphan nuclear receptor hepatocyte nuclear factor-4.

    PubMed

    Chou, Wan-Chih; Prokova, Vassiliki; Shiraishi, Keiko; Valcourt, Ulrich; Moustakas, Aristidis; Hadzopoulou-Cladaras, Margarita; Zannis, Vassilis I; Kardassis, Dimitris

    2003-03-01

    We have shown previously that the transforming growth factor-beta (TGFbeta)-regulated Sma-Mad (Smad) protein 3 and Smad4 proteins transactivate the apolipoprotein C-III promoter in hepatic cells via a hormone response element that binds the nuclear receptor hepatocyte nuclear factor 4 (HNF-4). In the present study, we show that Smad3 and Smad4 but not Smad2 physically interact with HNF-4 via their Mad homology 1 domains both in vitro and in vivo. The synergistic transactivation of target promoters by Smads and HNF-4 was shown to depend on the specific promoter context and did not require an intact beta-hairpin/DNA binding domain of the Smads. Using glutathione S-transferase interaction assays, we established that two regions of HNF-4, the N-terminal activation function 1 (AF-1) domain (aa 1-24) and the C-terminal F domain (aa 388-455) can mediate physical Smad3/HNF-4 interactions in vitro. In vivo, Smad3 and Smad4 proteins enhanced the transactivation function of various GAL4-HNF-4 fusion proteins via the AF-1 and the adjacent DNA binding domain, whereas a single tyrosine to alanine substitution in AF-1 abolished coactivation by Smads. The findings suggest that the transcriptional cross talk between the TGFbeta-regulated Smads and HNF-4 is mediated by specific functional domains in the two types of transcription factors. Furthermore, the specificity of this interaction for certain target promoters may play an important role in various hepatocyte functions, which are regulated by TGFbeta and the Smads. PMID:12631740

  13. The Molecular Chaperone GRP78 Contributes to Toll-like Receptor 3-mediated Innate Immune Response to Hepatitis C Virus in Hepatocytes.

    PubMed

    Wei, Dahai; Li, Nan L; Zeng, Yanli; Liu, Baoming; Kumthip, Kattareeya; Wang, Tony T; Huo, Dezheng; Ingels, Jesse F; Lu, Lu; Shang, Jia; Li, Kui

    2016-06-01

    Toll-like receptor-3 (TLR3) senses double-stranded RNA intermediates produced during hepatitis C virus (HCV) replication, leading to activation of interferon regulatory factor-3 (IRF3) and NF-κB and subsequent antiviral and proinflammatory responses. Yet, how this TLR3-dependent pathway operates in hepatocytes is unclear. Upon fractionating cultured hepatocytes into various cellular organelles, we observed that TLR3 predominantly resides in endolysosomes of hepatocytes. To determine the critical regulators of TLR3 signaling in response to HCV infection in human hepatocytes, we isolated endolysosome fractions from mock-infected and HCV-infected hepatoma Huh7.5 cells that had been reconstituted for TLR3 expression, separated these fractions on two-dimensional gels, and identified up-regulated/down-regulated proteins by mass spectrometry. Approximately a dozen of cellular proteins were found to be differentially expressed in endolysosome fractions following HCV infection. Of these, expression of several molecular chaperone proteins was elevated. Knockdown of one of these chaperones, glucose-regulated protein 78 kDa (GRP78), compromised TLR3-dependent induction of interferon-stimulated genes and chemokines following HCV infection or poly(I:C) stimulation in cultured hepatocytes. Consistent with this finding, GRP78 depletion impaired TLR3-mediated establishment of an antiviral state. Mechanistically, although TLR3 trafficking to endolysosomes was not affected, phosphorylated IRF3 diminished faster following GRP78 knockdown. Remarkably, GRP78 transcript was significantly up-regulated in liver biopsies of chronic hepatitis C patients as compared with normal liver tissues. Moreover, the GRP78 expression level correlated with that of RANTES (regulated upon activation, normal T-cell expressed and secreted) and CXCL10, two inflammatory chemokines most frequently elevated in HCV-infected liver. Altogether, our data suggest that GRP78 contributes to TLR3-mediated, IRF3

  14. Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition

    PubMed Central

    Ordovás, Laura; Boon, Ruben; Pistoni, Mariaelena; Chen, Yemiao; Wolfs, Esther; Guo, Wenting; Sambathkumar, Rangarajan; Bobis-Wozowicz, Sylwia; Helsen, Nicky; Vanhove, Jolien; Berckmans, Pieter; Cai, Qing; Vanuytsel, Kim; Eggermont, Kristel; Vanslembrouck, Veerle; Schmidt, Béla Z.; Raitano, Susanna; Van Den Bosch, Ludo; Nahmias, Yaakov; Cathomen, Toni; Struys, Tom; Verfaillie, Catherine M.

    2015-01-01

    Summary Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes. PMID:26455413

  15. Novel Role for γ-Catenin in the Regulation of Cancer Cell Migration via the Induction of Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1)*

    PubMed Central

    Sechler, Marybeth; Borowicz, Stanley; Van Scoyk, Michelle; Avasarala, Sreedevi; Zerayesus, Sereke; Edwards, Michael G.; Kumar Karuppusamy Rathinam, Manoj; Zhao, Xiangmin; Wu, Pei-Ying; Tang, Ke; Bikkavilli, Rama Kamesh; Winn, Robert A.

    2015-01-01

    γ-catenin (Plakoglobin), a well-described structural protein functioning at the adherens junctions and desmosomes, was shown to be either lost or weakly expressed in non-small cell lung cancer (NSCLC) cells and tumor tissues. However, the tumor suppressive affects of γ-catenin were not fully understood. In this study, we have identified a novel role for the affects of γ-catenin on non-small cell lung cancer (NSCLC) cell migration. Expression of γ-catenin in NSCLC cells resulted in reduced cell migration as determined by both scratch assays and trans-well cell migration assays. Moreover, the affects of γ-catenin on cell migration were observed to be p53-dependent. Mechanistically, the anti-migratory effects seen via γ-catenin were driven by the expression of hepatocyte growth factor activator inhibitor Type I (HAI-1 or SPINT-1), an upstream inhibitor of the c-MET signaling pathway. Furthermore, the re-expression of γ-catenin sensitized NSCLC cells to c-MET inhibitor-mediated growth inhibition. Taken together, we identify γ-catenin as a novel regulator of HAI-1, which is a critical regulator of HGF/c-MET signaling. Therefore, targeting γ-catenin-mediated HAI-1 expression might be a useful strategy to sensitize NSCLC to c-MET inhibitors. PMID:25925948

  16. Keratin impact on PKCδ- and ASMase-mediated regulation of hepatocyte lipid raft size - implication for FasR-associated apoptosis.

    PubMed

    Gilbert, Stéphane; Loranger, Anne; Omary, M Bishr; Marceau, Normand

    2016-09-01

    Keratins are epithelial cell intermediate filament (IF) proteins that are expressed as pairs in a cell-differentiation-regulated manner. Hepatocytes express the keratin 8 and 18 pair (denoted K8/K18) of IFs, and a loss of K8 or K18, as in K8-null mice, leads to degradation of the keratin partner. We have previously reported that a K8/K18 loss in hepatocytes leads to altered cell surface lipid raft distribution and more efficient Fas receptor (FasR, also known as TNFRSF6)-mediated apoptosis. We demonstrate here that the absence of K8 or transgenic expression of the K8 G62C mutant in mouse hepatocytes reduces lipid raft size. Mechanistically, we find that the lipid raft size is dependent on acid sphingomyelinase (ASMase, also known as SMPD1) enzyme activity, which is reduced in absence of K8/K18. Notably, the reduction of ASMase activity appears to be caused by a less efficient redistribution of surface membrane PKCδ toward lysosomes. Moreover, we delineate the lipid raft volume range that is required for an optimal FasR-mediated apoptosis. Hence, K8/K18-dependent PKCδ- and ASMase-mediated modulation of lipid raft size can explain the more prominent FasR-mediated signaling resulting from K8/K18 loss. The fine-tuning of ASMase-mediated regulation of lipid rafts might provide a therapeutic target for death-receptor-related liver diseases. PMID:27422101

  17. Elongation Factor 1A-1 Is a Mediator of Hepatocyte Lipotoxicity Partly through Its Canonical Function in Protein Synthesis

    PubMed Central

    Stoianov, Alexandra M.; Robson, Debra L.; Hetherington, Alexandra M.; Sawyez, Cynthia G.; Borradaile, Nica M.

    2015-01-01

    Elongation factor 1A-1 (eEF1A-1) has non-canonical functions in regulation of the actin cytoskeleton and apoptosis. It was previously identified through a promoter-trap screen as a mediator of fatty acid-induced cell death (lipotoxicity), and was found to participate in this process downstream of ER stress. Since ER stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), we investigated the mechanism of action of eEF1A-1 in hepatocyte lipotoxicity. HepG2 cells were exposed to excess fatty acids, followed by assessments of ER stress, subcellular localization of eEF1A-1, and cell death. A specific inhibitor of eEF1A-1 elongation activity, didemnin B, was used to determine whether its function in protein synthesis is involved in lipotoxicity. Within 6 h, eEF1A-1 protein was modestly induced by high palmitate, and partially re-localized from its predominant location at the ER to polymerized actin at the cell periphery. This early induction and subcellular redistribution of eEF1A-1 coincided with the onset of ER stress, and was later followed by cell death. Didemnin B did not prevent the initiation of ER stress by high palmitate, as indicated by eIF2α phosphorylation. However, consistent with sustained inhibition of eEF1A-1-dependent elongation activity, didemnin B prevented the recovery of protein synthesis and increase in GRP78 protein that are normally associated with later phases of the response to ongoing ER stress. This resulted in decreased palmitate-induced cell death. Our data implicate eEF1A-1, and its function in protein synthesis, in hepatocyte lipotoxicity. PMID:26102086

  18. Lipid modulatory activities of Cichorium glandulosum Boiss et Huet are mediated by multiple components within hepatocytes.

    PubMed

    Ding, Lin; Liu, Jun-Lin; Hassan, Waseem; Wang, Lu-Lu; Yan, Fang-Rong; Shang, Jing

    2014-01-01

    To investigate a possible methodology of exploiting herbal medicine and design polytherapy for the treatment of non-alcoholic fatty liver disease (NAFLD), we have made use of Cichorium glandulosum Boiss et Huet (CG), a traditional Chinese herbal medicine that has been proven to be effective in treating hepatic diseases. Here, we report that the extract of CG effectively reduced lipid accumulation under conditions of lipid overloading in vivo and in vitro (in a rat high-fat diet model and a hepG2 cell model of free fatty acid treatment). CG extract also protected hepatocytes from injury and inflammation to aid its lipid-lowering properties (in a rat high-fat diet model and a L02 cell model of acetaminophen treatment). Serum chemistry analysis accompanied by in vitro drug screening confirmed that CG-4, CG-10 and CG-14 are the lipo-effective components of CG. Western blotting analysis revealed that these components can regulate key lipid targets at the molecular level, including CD36, FATP5 and PPAR-α, thus the lipid oxidation and lipid absorption pathways. Finally, we adopted the experimental design and statistical method to calculate the best combination proportion (CG-4: CG-10: CG-14 = 2.065: 1.782: 2.153) to optimize its therapeutic effect. PMID:24797163

  19. Targeted Disruption of Heparan Sulfate Interaction with Hepatocyte and Vascular Endothelial Growth Factors Blocks Normal and Oncogenic Signaling

    PubMed Central

    Cecchi, Fabiola; Pajalunga, Deborah; Fowler, C. Andrew; Uren, Aykut; Rabe, Daniel C.; Peruzzi, Benedetta; MacDonald, Nicholas J.; Blackman, Davida K.; Stahl, Stephen J.; Byrd, R. Andrew; Bottaro, Donald P.

    2012-01-01

    Summary Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) regulate normal development and homeostasis, and drive disease progression in many forms of cancer. Both proteins signal by binding to receptor tyrosine kinases and heparan sulfate (HS) proteoglycans on target cell surfaces. Basic residues comprising the primary HS binding sites on HGF and VEGF provide similar surface charge distributions without underlying structural similarity. Combining three acidic amino acid substitutions in these sites in the HGF isoform NK1 or the VEGF isoform VEGF165 transformed each into potent, selective competitive antagonists of their respective normal and oncogenic signaling pathways. Our findings illustrate the importance of HS in growth factor driven cancer progression and reveal an efficient strategy for therapeutic antagonist development. PMID:22897854

  20. Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes

    SciTech Connect

    Latchoumycandane, Calivarathan; Seah, Quee Ming; Tan, Rachel C.H.; Sattabongkot, Jetsumon; Beerheide, Walter; Boelsterli, Urs A. . E-mail: phcbua@nus.edu.sg

    2006-11-15

    Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK signaling pathway. To this end, we exposed cultured immortalized human hepatocytes (HC-04) to the standard protoxicant drug acetaminophen (APAP), which induces CsA-sensitive mPT-mediated cell death. We determined the effects of leflunomide on the extent of APAP-induced hepatocyte injury and the upstream JNK-mediated mitochondrial signaling pathways. We found that leflunomide or A77 1726 concentration-dependently protected hepatocytes from APAP (1 mM)-induced mitochondrial permeabilization and lethal cell injury. This was not due to proximal inhibition of CYP-catalyzed APAP bioactivation to its thiol-reactive metabolite. Instead, we demonstrate that leflunomide (20 {mu}M) inhibited the APAP-induced early (3 h) activation (phosphorylation) of JNK1/2, thus inhibiting phosphorylation of the anti-apoptotic protein Bcl-2 and preventing P-Bcl-2-mediated induction of the mPT. This greatly attenuated mitochondrial cytochrome c release, which we used as a marker for mitochondrial permeabilization. The specific JNK2 inhibitor SP600125 similarly protected from APAP-induced cell death. In conclusion, these findings are consistent with our hypothesis that leflunomide protects from protoxicant-induced hepatocyte injury by inhibiting JNK signaling and preventing mPT induction.

  1. Mechanistic pharmacokinetic modeling for the prediction of transporter-mediated disposition in humans from sandwich culture human hepatocyte data.

    PubMed

    Jones, Hannah M; Barton, Hugh A; Lai, Yurong; Bi, Yi-An; Kimoto, Emi; Kempshall, Sarah; Tate, Sonya C; El-Kattan, Ayman; Houston, J Brian; Galetin, Aleksandra; Fenner, Katherine S

    2012-05-01

    With efforts to reduce cytochrome P450-mediated clearance (CL) during the early stages of drug discovery, transporter-mediated CL mechanisms are becoming more prevalent. However, the prediction of plasma concentration-time profiles for such compounds using physiologically based pharmacokinetic (PBPK) modeling is far less established in comparison with that for compounds with passively mediated pharmacokinetics (PK). In this study, we have assessed the predictability of human PK for seven organic anion-transporting polypeptide (OATP) substrates (pravastatin, cerivastatin, bosentan, fluvastatin, rosuvastatin, valsartan, and repaglinide) for which clinical intravenous data were available. In vitro data generated from the sandwich culture human hepatocyte system were simultaneously fit to estimate parameters describing both uptake and biliary efflux. Use of scaled active uptake, passive distribution, and biliary efflux parameters as inputs into a PBPK model resulted in the overprediction of exposure for all seven drugs investigated, with the exception of pravastatin. Therefore, fitting of in vivo data for each individual drug in the dataset was performed to establish empirical scaling factors to accurately capture their plasma concentration-time profiles. Overall, active uptake and biliary efflux were under- and overpredicted, leading to average empirical scaling factors of 58 and 0.061, respectively; passive diffusion required no scaling factor. This study illustrates the mechanistic and model-driven application of in vitro uptake and efflux data for human PK prediction for OATP substrates. A particular advantage is the ability to capture the multiphasic plasma concentration-time profiles for such compounds using only preclinical data. A prediction strategy for novel OATP substrates is discussed. PMID:22344703

  2. Cultivating Hepatocytes on Printed Arrays of HGF and BMP7 to Characterize Protective Effects of These Growth Factors During In Vitro Alcohol Injury

    PubMed Central

    Jones, Caroline N.; Tuleuova, Nazgul; Lee, Ji Youn; Ramanculov, Erlan; Reddi, A. Hari; Zern, Mark A.; Revzin, Alexander

    2010-01-01

    The goal of the present study was to investigate hepato-protective effects of growth factor (GF) arrays during alcohol injury. Hepatocyte growth factor (HGF) and bone morphogenetic protein (BMP)7 were mixed with collagen (I) and robotically printed onto standard glass slides to create arrays of 500 μm diameter spots. Primary rat hepatocytes were seeded on top of the arrays forming clusters corresponding in size to the underlying protein spots. Cell arrays were then injured in culture by exposure to 100 mM ethanol for 48h. Hepatocytes residing on GF spots were found to have less apoptosis then cells cultured on collagen-only spots. Least apoptosis (0.3 % as estimated by TUNEL assay) was observed on HGF/BMP7/collagen spots whereas most apoptosis (17.3%) was seen on collagen-only arrays. Interestingly, the extent of alcohol-induced apoptosis in hepatocytes varied based on the concentration of printed GF. In addition to preventing apoptosis, printed GFs contributed to maintenance of epithelial phenotype during alcohol injury as evidenced by higher levels of E-cadherin expression in HGF-protected hepatocytes. Importantly, GF microarrays could be used to investigate heterotypic interactions in the context of liver injury. To highlight this, stellate cells - nonparenchymal liver cells involved in fibrosis - were added to hepatocytes residing on arrays of either HGF/collagen or collagen-only spots. Exposure of these cocultures to ethanol followed by RT-PCR analysis revealed that stellate cells residing alongside HGF-protected hepatocytes were significantly less activated (less fibrotic) compared to controls. Overall, our results demonstrate that GF microarray format can be used to screen anti-fibrotic and anti-apoptotic effects of growth factors as well as to investigate how signals delivered to a specific cell type modulate heterotypic cellular interactions. PMID:20488537

  3. Interface-mediated growth of monodispersed nanostructures.

    PubMed

    Wang, Xun; Peng, Qing; Li, Yadong

    2007-08-01

    This Account focuses on the recent development of interface-mediated growth of monodispersed nanostructures in our laboratory. By rationally tuning the chemical reactions at various gas-liquid, solid-solid, liquid-liquid, and liquid-solid-solution interfaces, we could readily synthesize nanostructures such as hollow microspheres, core-shell nanoparticles, and monodispersed nanocrystals. These advances in interface-mediated synthesis could lead to progress in the development of nanocrystal crystallography and encourage some more unique and exciting research and applications to nanoscience and nanotechnology. PMID:17500508

  4. Hepatocyte growth factor suppresses hypoxia/reoxygenation-induced XO activation in cardiac microvascular endothelial cells.

    PubMed

    Zhang, Yingqian; Hu, Shunying; Chen, Yundai

    2015-07-01

    Hypoxia/reoxygenation (H/R) is one of the cellular stresses in pathological conditions, such as myocardial infarction, stroke and organ transplantation. Oxidative stress caused by reactive oxygen species (ROS) is a crucial element of H/R injury in vascular endothelial cells (ECs). Xanthine oxidase (XO) has been recognized to contribute to H/R injury. Of note, xanthine oxidoreductase is synthesized as xanthine dehydrogenase (XDH) and needs to be converted to XO to become a source of superoxide. Hepatocyte growth factor (HGF) has been found to protect ECs against H/R injury. The relation, however, between HGF and XO in ECs under H/R conditions remains to be determined. Primary cultured rat cardiac microvascular endothelial cells (CMECs) were exposed to 4 h of hypoxia and followed by 1 h of reoxygenation. Generation of ROS and cytosolic Ca2+ concentration was measured by flow cytometry qualification of DCFHDA and fluo-3 AM staining cells, respectively. XDH mRNA was qualified by qRT-PCR analysis. XO activity was determined by colorimetric assay and XO protein levels were determined by Western blot. Cell apoptosis was assessed by caspase-3 activity and Annexin V/PI staining. After H/R, cellular ROS production significantly increased. Both XO activity and XO protein increased after H/R. Cellular ROS elevation was inhibited by allopurinol (a potent XO inhibitor), indicting XO accounting for the generation of ROS after H/R. In addition, XDH mRNA increased after H/R, indicating a de novo XDH synthesis, which needs to be converted to XO to become a source of superoxide. Pretreatment of HGF inhibited the elevation of XO activity and XO protein level after H/R; however, HGF has no effect on the increase of XDH mRNA. We also find an increase of the cytosolic Ca2+ in CMECs after H/R. BAPTA-AM, a cell-permeable Ca2+ chelator, prevented the increase of XO activity and XO protein levels, implicating the elevated cytosolic Ca2+ concentration involvement in XO conversion and XO

  5. Interleukin-6-Specific Activation of the C/EBPδ Gene in Hepatocytes Is Mediated by Stat3 and Sp1

    PubMed Central

    Cantwell, Carrie A.; Sterneck, Esta; Johnson, Peter F.

    1998-01-01

    C/EBPδ (CCAAT/enhancer binding protein δ) has been implicated as a regulator of acute-phase response (APR) genes in hepatocytes. Its expression increases dramatically in liver during the APR and can be induced in hepatic cell lines by interleukin-6 (IL-6), an acute-phase mediator that activates transcription of many APR genes. Here we have investigated the mechanism by which C/EBPδ expression is regulated by IL-6 in hepatoma cells. C/EBPδ promoter sequences to −125 bp are sufficient for IL-6 inducibility of a reporter gene and include an APR element (APRE) that is essential for IL-6 responsiveness. DNA binding experiments and transactivation assays demonstrate that Stat3, but not Stat1, interacts with this APRE. Two Sp1 sites, one of which is adjacent to the APRE, are required for IL-6 induction and transactivation by Stat3. Thus, Stat3 and Sp1 function cooperatively to activate the C/EBPδ promoter. Replacement of the APRE with Stat binding elements (SBEs) from the ICAM-1 or C/EBPβ promoter, both of which recognize both Stat1 and Stat3, confers responsiveness to gamma interferon, a cytokine that selectively activates Stat1. Sequence comparisons suggest that the distinct Stat binding specificities of the C/EBPδ and C/EBPβ SBEs are determined primarily by a single base pair difference. Our findings indicate that the cytokine specificity of C/EBPδ gene expression is governed by the APRE sequence. PMID:9528783

  6. Overexpression of hepatocyte growth factor in SBMA model mice has an additive effect on combination therapy with castration.

    PubMed

    Ding, Ying; Adachi, Hiroaki; Katsuno, Masahisa; Huang, Zhe; Jiang, Yue-Mei; Kondo, Naohide; Iida, Madoka; Tohnai, Genki; Nakatsuji, Hideaki; Funakoshi, Hiroshi; Nakamura, Toshikazu; Sobue, Gen

    2015-12-25

    Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine (polyQ)-encoding tract within the androgen receptor (AR) gene. The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem and diffuse nuclear accumulation and nuclear inclusions of mutant AR in residual motor neurons and certain visceral organs. Hepatocyte growth factor (HGF) is a polypeptide growth factor which has neuroprotective properties. To investigate whether HGF overexpression can affect disease progression in a mouse model of SBMA, we crossed SBMA transgenic model mice expressing an AR gene with an expanded CAG repeat with mice overexpressing HGF. Here, we report that high expression of HGF induces Akt phosphorylation and modestly ameliorated motor symptoms in an SBMA transgenic mouse model treated with or without castration. These findings suggest that HGF overexpression can provide a potential therapeutic avenue as a combination therapy with disease-modifying therapies in SBMA. PMID:26551462

  7. The fate of (125I)iodoepidermal growth factor in isolated hepatocytes: a quantitative electron microscopic autoradiographic study

    SciTech Connect

    Carpentier, J.L.; Gorden, P.; Freychet, P.; Canivet, B.; Orci, L.

    1981-09-01

    When (125I)iodoepidermal growth factor is incubated with freshly isolated rat hepatocytes, cell-associated radioactivity reaches apparent steady state by 60 min at 20 C and by 30 min of incubation at 37 C. When the distribution of cell-associated radioactivity is studied at different times of incubation by quantitative electron microscopic autoradiography, the ligand initially associates with the plasma membrane and is progressively internalized as a function of time. The internalized ligand preferentially associates with lysosome-like structures. Qualitatively, these events are similar to those previously obtained with labeled insulin and glucagon in this cell, but quantitatively, the internalization of epidermal growth factor is much greater. The data suggest that the ligand or its specific receptor rather than the cell type is the major determinant of the rate of internalization.

  8. Transforming Growth Factor β1 (TGF-β1) Activates Hepcidin mRNA Expression in Hepatocytes.

    PubMed

    Chen, Simeng; Feng, Teng; Vujić Spasić, Maja; Altamura, Sandro; Breitkopf-Heinlein, Katja; Altenöder, Jutta; Weiss, Thomas S; Dooley, Steven; Muckenthaler, Martina U

    2016-06-17

    The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-β1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-β1-induced hepcidin expression are still unclear. Here we show that TGF-β1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-β1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-β1 depends on functional TGF-β1 type I receptor (ALK5) and TGF-β1 type II receptor (TβRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-β1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5-mediated hepcidin response to TGF-β1. TGF-β1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-β1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-β1 and BMP6 may control the sensing of systemic and/or hepatic iron levels. PMID:27129231

  9. Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes.

    PubMed

    Xu, Wenyi; Wu, Lizhen; Yu, Miao; Chen, Feng-Jung; Arshad, Muhammad; Xia, Xiayu; Ren, Hao; Yu, Jinhai; Xu, Li; Xu, Dijin; Li, John Zhong; Li, Peng; Zhou, Linkang

    2016-02-26

    Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions. PMID:26733203

  10. Dual Delivery of Hepatocyte and Vascular Endothelial Growth Factors via a Protease-Degradable Hydrogel Improves Cardiac Function in Rats

    PubMed Central

    Boopathy, Archana V.; Che, Pao-lin; Brown, Milton; García, Andrés J.; Davis, Michael E.

    2012-01-01

    Acute myocardial infarction (MI) caused by ischemia and reperfusion (IR) is the most common cause of cardiac dysfunction due to local cell death and a temporally regulated inflammatory response. Current therapeutics are limited by delivery vehicles that do not address spatial and temporal aspects of healing. The aim of this study was to engineer biotherapeutic delivery materials to harness endogenous cell repair to enhance myocardial repair and function. We have previously engineered poly(ethylene glycol) (PEG)-based hydrogels to present cell adhesive motifs and deliver VEGF to promote vascularization in vivo. In the current study, bioactive hydrogels with a protease-degradable crosslinker were loaded with hepatocyte and vascular endothelial growth factors (HGF and VEGF, respectively) and delivered to the infarcted myocardium of rats. Release of both growth factors was accelerated in the presence of collagenase due to hydrogel degradation. When delivered to the border zones following ischemia-reperfusion injury, there was no acute effect on cardiac function as measured by echocardiography. Over time there was a significant increase in angiogenesis, stem cell recruitment, and a decrease in fibrosis in the dual growth factor delivery group that was significant compared with single growth factor therapy. This led to an improvement in chronic function as measured by both invasive hemodynamics and echocardiography. These data demonstrate that dual growth factor release of HGF and VEGF from a bioactive hydrogel has the capacity to significantly improve cardiac remodeling and function following IR injury. PMID:23226440

  11. Hepatocyte growth factor increases the invasive potential of PC-3 human prostate cancer cells via an ERK/MAPK and Zeb-1 signaling pathway

    PubMed Central

    HAN, YILI; LUO, YONG; WANG, YONGXING; CHEN, YATONG; LI, MINGCHUAN; JIANG, YONGGUANG

    2016-01-01

    Hepatocyte growth factor (HGF) has been implicated in epithelial-mesenchymal transition (EMT) in numerous types of cancer. However, to the best of our knowledge, there has been no previous evidence that HGF has a role in prostate cancer. The present study aimed to investigate the effect of HGF on EMT and invasive potential, as well as the underlying molecular mechanisms, in a human prostate cancer cell line. Therefore, PC-3 cells were treated with various concentrations of HGF for varying durations. EMT-associated proteins, including E-cadherin and vimentin, were examined by western blot analysis. The effects of HGF on cell proliferation, migration, invasion and tumorigenicity were assessed using MTT, wound-healing, Transwell and soft-agar assays. Subsequently, the role of c-Met in the mediation of EMT-like changes was investigated using reverse transcription-polymerase chain reaction, western blot analysis and gene knockdown by small interfering RNA. Finally, western blot analysis was used to quantify the expression of a downstream transcription factor and extracellular signal-related kinase/mitogen activated protein kinase (ERK/MAPK) signaling pathway proteins. The results indicated that treatment with HGF induced EMT-like changes and enhanced the invasive potential of PC-3 cells. There was an increase in the expression of ERK, phosphorylated-ERK and zinc finger E-box binding homeobox-1 (Zeb-1), suggesting that EMT-like changes may be mediated through the ERK/MAPK and Zeb-1 signaling pathway. Furthermore, HGF-mediated EMT-like changes were associated with c-Met activation, and these changes were able to be blocked by c-Met knockdown. The present study demonstrated that HGF-induced EMT increased the invasive potential of PC-3 human prostate cancer cells through activating the ERK/MAPK and Zeb-1 signaling pathway. PMID:26870279

  12. Involvement of activator protein 1 complexes in the epithelium-specific activation of the laminin gamma2-chain gene promoter by hepatocyte growth factor (scatter factor).

    PubMed Central

    Olsen, J; Lefebvre, O; Fritsch, C; Troelsen, J T; Orian-Rousseau, V; Kedinger, M; Simon-Assmann, P

    2000-01-01

    Laminin-5 is a trimer of laminin alpha3, beta3 and gamma2 chains that is found in the intestinal basement membrane. Deposition of the laminin gamma2 chain at the basement membrane is of great interest because it undergoes a developmental shift in its cellular expression. Here we study the regulatory elements that control basal and cytokine-activated transcriptional expression of the LAMC2 gene, which encodes the laminin gamma2 chain. By using transient transfection experiments we demonstrated the presence of constitutive and cytokine-responsive cis-elements. Comparison of the transcriptional activity of the LAMC2 promoter in the epithelial HT29mtx cells with that in small-intestinal fibroblastic cells (C20 cells) led us to conclude that two regions with constitutive epithelium-specific activity are present between positions -1.2 and -0.12 kb. This was further validated by transfections of primary foetal intestinal endoderm and mesenchyme. A 2.5 kb portion of the LAMC2 5' flanking region was equally responsive to PMA and hepatocyte growth factor (HGF), whereas it was less responsive to transforming growth factor beta1. A minimal promoter limited to the initial 120 bp upstream of the transcriptional start site maintained inducibility by PMA and HGF. This short promoter fragment contains two activator protein 1 (AP-1) elements and the 5'-most of these is a composite AP-1/Sp1 element. The 5'AP-1 element is crucial to the HGF-mediated activity of the promoter; analysis of interacting nuclear proteins demonstrated that AP-1 proteins containing JunD mediate the response to HGF. PMID:10749670

  13. Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer.

    PubMed

    Bierwolf, Jeanette; Volz, Tassilo; Lütgehetmann, Marc; Allweiss, Lena; Riecken, Kristoffer; Warlich, Michael; Fehse, Boris; Kalff, Joerg C; Dandri, Maura; Pollok, Joerg-Matthias

    2016-05-01

    Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo. PMID:27068494

  14. Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer

    PubMed Central

    Bierwolf, Jeanette; Volz, Tassilo; Lütgehetmann, Marc; Allweiss, Lena; Riecken, Kristoffer; Warlich, Michael; Fehse, Boris; Kalff, Joerg C.; Dandri, Maura

    2016-01-01

    Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo. PMID:27068494

  15. Priming Dental Pulp Stem Cells With Fibroblast Growth Factor-2 Increases Angiogenesis of Implanted Tissue-Engineered Constructs Through Hepatocyte Growth Factor and Vascular Endothelial Growth Factor Secretion.

    PubMed

    Gorin, Caroline; Rochefort, Gael Y; Bascetin, Rumeyza; Ying, Hanru; Lesieur, Julie; Sadoine, Jérémy; Beckouche, Nathan; Berndt, Sarah; Novais, Anita; Lesage, Matthieu; Hosten, Benoit; Vercellino, Laetitia; Merlet, Pascal; Le-Denmat, Dominique; Marchiol, Carmen; Letourneur, Didier; Nicoletti, Antonino; Vital, Sibylle Opsahl; Poliard, Anne; Salmon, Benjamin; Muller, Laurent; Chaussain, Catherine; Germain, Stéphane

    2016-03-01

    Tissue engineering strategies based on implanting cellularized biomaterials are promising therapeutic approaches for the reconstruction of large tissue defects. A major hurdle for the reliable establishment of such therapeutic approaches is the lack of rapid blood perfusion of the tissue construct to provide oxygen and nutrients. Numerous sources of mesenchymal stem cells (MSCs) displaying angiogenic potential have been characterized in the past years, including the adult dental pulp. Establishment of efficient strategies for improving angiogenesis in tissue constructs is nevertheless still an important challenge. Hypoxia was proposed as a priming treatment owing to its capacity to enhance the angiogenic potential of stem cells through vascular endothelial growth factor (VEGF) release. The present study aimed to characterize additional key factors regulating the angiogenic capacity of such MSCs, namely, dental pulp stem cells derived from deciduous teeth (SHED). We identified fibroblast growth factor-2 (FGF-2) as a potent inducer of the release of VEGF and hepatocyte growth factor (HGF) by SHED. We found that FGF-2 limited hypoxia-induced downregulation of HGF release. Using three-dimensional culture models of angiogenesis, we demonstrated that VEGF and HGF were both responsible for the high angiogenic potential of SHED through direct targeting of endothelial cells. In addition, FGF-2 treatment increased the fraction of Stro-1+/CD146+ progenitor cells. We then applied in vitro FGF-2 priming to SHED before encapsulation in hydrogels and in vivo subcutaneous implantation. Our results showed that FGF-2 priming is more efficient than hypoxia at increasing SHED-induced vascularization compared with nonprimed controls. Altogether, these data demonstrate that FGF-2 priming enhances the angiogenic potential of SHED through the secretion of both HGF and VEGF. PMID:26798059

  16. The Hepatocyte Growth Factor/c-Met Antagonist, Divalinal-Angiotensin IV, Blocks the Acquisition of Methamphetamine Dependent Conditioned Place Preference in Rats

    PubMed Central

    Wright, John W.; Wilson, Wendy L.; Wakeling, Vanessa; Boydstun, Alan S.; Jensen, Audrey; Kawas, Leen; Harding, Joseph W.

    2012-01-01

    The use of methamphetamine (MA) is increasing in the U.S. and elsewhere around the world. MA’s capacity to cause addiction significantly exceeds other psychostimulant drugs, and its use negatively impacts learning and memory. Recently, attempts have been made to interfere with the presumed mechanism(s) underlying the establishment of drug-induced memory consolidation. The majority of these studies have employed matrix metalloproteinase (MMP) inhibitors to disrupt MMP-induced extracellular matrix molecule dependent synaptic reconfiguration, or GABA receptor agonists. The present investigation utilized an angiotensin IV (AngIV) analogue, Divalinal-AngIV (divalinal), to disrupt acquisition of MA-induced dependence in rats as measured using the conditioned place preference paradigm. Results indicate that both acute and chronic intracerebroventricular infusion of divalinal prior to each daily subcutaneous injection of MA prevented acquisition. However, divalinal was unable to prevent MA-induced reinstatement after prior acquisition followed by extinction trials. These results indicate that prevention of MA dependence can be accomplished by blockade of the brain AT4 receptor subtype. On the other hand, once MA-induced memory consolidation is in place divalinal appears to be ineffective. Mechanistic studies indicated that divalinal is a potent inhibitor of the hepatocyte growth factor (HGF)/c-Met receptor system, and thus it appears that a functional HGF/c-Met system is required for the acquisition of MA-mediated conditioned place preference. PMID:24961196

  17. Analogs of the hepatocyte growth factor and macrophage-stimulating protein hinge regions act as Met and Ron dual inhibitors in pancreatic cancer cells.

    PubMed

    Church, Kevin J; Vanderwerff, Brett R; Riggers, Rachelle R; McMicheal, Michelle D; Mateo-Victoriano, Beatriz; Sukumar, Sudharsan R; Harding, Joseph W

    2016-09-01

    Pancreatic cancer is among the leading causes of cancer death in the USA, with limited effective treatment options. A major contributor toward the formation and persistence of pancreatic cancer is the dysregulation of the hepatocyte growth factor (HGF)/Met (HGF receptor) and the macrophage-stimulating protein (MSP)/Ron (MSP receptor) systems. These systems normally mediate a variety of cellular behaviors including proliferation, survival, and migration, but are often overactivated in pancreatic cancer and contribute toward cancer progression. Previous studies have shown that HGF must dimerize to activate Met. Small-molecule antagonists with homology to a 'hinge' region within the putative dimerization domain of HGF have been developed that bind to HGF and block dimerization, therefore inhibiting Met signaling. Because of the structural and sequence homology between MSP and HGF, we hypothesized that the inhibition of HGF by the hinge analogs may extend to MSP. The primary aim of this 'proof-of-concept' study was to determine whether hinge analogs could inhibit cellular responses to both HGF and MSP in pancreatic cancer cells. Our results showed that these compounds inhibited HGF and MSP activity. Hinge analog treatment resulted in decreased Met and Ron activation, and suppressed malignant cell behaviors including proliferation, migration, and invasion in pancreatic cancer cells in vitro. These results suggest that the hinge analogs represent a novel group of molecules that may offer a therapeutic approach for the treatment of pancreatic cancer and warrant further development and optimization. PMID:27314431

  18. Expression and characterization of biologically active human hepatocyte growth factor (HGF) by insect cells infected with HGF-recombinant baculovirus.

    PubMed

    Yee, C J; DeFrances, M C; Bell, A; Bowen, W; Petersen, B; Michalopoulos, G K; Zarnegar, R

    1993-08-10

    A cDNA containing the entire coding sequence of human hepatocyte growth factor (HGF) [also known as scatter factor (SF)] was inserted into the genome of Autographa california nuclear polyhedrosis virus (baculovirus) adjacent to the polyhedrin promoter by homologous recombination. Insect cells (Spodoptera frugiperda) infected with the recombinant virus secrete relatively high levels (3-8 mg/L) of biologically active HGF into the culture medium. The recombinant HGF induces pronounced morphological changes and scattering of primary cultures of rat, mouse, and human hepatocytes within 24 h after plating and stimulates DNA synthesis in these cells with the same magnitude as native HGF derived from human placenta or rabbit serum. The human recombinant HGF produced by the insect cells is N-glycosylated, binds to heparin like native HGF, and is recognized by polyclonal antiserums raised against human or rabbit HGF as assessed by immunoblot, ELISA, and immunoneutralization experiments. Metabolic radiolabeling with L-[35S]methionine (pulse-chase experiments) as well as Western blot analysis indicates that the recombinant HGF is synthesized and secreted by the infected insect cells as the unprocessed single-chain form (pro-HGF) when the cells are cultured in serum-free medium. However, when the infected insect cells are cultured in insect culture medium (Grace's medium) containing fetal bovine serum, the secreted HGF is present mainly in the mature heterodimeric form. Addition of serum to the baculovirus-expressed single-chain [125I]HGF in a cell-free system results in conversion to the heterodimeric two-chain form, and the activation is prevented by the serine protease inhibitor PMSF. Incubation of 125I-labeled pro-HGF with rat liver or spleen extracts resulted in conversion of pro-HGF to the heterodimeric two-chain form. A truncated form of HGF containing the N-terminal portion of HGF (kringles 1-3) was also produced in the same expression system. This deleted HGF, by

  19. Usage of adenovirus expressing thymidine kinase mediated hepatocellular damage for enabling mouse liver repopulation with allogenic or xenogenic hepatocytes.

    PubMed

    Moreno, Daniel; Balasiddaiah, Anangi; Lamas, Oscar; Duret, Cedric; Neri, Leire; Guembe, Laura; Galarraga, Miguel; Larrea, Esther; Daujat-Chavanieu, Martine; Muntane, Jordi; Maurel, Patrick; Riezu, Jose Ignacio; Prieto, Jesus; Aldabe, Rafael

    2013-01-01

    It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2(-/-) γc(-/-) mice using an adenovirus encoding herpes virus thymidine kinase (AdTk) and two consecutive doses of ganciclovir (GCV). We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line. PMID:24086405

  20. [Differences in dynamics of insulin and insulin-like growth I (IGF-I) receptors internalization in isolated rat hepatocytes].

    PubMed

    2013-01-01

    Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is the most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones, the internalization dynamics of 125I-insulin and 125I-IGF-I was traced in isolated rat hepatocytes at 37 and 12 degrees C. There were established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37 degrees C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. However, essential differences in the internalization course of these two related peptide were obvious at the temperature of 12 degrees C. The internalization level of insulin receptors at 12 degrees C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocytes plasma membrane. At 12 degrees C a slight decrease of the proportion of intracellular 125I-IGF-I correlated with a decrease in the 125I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12 degrees C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar "inhibition mechanism" of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates. PMID:25509050

  1. [Differences in dynamics of insulin and insulin-like growth I (IGF-I) receptors internalization in isolated rat hepatocytes].

    PubMed

    Kolychev, A P; Ternovskaya, E E; Arsenieva, A V; Shapkina, E V

    2013-01-01

    Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is the most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones, the internalization dynamics of 125I-insulin and 125I-IGF-I was traced in isolated rat hepatocytes at 37 and 12 degrees C. There were established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37 degrees C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. However, essential differences in the internalization course of these two related peptide were obvious at the temperature of 12 degrees C. The internalization level of insulin receptors at 12 degrees C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocytes plasma membrane. At 12 degrees C a slight decrease of the proportion of intracellular 125I-IGF-I correlated with a decrease in the 125I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12 degrees C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar "inhibition mechanism" of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates. PMID:25490849

  2. Cross-talk between glucagon- and adenosine-mediated signalling systems in rat hepatocytes: effects on cyclic AMP-phosphodiesterase activity.

    PubMed Central

    Robles-Flores, M; Allende, G; Piña, E; García-Sáinz, J A

    1995-01-01

    The effect of adenosine analogues on glucagon-stimulated cyclic AMP accumulation in rat hepatocytes was explored. N6-Cyclopentyladenosine (CPA), 5'-N-ethylcarboxamidoadenosine and N6-(R-phenylisopropyl)adenosine inhibited in a dose-dependent manner the cyclic AMP accumulation induced by glucagon. This effect seems to be mediated through A1 adenosine receptors. Pertussis toxin completely abolished the effect of CPA on glucagon-stimulated cyclic AMP accumulation in whole cells which suggested that a pertussis-toxin-sensitive G-protein was involved. On the other hand, this action of adenosine analogues on glucagon-induced cyclic AMP accumulation was reverted by the selective low-Km cyclic AMP-phosphodiesterase inhibitor Ro 20-1724. Analysis of cyclic AMP-phosphodiesterase activity in purified hepatocyte plasma membranes showed that glucagon in the presence of GTP inhibited basal PDE activity by 45% and that CPA reverted this inhibition in dose-dependent manner. In membranes derived from pertussis-toxin-treated rats, we observed no inhibition of cyclic AMP-phosphodiesterase activity by glucagon in the absence or presence of CPA. Our results indicate that in hepatocyte plasma membranes, stimulation of adenylate cyclase activity and inhibition of a low-Km cyclic AMP phosphodiesterase activity are co-ordinately regulated by glucagon, and that A1 adenosine receptors can inhibit glucagon-stimulated cyclic AMP accumulation by blocking glucagon's effect on phosphodiesterase activity. Images Figure 2 PMID:8554517

  3. Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

    SciTech Connect

    Ji Lili; Chen Ying; Liu Tianyu; Wang Zhengtao

    2008-09-15

    Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 {mu}M)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 {mu}M clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 {mu}M) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway.

  4. Fructose Mediated Non-Alcoholic Fatty Liver Is Attenuated by HO-1-SIRT1 Module in Murine Hepatocytes and Mice Fed a High Fructose Diet

    PubMed Central

    Sodhi, Komal; Puri, Nitin; Favero, Gaia; Stevens, Sarah; Meadows, Charles; Abraham, Nader G.; Rezzani, Rita; Ansinelli, Hayden; Lebovics, Edward; Shapiro, Joseph I.

    2015-01-01

    Background Oxidative stress underlies the etiopathogenesis of nonalcoholic fatty liver disease (NAFLD), obesity and cardiovascular disease (CVD). Heme Oxygenase-1 (HO-1) is a potent endogenous antioxidant gene that plays a key role in decreasing oxidative stress. Sirtuin1 (SIRT1) belongs to the family of NAD-dependent de-acyetylases and is modulated by cellular redox. Hypothesis We hypothesize that fructose-induced obesity creates an inflammatory and oxidative environment conducive to the development of NAFLD and metabolic syndrome. The aim of this study is to determine whether HO-1 acts through SIRT1 to form a functional module within hepatocytes to attenuate steatohepatitis, hepatic fibrosis and cardiovascular dysfunction. Methods and Results We examined the effect of fructose, on hepatocyte lipid accumulation and fibrosis in murine hepatocytes and in mice fed a high fructose diet in the presence and absence of CoPP, an inducer of HO-1, and SnMP, an inhibitor of HO activity. Fructose increased oxidative stress markers and decreased HO-1 and SIRT1 levels in hepatocytes (p<0.05). Further fructose supplementation increased FAS, PPARα, pAMPK and triglycerides levels; CoPP negated this increase. Concurrent treatment with CoPP and SIRT1 siRNA in hepatocytes increased FAS, PPARα, pAMPK and triglycerides levels suggesting that HO-1 is upstream of SIRT1 and suppression of SIRT1 attenuates the beneficial effects of HO-1. A high fructose diet increased insulin resistance, blood pressure, markers of oxidative stress and lipogenesis along with fibrotic markers in mice (p<0.05). Increased levels of HO-1 increased SIRT1 levels and ameliorated fructose-mediated lipid accumulation and fibrosis in liver along with decreasing vascular dysfunction (p<0.05 vs. fructose). These beneficial effects of CoPP were reversed by SnMP. Conclusion Taken together, our study demonstrates, for the first time, that HO-1 induction attenuates fructose-induced hepatic lipid deposition, prevents the

  5. miR-223 Deficiency Protects against Fas-Induced Hepatocyte Apoptosis and Liver Injury through Targeting Insulin-Like Growth Factor 1 Receptor.

    PubMed

    Qadir, Ximena V; Chen, Weina; Han, Chang; Song, Kyoungsub; Zhang, Jinqiang; Wu, Tong

    2015-12-01

    The biological functions and molecular mechanisms of miR-223 action in liver cells and liver diseases remain unclear. We therefore determined the effect and mechanism of action of miR-233 in Fas-induced hepatocyte apoptosis and liver injury. Wild-type (WT) and miR-223 knockout (KO) mice were treated i.p. with 0.5 μg/g body weight anti-Fas antibody Jo2, and the animals were monitored for survival and the extent of liver injury. Although WT mice died 4 to 6 hours after Jo2 injection (n = 6), all of the miR-223 KO mice (n = 6) survived. In comparison to WT mice, the miR-223 KO mice showed resistance to Fas-induced liver injury, as indicated by less tissue damage under histopathological examination, fewer apoptotic hepatocytes under caspase-3 immunostaining, and less elevation of serum transaminases. miR-223 KO livers showed less caspase-3, caspase-8, and caspase-9 activation and less poly (ADP-ribose) polymerase cleavage compared with WT livers (P < 0.05). Furthermore, tail vein injection of miR-223 lentiviral vector to miR-223 KO mice restored Jo2-induced liver injury. Transfection of miR-223 KO hepatocytes with miR-223 mimic enhanced Jo2-induced activation of caspase-3, caspase-8, and caspase-9, whereas transfection of WT hepatocytes with the miR-223 inhibitor attenuated Jo2-induced apoptosis. These findings demonstrate that miR-223 deficiency protects against Fas-induced hepatocyte apoptosis and liver injury. Further in vitro and in vivo data indicate that miR-223 regulates Fas-induced hepatocyte apoptosis and liver injury by targeting the insulin-like growth factor 1 receptor. PMID:26598234

  6. The hepatocyte growth factor (HGF)-MET receptor tyrosine kinase signaling pathway: Diverse roles in modulating immune cell functions.

    PubMed

    Ilangumaran, Subburaj; Villalobos-Hernandez, Alberto; Bobbala, Diwakar; Ramanathan, Sheela

    2016-06-01

    Hepatocyte growth factor (HGF) signaling via the MET receptor is essential for embryonic development and tissue repair. On the other hand, deregulated MET signaling promotes tumor progression in diverse types of cancers. Even though oncogenic MET signaling remains the major research focus, the HGF-MET axis has also been implicated in diverse aspects of immune cell development and functions. In the presence of other hematopoietic growth factors, HGF promotes the development of erythroid, myeloid and lymphoid lineage cells and thrombocytes. In monocytes and macrophages responding to inflammatory stimuli, induction of autocrine HGF-MET signaling can contribute to tissue repair via stimulating anti-inflammatory cytokine production. HGF-MET signaling can also modulate adaptive immune response by facilitating the migration of Langerhans cells and dendritic cells to draining lymph nodes. However, MET signaling has also been shown to induce tolerogenic dendritic cells in mouse models of graft-versus-host disease and experimental autoimmune encephalomyelitis. HGF-MET axis is also implicated in promoting thymopoiesis and the survival and migration of B lymphocytes. Recent studies have shown that MET signaling induces cardiotropism in activated T lymphocytes. Further understanding of the HGF-MET axis in the immune system would allow its therapeutic manipulation to improve immune cell reconstitution, restore immune homeostasis and to treat immuno-inflammatory diseases. PMID:26822708

  7. The Hepatocyte Growth Factor (HGF)/Met Axis: A Neglected Target in the Treatment of Chronic Myeloproliferative Neoplasms?

    PubMed Central

    Boissinot, Marjorie; Vilaine, Mathias; Hermouet, Sylvie

    2014-01-01

    Met is the receptor of hepatocyte growth factor (HGF), a cytoprotective cytokine. Disturbing the equilibrium between Met and its ligand may lead to inappropriate cell survival, accumulation of genetic abnormalities and eventually, malignancy. Abnormal activation of the HGF/Met axis is established in solid tumours and in chronic haematological malignancies, including myeloma, acute myeloid leukaemia, chronic myelogenous leukaemia (CML), and myeloproliferative neoplasms (MPNs). The molecular mechanisms potentially responsible for the abnormal activation of HGF/Met pathways are described and discussed. Importantly, inCML and in MPNs, the production of HGF is independent of Bcr-Abl and JAK2V617F, the main molecular markers of these diseases. In vitro studies showed that blocking HGF/Met function with neutralizing antibodies or Met inhibitors significantly impairs the growth of JAK2V617F-mutated cells. With personalised medicine and curative treatment in view, blocking activation of HGF/Met could be a useful addition in the treatment of CML and MPNs for those patients with high HGF/MET expression not controlled by current treatments (Bcr-Abl inhibitors in CML; phlebotomy, hydroxurea, JAK inhibitors in MPNs). PMID:25119536

  8. Hepatocyte growth factor regulates the TGF-β1-induced proliferation, differentiation and secretory function of cardiac fibroblasts

    PubMed Central

    YI, XIN; LI, XIAOYAN; ZHOU, YANLI; REN, SHAN; WAN, WEIGUO; FENG, GAOKE; JIANG, XUEJUN

    2014-01-01

    Cardiac fibroblast (CF) proliferation and transformation into myofibroblasts play important roles in cardiac fibrosis during pathological myocardial remodeling. In this study, we demonstrate that hepatocyte growth factor (HGF), an antifibrotic factor in the process of pulmonary, renal and liver fibrosis, is a negative regulator of cardiac fibroblast transformation in response to transforming growth factor-β1 (TGF-β1). HGF expression levels were significantly reduced in the CFs following treatment with 5 ng/ml TGF-β1 for 48 h. The overexpression of HGF suppressed the proliferation, transformation and the secretory function of the CFs following treatment with TGF-β1, as indicated by the attenuated expression levels of α-smooth muscle actin (α-SMA) and collagen I and III, whereas the knockdown of HGF had the opposite effect. Mechanistically, we identified that the phosphorylation of c-Met, Akt and total protein of TGIF was significantly inhibited by the knockdown of HGF, but was significantly enhanced by HGF overexpression. Collectively, these results indicate that HGF activates the c-Met-Akt-TGIF signaling pathway, inhibiting CF proliferation and transformation in response to TGF-β1 stimulation. PMID:24840640

  9. Hepatocyte growth factor (HGF) enhances cardiac commitment of differentiating embryonic stem cells by activating PI3 kinase

    SciTech Connect

    Roggia, Cristiana; Ukena, Christian; Boehm, Michael; Kilter, Heiko . E-mail: kilter@med-in.uni-saarland.de

    2007-03-10

    Hepatocyte growth factor (HGF) is a pleiotropic cytokine promoting proliferation, migration and survival in several cell types. HGF and its cognate receptor c-Met are expressed in cardiac cells during early cardiogenesis, but data concerning its role in cardiac differentiation of embryonic stem cells (ESCs) and the underlying molecular mechanisms involved are limited. In the present study we show that HGF significantly increases the number of beating embryoid bodies of differentiating ESCs without affecting beating frequency. Furthermore, HGF up-regulates the expression of the cardiac-specific transcription factors Nkx 2.5 and GATA-4 and of markers of differentiated cardiomyocytes, i.e. {alpha}-MHC, {beta}-MHC, ANF, MLC2v and Troponin T. The HGF-induced increase in Nkx 2.5 expression was inhibited by co-treatment with the PI3 kinase inhibitors Wortmannin and LY294002, but not by its inactive homolog LY303511, suggesting an involvement of the PI3 kinase/Akt pathway in this effect. We conclude that HGF is an important growth factor involved in cardiac differentiation and/or proliferation of ESCs and may therefore be critical for the in vitro generation of pre- or fully differentiated cardiomyocytes as required for clinical use of embryonic stem cells in cardiac diseases.

  10. Accelerated adhesion of grafted skin by laser-induced stress wave-based gene transfer of hepatocyte growth factor

    NASA Astrophysics Data System (ADS)

    Aizawa, Kazuya; Sato, Shunichi; Terakawa, Mitsuhiro; Saitoh, Daizoh; Tsuda, Hitoshi; Ashida, Hiroshi; Obara, Minoru

    2009-11-01

    Gene therapy using wound healing-associated growth factor gene has received much attention as a new strategy for improving the outcome of tissue transplantation. We delivered plasmid DNA coding for human hepatocyte growth factor (hHGF) to rat free skin grafts by the use of laser-induced stress waves (LISWs); autografting was performed with the grafts. Systematic analysis was conducted to evaluate the adhesion properties of the grafted tissue; angiogenesis, cell proliferation, and reepithelialization were assessed by immunohistochemistry, and reperfusion was measured by laser Doppler imaging as a function of time after grafting. Both the level of angiogenesis on day 3 after grafting and the increased ratio of blood flow on day 4 to that on day 3 were significantly higher than those in five control groups: grafting with hHGF gene injection alone, grafting with control plasmid vector injection alone, grafting with LISW application alone, grafting with LISW application after control plasmid vector injection, and normal grafting. Reepithelialization was almost completed on day 7 even at the center of the graft with LISW application after hHGF gene injection, while it was not for the grafts of the five control groups. These findings demonstrate the validity of our LISW-based HGF gene transfection to accelerate the adhesion of grafted skins.

  11. Hepatocyte growth factor stimulates motility, chemotaxis and mitogenesis in ovarian carcinoma cells expressing high levels of c-met.

    PubMed

    Corps, A N; Sowter, H M; Smith, S K

    1997-09-26

    A proportion of ovarian carcinomas markedly overexpress the proto-oncogene c-met, which encodes the receptor for hepatocyte growth factor (HGF). HGF may either stimulate or inhibit the multiplication of its target cells, and may also promote motogenesis and morphogenesis. In this study, we established that the ovarian carcinoma-derived cell-line SK-OV-3 expressed about 20-fold higher levels of c-met protein than are expressed by a second line, CH1. This enabled us to test functional consequences of high-level expression of c-met in ovarian carcinoma cells. The addition of HGF to attached cultures of SK-OV-3 cells caused a change to a motile phenotype, that was evident after 4-6 hr and affected essentially all of the cells by 24 hr. When HGF was placed in the lower compartment of a migration chamber, it induced a 17-fold increase in the migration of SK-OV-3 cells to the lower surface of the filter. Finally, HGF stimulated the incorporation of [3H]-thymidine by cultures of SK-OV-3 cells incubated in medium containing either low (0.2%) or full (10%) FCS. None of these responses were obtained when HGF was added to CH1 cells. We conclude that high levels of c-met expression in ovarian cancer cells may lead to a range of responses to HGF that would promote tumour growth and dissemination. PMID:9334823

  12. HALOGENATED AROMATIC HYDROCARBON-MEDIATED PORPHYRIN ACCUMULATION AND INDUCTION OF CYTOCHROME P4501A IN CHICKEN EMBRYO HEPATOCYTES. (R823889)

    EPA Science Inventory

    Concentration-dependent induction of cytochrome P4501A (CYP1A) and intracellular porphyrin accumulation were observed following treatment of chicken embryo hepatocyte (CEH) cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4'...

  13. Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Facilitate Tubular Epithelial Cell Dedifferentiation and Growth via Hepatocyte Growth Factor Induction

    PubMed Central

    Wu, Shuai; Zou, Xiang-yu; Zhang, Guang-yuan; Gu, Di; Miao, Shuai; Zhu, Ying-jian; Sun, Jie; Du, Tao

    2015-01-01

    During acute kidney injury (AKI), tubular cell dedifferentiation initiates cell regeneration; hepatocyte growth factor (HGF) is involved in modulating cell dedifferentiation. Mesenchymal stem cell (MSC)-derived microvesicles (MVs) deliver RNA into injured tubular cells and alter their gene expression, thus regenerating these cells. We boldly speculated that MVs might induce HGF synthesis via RNA transfer, thereby facilitating tubular cell dedifferentiation and regeneration. In a rat model of unilateral AKI, the administration of MVs promoted kidney recovery. One of the mechanisms of action is the acceleration of tubular cell dedifferentiation and growth. Both in vivo and in vitro, rat HGF expression in damaged rat tubular cells was greatly enhanced by MV treatment. In addition, human HGF mRNA present in MVs was delivered into rat tubular cells and translated into the HGF protein as another mechanism of HGF induction. RNase treatment abrogated all MV effects. In the in vitro experimental setting, the conditioned medium of MV-treated injured tubular cells, which contains a higher concentration of HGF, strongly stimulated cell dedifferentiation and growth, as well as Erk1/2 signaling activation. Intriguingly, these effects were completely abrogated by either c-Met inhibitor or MEK inhibitor, suggesting that HGF induction is a crucial contributor to the acceleration of cell dedifferentiation and growth. All these findings indicate that MV-induced HGF synthesis in damaged tubular cells via RNA transfer facilitates cell dedifferentiation and growth, which are important regenerative mechanisms. PMID:25793303

  14. Glutamine inhibits CCl4 induced liver fibrosis in mice and TGF-β1 mediated epithelial-mesenchymal transition in mouse hepatocytes.

    PubMed

    Shrestha, Nirajan; Chand, Lokendra; Han, Myung Kwan; Lee, Seung Ok; Kim, Chan Young; Jeong, Yeon Jun

    2016-07-01

    Glutamine, traditionally a non-essential amino acid, now has been considered as essential in serious illness and injury. It is a major precursor for glutathione synthesis. However, the anti-fibrotic effect of glutamine and its molecular mechanism in experimental liver fibrosis have not been explored. In the present study we aimed to examine the potential role of glutamine in carbon tetrachloride (CCl4) induced liver fibrosis and TGF-β1 mediated epithelial mesenchymal transition (EMT) and apoptosis in mouse hepatocytes. Liver fibrosis was induced by intraperitoneal injection of CCl4 three times a week for 10 weeks. Glutamine treatment effectively attenuated liver injury and oxidative stress. Collagen content was significantly decreased in liver sections of glutamine treated mice compared to CCl4 model mice. Furthermore, glutamine decreased expression level of α-SMA and TGF-β in liver tissue. Our in vitro study showed that TGF-β1 treatment in hepatocytes resulted in loss of E-cadherin and increased expression of mesenchymal markers and EMT related transcription factor. In addition, TGF-β1 increased the expression of apoptotic markers. However, glutamine interestingly suppressed TGF-β1 mediated EMT and apoptosis. In conclusion, our results suggest that glutamine ameliorates CCl4 induced liver fibrosis and suppresses TGF-β1 induced EMT progression and apoptosis. PMID:27137983

  15. Growth-dependent inhibition of CCAAT enhancer-binding protein (C/EBP alpha) gene expression during hepatocyte proliferation in the regenerating liver and in culture.

    PubMed Central

    Mischoulon, D; Rana, B; Bucher, N L; Farmer, S R

    1992-01-01

    As an approach to understanding physiological mechanisms that control the proliferation of highly differentiated cells, we are addressing whether certain hepatic transcription factors participate in mechanisms that control the growth of hepatocytes. We have focused on CCAAT enhancer-binding protein (C/EBP alpha), a transcription factor which is highly abundant in normal liver and is considered to regulate expression of many genes, including some involved in energy metabolism (S. L. McKnight, M. D. Lane, and S. Gluecksohn-Walsh. Genes Dev. 3:2021-2024, 1989). Using Northern (RNA) blot analysis, we have examined the expression of C/EBP alpha mRNA during liver regeneration and in primary cultures of hepatocytes. C/EBP alpha mRNA levels decrease 60 to 80% within 1 to 3 h after partial hepatectomy as the cells move from G0 to G1 and decrease further when cells progress into S phase. Run-on transcription analysis is in agreement with the Northern blot data, thus suggesting that C/EBP alpha is transcriptionally regulated in regenerating liver. C/EBP alpha mRNA expression also decreases dramatically during the growth of freshly isolated normal hepatocytes cultured under conventional conditions (on dried rat tail collagen; stimulated to proliferate by epidermal growth factor [EGF] and insulin). Cultures of hepatocytes on rat tail collagen in the presence or absence of EGF clearly show that within 3 h, EGF depresses C/EBP alpha mRNA expression and that this effect is substantially greater by 4 h. Inhibition of protein synthesis in the liver by cycloheximide or in cultured hepatocytes by puromycin or cycloheximide effectively blocks the down-regulation of C/EBP alpha gene expression, apparently by stabilizing the normal rapid turnover of the C/EBP alpha mRNA (half-life of <2 h). This drop in C/EBP alpha gene expression in response to activation of hepatocyte growth is consistent with the proposal that C/EBP alpha has an antiproliferative role to play in highly differentiated

  16. A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

    SciTech Connect

    Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen

    2015-08-14

    The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.

  17. Safety and efficacy of plasmid DNA expressing two isoforms of hepatocyte growth factor in patients with critical limb ischemia.

    PubMed

    Kibbe, M R; Hirsch, A T; Mendelsohn, F O; Davies, M G; Pham, H; Saucedo, J; Marston, W; Pyun, W-B; Min, S-K; Peterson, B G; Comerota, A; Choi, D; Ballard, J; Bartow, R A; Losordo, D W; Sherman, W; Driver, V; Perin, E C

    2016-03-01

    VM202, a plasmid DNA that expresses two isoforms of hepatocyte growth factor, may elicit angiogenic effects that could benefit patients with critical limb ischemia (CLI). In a phase 2, double-blind trial in 52 CLI patients, we examined the safety and potential efficacy of intramuscular injections of low-dose (n=21) or high-dose (n=20) VM202 or placebo (n=11) in the affected limb (days 0, 14, 28 and 42). Adverse events and serious adverse events were similar among the groups; no malignancy or proliferative retinopathy was seen. In exploratory efficacy analyses, we found no differences in ankle or toe-brachial index, VAS, VascuQuol or amputation rate among the groups. Complete ulcer healing was significantly better in high-dose (8/13 ulcers; P<0.01) versus placebo (1/9) patients. Clinically meaningful reductions (>50%) in ulcer area occurred in high-dose (9/13 ulcers) and low-dose (19/27) groups versus placebo (1/9; P<0.05 and P<0.005, respectively). At 12 months, significant differences were seen in TcPO2 between the high-dose and placebo groups (47.5 ± 17.8 versus 36.6 ± 24.0 mm Hg, respectively; P<0.05) and in the change from baseline among the groups (P<0.05). These data suggest that VM202 is safe and may provide therapeutic bioactivity in CLI patients. PMID:26649448

  18. Regenerative and fibrotic pathways in canine hepatic portosystemic shunt and portal vein hypoplasia, new models for clinical hepatocyte growth factor treatment

    PubMed Central

    Spee, Bart; Penning, Louis C; van den Ingh, Ted SGAM; Arends, Brigitte; IJzer, Jooske; van Sluijs, Frederik J; Rothuizen, Jan

    2005-01-01

    Background We analyzed two spontaneous dog diseases characterized by subnormal portal perfusion and reduced liver growth: (i) congenital portosystemic shunts (CPSS) without fibrosis and (ii) primary portal vein hypoplasia (PPVH), a disease associated with fibrosis. These pathologies, that lack inflammation or cholestasis, may represent simplified models to study liver growth and fibrosis. To investigate the possible use of those models for hepatocyte growth factor (HGF) treatment, we studied the functionality of HGF signaling in CPSS and PPVH dogs and compared this to aged-matched healthy controls. Results We used quantitative real-time polymerase chain reaction (Q-PCR) to analyze the mRNA expression of HGF, transforming growth factor β1 (TGF-β1), and relevant mediators in liver biopsies from cases with CPSS or PPVH, in comparison with healthy control dogs. CPSS and PPVH were associated with a decrease in mRNA expression of HGF and of MET proto-oncogene (c-MET). Western blot analysis confirmed the Q-PCR results and showed that intracellular signaling components (protein kinase B/Akt, ERK1/2, and STAT3) were functional. The TGF-β1 mRNA levels were unchanged in CPSS whereas there was a 2-fold increase in PPVH indicating an active TGF-β1 pathway, consistent with the observation of fibrosis seen in PPVH. Western blots on TGF-β1 and phosphorylated Smad2 confirmed an activated pro-fibrotic pathway in PPVH. Furthermore, Q-PCR showed an increase in the amount of collagen I present in PPVH compared to CPSS and control, which was confirmed by Western blot analysis. Conclusion The pathophysiological differences between CPSS and PPVH can adequately be explained by the Q-PCR measurements and Western blots. Although c-MET levels were reduced, downstream signaling seemed to be functional and provides a rational for HGF-supplementation in controlled studies with CPSS and PPVH. Furthermore both diseases may serve as simplified models for comparison with more complex chronic

  19. Differential Effects of Hepatocyte Nuclear Factor 4α Isoforms on Tumor Growth and T-Cell Factor 4/AP-1 Interactions in Human Colorectal Cancer Cells

    PubMed Central

    Vuong, Linh M.; Chellappa, Karthikeyani; Dhahbi, Joseph M.; Deans, Jonathan R.; Fang, Bin; Bolotin, Eugene; Titova, Nina V.; Hoverter, Nate P.; Spindler, Stephen R.; Waterman, Marian L.

    2015-01-01

    The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) is tumor suppressive in the liver but amplified in colon cancer, suggesting that it also might be oncogenic. To investigate whether this discrepancy is due to different HNF4α isoforms derived from its two promoters (P1 and P2), we generated Tet-On-inducible human colon cancer (HCT116) cell lines that express either the P1-driven (HNF4α2) or P2-driven (HNF4α8) isoform and analyzed them for tumor growth and global changes in gene expression (transcriptome sequencing [RNA-seq] and chromatin immunoprecipitation sequencing [ChIP-seq]). The results show that while HNF4α2 acts as a tumor suppressor in the HCT116 tumor xenograft model, HNF4α8 does not. Each isoform regulates the expression of distinct sets of genes and recruits, colocalizes, and competes in a distinct fashion with the Wnt/β-catenin mediator T-cell factor 4 (TCF4) at CTTTG motifs as well as at AP-1 motifs (TGAXTCA). Protein binding microarrays (PBMs) show that HNF4α and TCF4 share some but not all binding motifs and that single nucleotide polymorphisms (SNPs) in sites bound by both HNF4α and TCF4 can alter binding affinity in vitro, suggesting that they could play a role in cancer susceptibility in vivo. Thus, the HNF4α isoforms play distinct roles in colon cancer, which could be due to differential interactions with the Wnt/β-catenin/TCF4 and AP-1 pathways. PMID:26240283

  20. The synergistic therapeutic effect of hepatocyte growth factor and granulocyte colony-stimulating factor on pulmonary hypertension in rats.

    PubMed

    Guo, Yinghua; Su, Longxiang; Li, Yinghui; Guo, Na; Xie, Lixin; Zhang, Dong; Zhang, Xiaojun; Li, Hongxia; Zhang, Guizhi; Wang, Yajuan; Liu, Changting

    2014-07-01

    Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary arterial pressure and vascular resistance. Despite advances in therapy for PAH, its treatment and prognosis remain poor. We aimed to investigate whether the transplantation of bone marrow mesenchymal stem cells (MSCs) overexpressing hepatocyte growth factor (HGF), alone or in combination with granulocyte colony-stimulating factor (G-CSF), attenuates the development of experimental monocrotaline (MCT)-induced PAH. Three weeks after MCT administration, rats were divided into the following groups: (1) untreated (PAH); (2) HGF treated; (3) MSCs administered; (4) HGF-MSCs treated; and (5) HGF-MSCs plus G-CSF treated. After 3 weeks, hemodynamic changes, histomorphology, and angiogenesis were evaluated. To elucidate the molecular mechanisms of vascular remodeling and angiogenesis, serum levels of transforming growth factor (TGF)-β and endothelin-1 (ET-1) were measured, and the gene and protein expression levels of vascular cell adhesion molecule-1 (VCAM-1) and matrix metalloproteinase-9 (MMP-9) were determined. Compared with the PAH, MSC, and G-CSF groups, the HGF and HGF+G-CSF groups exhibited significantly reduced right ventricular hypertrophy and mean pulmonary arterial pressure (P < 0.05). Histologically, vessel muscularization or thickening and collagen deposition were also significantly decreased (P < 0.05). The number of vessels in the HGF+G-CSF group was higher than that in the other groups (P < 0.05). The TGF-β and ET-1 concentrations in the plasma of pulmonary hypertensive rats were markedly lower in the HGF and HGF+G-CSF groups (P < 0.05). Furthermore, HGF induced the expression of VCAM-1, and HGF treatment together with G-CSF synergistically stimulated MMP-9 expression. Transplanted HGF-MSCs combined with G-CSF potentially offer synergistic therapeutic benefit for the treatment of PAH. PMID:23933910

  1. Increased production of interleukin 1 beta and hepatocyte growth factor may contribute to foveolar hyperplasia in enlarged fold gastritis.

    PubMed Central

    Yasunaga, Y; Shinomura, Y; Kanayama, S; Higashimoto, Y; Yabu, M; Miyazaki, Y; Kondo, S; Murayama, Y; Nishibayashi, H; Kitamura, S; Matsuzawa, Y

    1996-01-01

    BACKGROUND AND AIMS: It has been reported that eradication of Helicobacter pylori improves fold width in H pylori associated enlarged fold gastritis. The aim of this study was to clarify the mechanism of fold thickening in this condition. PATIENTS AND METHODS: In eight patients with enlarged fold gastritis and 13 patients without enlarged folds, the presence of H pylori infection, inflammatory infiltrates, mucosal plasia, and epithelial cell proliferation in the body mucosa were investigated, and production of transforming growth factor alpha (TGF alpha), hepatocyte growth factor (HGF), and interleukin 1 beta (IL 1 beta) was determined by a competitive reverse transcription/polymerase chain reaction method and in vitro short-term culture of biopsy specimens. RESULTS: In the patients with enlarged fold gastritis, inflammatory infiltrates including macrophages increased with H pylori colonisation in the body. Foveolar thickness and proliferating cell nuclear antigen (PCNA) labelling index were increased. Messenger RNA levels of HGF, but not TGF alpha, were increased, and release of HGF and IL 1 beta was increased. HGF release, which was positively correlated with IL 1 beta release and foveolar thickness, decreased in the presence of IL 1 receptor antagonist. After eradication of H pylori, inflammatory infiltrates, IL 1 beta and HGF release decreased with concomitant decreases in PCNA labelling index, foveolar thickness and fold width. CONCLUSIONS: Increased IL 1 beta and HGF production caused by H pylori infection may contribute to fold thickening of the stomach by stimulating epithelial cell proliferation and foveolar hyperplasia in patients with enlarged fold gastritis. Images Figure 1 Figure 2 Figure 7 PMID:9038658

  2. Lung fibroblasts accelerate wound closure in human alveolar epithelial cells through hepatocyte growth factor/c-Met signaling

    PubMed Central

    Correll, Kelly; Schiel, John A.; Finigan, Jay H.; Prekeris, Rytis; Mason, Robert J.

    2014-01-01

    There are 190,600 cases of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) each year in the United States, and the incidence and mortality of ALI/ARDS increase dramatically with age. Patients with ALI/ARDS have alveolar epithelial injury, which may be worsened by high-pressure mechanical ventilation. Alveolar type II (ATII) cells are the progenitor cells for the alveolar epithelium and are required to reestablish the alveolar epithelium during the recovery process from ALI/ARDS. Lung fibroblasts (FBs) migrate and proliferate early after lung injury and likely are an important source of growth factors for epithelial repair. However, how lung FBs affect epithelial wound healing in the human adult lung has not been investigated in detail. Hepatocyte growth factor (HGF) is known to be released mainly from FBs and to stimulate both migration and proliferation of primary rat ATII cells. HGF is also increased in lung tissue, bronchoalveolar lavage fluid, and serum in patients with ALI/ARDS. Therefore, we hypothesized that HGF secreted by FBs would enhance wound closure in alveolar epithelial cells (AECs). Wound closure was measured using a scratch wound-healing assay in primary human AEC monolayers and in a coculture system with FBs. We found that wound closure was accelerated by FBs mainly through HGF/c-Met signaling. HGF also restored impaired wound healing in AECs from the elderly subjects and after exposure to cyclic stretch. We conclude that HGF is the critical factor released from FBs to close wounds in human AEC monolayers and suggest that HGF is a potential strategy for hastening alveolar repair in patients with ALI/ARDS. PMID:24748602

  3. Hepatocyte Growth Factor Modification Enhances the Anti-Arrhythmic Properties of Human Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Du, Wei; Yu, Yi-Chao; Ju, Wei-Zhu; Man, Yi-Long; Li, Xiao-Rong; Chen, Yan; Wang, Zi-Dun; Gu, Wei-Juan; Zhang, Feng-Xiang; Wang, Hua; Wu, Chu-Tse; Cao, Ke-Jiang

    2014-01-01

    Background/Aims Chronic myocardial infarction (MI) results in the formation of arrhythmogenic substrates, causing lethal ventricular arrhythmia (VA). We aimed to determine whether mesenchymal stem cells (MSCs) carrying a hepatocyte growth factor (HGF) gene modification (HGF-MSCs) decrease the levels of arrhythmogenic substrates and reduce the susceptibility to developing VA compared with unmodified MSCs and PBS in a swine infarction model. Methods The left descending anterior artery was balloon-occluded to establish an MI model. Four weeks later, the randomly grouped pigs were administered MSCs, PBS or HGF-MSCs via thoracotomy. After an additional four weeks, dynamic electrocardiography was performed to assess heart rate variability, and programmed electrical stimulation was conducted to evaluate the risk for VA. Then, the pigs were euthanized for morphometric, immunofluorescence and western blot analyses. Results: The HGF-MSC group displayed the highest vessel density and Cx43 expression levels, and the lowest levels of apoptosis, and tyrosine hydroxylase (TH) and growth associated protein 43 (GAP43) expression. Moreover, the HGF-MSC group exhibited a decrease in the number of sympathetic nerve fibers, substantial decreases in the low frequency and the low-/high- frequency ratio and increases in the root mean square of successive differences (rMSSD) and the percentage of successive normal sinus R-R intervals longer than 50 ms (pNN50), compared with the other two groups. Finally, the HGF-MSC group displayed the lowest susceptibility to developing VA. Conclusion HGF-MSCs displayed potent antiarrhythmic effects, reducing the risk for VA. PMID:25360679

  4. Hepatocyte Transplantation

    PubMed Central

    Mitry, Ragai R; Hughes, Robin D; Dhawan, Anil

    2011-01-01

    Hepatocyte transplantation (HTx) has been developed for use in liver-based metabolic disorders and in acute liver failure. Worldwide, there are around 80 patients that have been transplanted with hepatocytes. Almost all reported studies prove feasibility and safety of the procedure with short- to medium-term success. Availability of good quality hepatocytes (HCs) is the main limiting factor, and therefore alternative sources of cells such as stem cells are being investigated. Other limiting factors include cell engraftment, survival, and function of transplanted cells. It remains to be seen if progress in HTx research can overcome these hurdles leading to the wider use of the technique as an alternative to liver transplantation in the future. PMID:25755322

  5. Compensatory adrenal growth - A neurally mediated reflex

    NASA Technical Reports Server (NTRS)

    Dallman, M. F.; Engeland, W. C.; Shinsako, J.

    1976-01-01

    The responses of young rats to left adrenalectomy or left adrenal manipulation were compared to surgical sham adrenalectomy in which adrenals were observed but not touched. At 12 h right adrenal wet weight, dry weight, DNA, RNA, and protein content were increased (P less than 0.05) after the first two operations. Left adrenal manipulation resulted in increased right adrenal weight at 12 h but no change in left adrenal weight. Sequential manipulation of the left adrenal at time 0 and the right adrenal at 12 h resulted in an enlarged right adrenal at 12 h (P less than 0.01), and an enlarged left adrenal at 24 h (P less than 0.05), showing that the manipulated gland was capable of response. Bilateral adrenal manipulation of the adrenal glands resulted in bilateral enlargement of 12 h (P less than 0.01). Taken together with previous results, these findings strongly suggest that compensatory adrenal growth is a neurally mediated reflex.

  6. Hepatocyte growth factor (HGF) signals through SHP2 to regulate primary mouse myoblast proliferation

    SciTech Connect

    Li, Ju; Reed, Sarah A.; Johnson, Sally E.

    2009-08-01

    Niche localized HGF plays an integral role in G{sub 0} exit and the return to mitotic activity of adult skeletal muscle satellite cells. HGF actions are regulated by MET initiated intracellular signaling events that include recruitment of SHP2, a protein tyrosine phosphatase. The importance of SHP2 in HGF-mediated signaling was examined in myoblasts and primary cultures of satellite cells. Myoblasts stably expressing SHP2 (23A2-SHP2) demonstrate increased proliferation rates by comparison to controls or myoblasts expressing a phosphatase-deficient SHP2 (23A2-SHP2DN). By comparison to 23A2 myoblasts, treatment of 23A2-SHP2 cells with HGF does not further increase proliferation rates and 23A2-SHP2DN myoblasts are unresponsive to HGF. Importantly, the effects of SHP2 are independent of downstream ERK1/2 activity as inclusion of PD98059 does not blunt the HGF-induced proliferative response. SHP2 function was further evaluated in primary satellite cell cultures. Ectopic expression of SHP2 in satellite cells tends to decrease proliferation rates and siSHP2 causes an increase the percentage of dividing myogenic cells. Interestingly, treatment of satellite cells with high concentrations of HGF (50 ng/ml) inhibits proliferation, which can be overcome by knockdown of SHP2. From these results, we conclude that HGF signals through SHP2 in myoblasts and satellite cells to directly alter proliferation rates.

  7. Hepatocyte growth factor reduces sensitivity to the epidermal growth factor receptor-tyrosine kinase inhibitor, gefitinib, in lung adenocarcinoma cells harboring wild-type EGFR

    PubMed Central

    Yang, Hua; Wang, Rong; Peng, Shunli; Chen, Longhua; Li, Qi; Wang, Wei

    2016-01-01

    Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy is an option for lung cancers harboring wild-type EGFR when chemotherapeutic reagents have failed. In this study, we found that the EGFR-TKI, gefitinib, modestly suppressed proliferation of the lung cancer cell lines, A549 and H358, which both harbor wild-type EGFR. Treatment with hepatocyte growth factor (HGF) reduced the sensitivity to gefitinib, whereas sensitivity was restored by treatment with an HGF antibody, a MET inhibitor, or depletion of MET but not ErbB3 gene. Moreover, both PI3K/mTOR inhibitors and MEK inhibitors suppressed proliferation of A549 cells, whereas only PI3K/mTOR inhibitors effectively suppressed cell viability of EGFR mutant PC-9 cells. Our findings suggest that HGF reduced the gefitinib sensitivity through MET and downstream PI3K and MAPK pathways. Combined use of EGFR-TKI and MET inhibitors or inhibition of downstream signaling molecules might be a better second or third line choice for a group of patients with advanced lung cancer harboring wild-type EGFR. PMID:26919104

  8. Liver Failure Impairs the Intrahepatic Elimination of Interleukin-6, Tumor Necrosis Factor-Alpha, Hepatocyte Growth Factor, and Transforming Growth Factor-Beta.

    PubMed

    Porowski, Dawid; Wirkowska, Agnieszka; Hryniewiecka, Ewa; Wyzgał, Janusz; Pacholczyk, Marek; Pączek, Leszek

    2015-01-01

    The strategic location of the liver and its metabolic activity make it a key organ regulating homeostasis. Our purpose was to examine its participation in removal of cytokines: interleukin-6 (Il-6), tumor necrosis factor-alpha (TNF-α), hepatocyte growth factor (HGF), and transforming growth factor-beta (TGF-β) from the portal circulation in human. 20 liver donors and 20 patients with end-stage liver failure were included in the study. Their blood was collected during liver transplantation from the portal, hepatic, and peripheral vein, and the hepatic artery and cytokines' concentrations were determined. Using the results the mathematical model of cytokine elimination by the liver was developed. In donors significantly lower levels of IL-6, TNF-α, HGF, and TGF-β were detected in portal blood compared to hepatic vein. In patients with cirrhosis there were no significant differences of IL-6, TNF-α, and TGF-β levels between portal and hepatic veins. Significantly higher level of HGF in hepatic compared to portal vein was observed. In healthy liver elimination of the cytokines prevailed over their synthesis, as reflected by the positive values of the elimination ratios. In the cirrhotic liver elimination ratios of Il-6, HGF, and TGF-β were negative indicating the prevalence of intrahepatic synthesis of cytokines over their removal. PMID:26090463

  9. Recombinant adenovirus containing hyper-interleukin-6 and hepatocyte growth factor ameliorates acute-on-chronic liver failure in rats

    PubMed Central

    Gao, Dan-Dan; Fu, Jia; Qin, Bo; Huang, Wen-Xiang; Yang, Chun; Jia, Bei

    2016-01-01

    AIM: To investigate the protective efficacy of recombinant adenovirus containing hyper-interleukin-6 (Hyper-IL-6, HIL-6) and hepatocyte growth factor (HGF) (Ad-HGF-HIL-6) compared to that of recombinant adenovirus containing either HIL-6 or HGF (Ad-HIL-6 or Ad-HGF) in rats with acute-on-chronic liver failure (ACLF). METHODS: The recombinant adenoviruses containing HIL-6 and/or HGF were constructed. We established an ACLF model, and rats were randomly assigned to control, model, Ad-GFP, Ad-HIL-6, Ad-HGF or Ad-HGF-HIL-6 group. We collected serum and liver tissue samples to test pathological changes, biochemical indexes and molecular biological indexes. RESULTS: Attenuated alanine aminotransferase, prothrombin time, high-mobility group box 1 (HMGB1), endotoxin, tumour necrosis factor (TNF)-α and interferon-γ were observed in the Ad-HGF-, Ad-HIL-6- and Ad-HGF-HIL-6-treated rats with ACLF. Likewise, reduced hepatic damage and apoptotic activity, as well as reduced HMGB1 and Bax proteins, but raised expression of Ki67 and Bcl-2 proteins and Bcl-2/Bax ratio were also observed in the Ad-HGF-, Ad-HIL-6- and Ad-HGF-HIL-6-treated rats with ACLF. More significant changes were observed in the Ad-HGF-HIL-6 treatment group without obvious side effects. Furthermore, caspase-3 at the protein level decreased in the Ad-HIL-6 and Ad-HGF-HIL-6 treatment groups, more predominantly in the latter group. CONCLUSION: This study identifies that the protective efficacy of Ad-HGF-HIL-6 is more potent than that of Ad-HGF or Ad-HIL-6 in ACLF rats, with no significant side effects. PMID:27122664

  10. Met inactivation by S-allylcysteine suppresses the migration and invasion of nasopharyngeal cancer cells induced by hepatocyte growth factor

    PubMed Central

    Cho, Oyeon; Hwang, Hye-Sook; Lee, Bok-Soon; Oh, Young-Taek

    2015-01-01

    Purpose Past studies have reported that S-allylcysteine (SAC) inhibits the migration and invasion of cancer cells through the restoration of E-cadherin, the reduction of matrix metalloproteinase (MMP) and Slug protein expression, and inhibition of the production of reactive oxygen species (ROS). Furthermore, evidence is emerging that shows that ROS induced by radiation could increase Met activation. Following on these reports of SAC and Met, we investigated whether SAC could suppress Met activation. Materials and Methods Wound healing, invasion, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT), soft agar colony forming, western blotting, and gelatin zymography assays were performed in the human nasopharyngeal cancer cell lines HNE1 and HONE1 treated with SAC (0, 10, 20, or 40 mM) and hepatocyte growth factor (HGF). Results This study showed that SAC could suppress the migration and invasion of HNE1 and HONE1 cell lines by inhibiting p-Met. An increase of migration and invasion induced by HGF and its decrease in a dose dependent manner by SAC in wound healing and invasion assays was observed. The reduction of p-Met by SAC was positively correlated with p-focal adhesion kinase (p-FAK) and p-extracellular related kinase (p-ERK in both cell lines). SAC reduced Slug, MMP2, and MMP9 involved in migration and invasion with the inhibition of Met-FAK signaling. Conclusion These results suggest that SAC inhibited not only Met activation but also the downstream FAK, Slug, and MMP expression. Finally, SAC may be a potent anticancer compound for nasopharyngeal cancer treated with radiotherapy. PMID:26756033

  11. Involvement of PI3K and ERK1/2 pathways in hepatocyte growth factor-induced cholangiocarcinoma cell invasion

    PubMed Central

    Menakongka, Apaporn; Suthiphongchai, Tuangporn

    2010-01-01

    AIM: To investigate the role of hepatocyte growth factor (HGF) in cholangiocarcinoma (CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS: Effects of HGF on cell invasion and motility were investigated in two human CCA cell lines, HuCCA-1 and KKU-M213, using Transwell in vitro assay. Levels of proteins of interest and their phosphorylated forms were determined by Western blotting. Localization of E-cadherin was analyzed by immunofluorescence staining and visualized under confocal microscope. Activities of matrix degrading enzymes were determined by zymography. RESULTS: Both CCA cell lines expressed higher Met levels than the H69 immortalized cholangiocyte cell line. HGF induced invasion and motility of the cell lines and altered E-cadherin from membrane to cytoplasm localization, but did not affect the levels of secreted matrix metalloproteinase (MMP)-2, MMP-9 and urokinase plasminogen activator, key matrix degrading enzymes involved in cell invasion. Concomitantly, HGF stimulated Akt and extracellular signal-regulated kinase (ERK)1/2 phosphorylation but with slightly different kinetic profiles in the two cell lines. Inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway by the PI3K inhibitor, LY294002, markedly suppressed HGF-stimulated invasion of both CCA cell lines, and inhibition of the ERK pathway by U0126 suppressed HGF-induced invasion of the KKU-M213 cell line but had a moderate effect on HuCCA-1 cells. CONCLUSION: These data indicate that HGF promotes CCA cell invasiveness through dys-localization of E-cadherin and induction of cell motility by distinct signaling pathways depending on cell line type. PMID:20135719

  12. Effect of semisynthetic extracellular matrix-like hydrogel containing hepatocyte growth factor on repair of femoral neck defect in rabbits.

    PubMed

    Liu, Pengfei; Guo, Lin; Huang, Lanfeng; Zhao, Dewei; Zhen, Ruixin; Hu, Xiaoning; Yuan, Xiaolin

    2015-01-01

    Using tissue engineering technology research to develop organized artificial bone, then repair bone defect. This work aims to investigate the role of semisynthetic extracellular matrix-like hydrogel (sECMH) containing hepatocyte growth factor (HGF) on repair of femoral neck defect in rabbits. 18 New Zealand rabbits were used in this study. According to autologous paired comparison method, the left and right sides of rabbit were used as control and experimental side, respectively. The models of bilateral femoral neck bone defect were established. In experimental side, sECMH containing HGF was implanted in the defect area. In control side, no material was implanted in the defect area. At the 2nd, 4th and 8th week after surgery, the gross observation, histological examination and molybdenum target (Mo-target) X-ray examination were performed on the specimens to study the repair of femoral neck defect. In gross observation, there was no macroscopic difference of femoral neck specimen between the 2nd and 4th postoperative week. At the 8th week, the defect orifice was closed with immature cortical bone, with unblocked marrow cavity. HE staining results showed that, at the 4th week, there were more new vessels in defect area of experimental side, compared with control side. At the 8th week, in experimental side there was immature cortical bone connecting the fracture end in defect area, with visible bone marrow cells. Mo-target X-ray examination found that, at the 8th week, the bone tissue repair in experimental side was better than control side. As a new drug delivery system, sECMH containing HGF has good application prospect in bone tissue repair. PMID:26221278

  13. Enhanced cell survival and paracrine effects of mesenchymal stem cells overexpressing hepatocyte growth factor promote cardioprotection in myocardial infarction.

    PubMed

    Zhao, Liyan; Liu, Xiaolin; Zhang, Yuelin; Liang, Xiaoting; Ding, Yue; Xu, Yan; Fang, Zhen; Zhang, Fengxiang

    2016-05-15

    Poor cell survival post transplantation compromises the therapeutic benefits of mesenchymal stem cells (MSCs) in myocardial infarction (MI). Hepatocyte growth factor (HGF) is an important cytokine for angiogenesis, anti-inflammation and anti-apoptosis. This study aimed to evaluate the cardioprotective effects of MSCs overexpressing HGF in a mouse model of MI. The apoptosis of umbilical cord-derived MSCs (UC-MSCs) and HGF-UC-MSCs under normoxic and hypoxic conditions was detected. The conditioned medium (CdM) of UC-MSCs and HGF-UC-MSCs under a hypoxic condition was harvested and its protective effect on neonatal cardiomyocytes (NCMs) exposed to a hypoxic challenge was examined. UC-MSCs and HGF-UC-MSCs were transplanted into the peri-infarct region in mice following MI and heart function assessed 4 weeks post transplantation. The apoptosis of HGF-UC-MSCs under hypoxic conditions was markedly decreased compared with that of UC-MSCs. NCMs treated with HGF-UC-MSC hypoxic CdM (HGF-UC-MSCs-hy-CdM) exhibited less cell apoptosis in response to hypoxic challenge than those treated with UC-MSC hypoxic CdM (UC-MSCs-hy-CdM). HGF-UC-MSCs-hy-CdM released the inhibited p-Akt and lowered the enhanced ratio of Bax/Bcl-2 induced by hypoxia in the NCMs. HGF-UC-MSCs-hy-CdM expressed higher levels of HGF, EGF, bFGF and VEGF than UC-MSCs-hy-CdM. Transplantation of HGF-UC-MSCs or UC-MSCs greatly improved heart function in the mouse model of MI. Compared with UC-MSCs, transplantation of HGF-UC-MSCs was associated with less cardiomyocyte apoptosis, enhanced angiogenesis and increased proliferation of cardiomyocytes. This study may provide a novel therapeutic strategy for MSC-based therapy in cardiovascular disease. PMID:27025401

  14. The hepatocyte growth factor isoform NK2 activates motogenesis and survival but not proliferation due to lack of Akt activation.

    PubMed

    Mungunsukh, Ognoon; Lee, Young H; Bottaro, Donald P; Day, Regina M

    2016-08-01

    Hepatocyte growth factor (HGF) is a pleiotrophic factor involved in cellular proliferation, migration and morphogenesis. HGF is required for normal tissue and organ development during embryogenesis, but in the adult HGF has been demonstrated to drive normal tissue repair and inhibit fibrotic remodeling. HGF has two naturally occurring human isoforms as a result of alternative splicing, NK1 and NK2. While NK1 has been defined as an agonist for HGF receptor, Met, NK2 is defined as a partial Met antagonist. Furthermore, under conditions of fibrotic remodeling, NK2 is still expressed while full length HGF is suppressed. Furthermore, the mechanism by which NK2 partially signals through Met is not completely understood. Here, we investigated the mitogenic, motogenic, and anti-apoptotic activities of NK2 compared with full length HGF in primary human bronchial epithelial cells (BEpC) and bovine pulmonary artery endothelial cells (PAEC). In human BEpC, NK2 partial activated Met, inducing Met phosphorylation at Y1234/1235 in the tyrosine-kinase domain but not at Y1349 site in the multifunctional docking domain. Partial phosphorylation of Met by NK2 resulted in activation of MAPK and STAT3, but not AKT. This correlated with motogenesis and survival in a MAPK-dependent manner, but not cell proliferation. Overexpression of a constitutively active AKT complemented NK2 signaling, allowing NK2 to induce cell proliferation. These data indicate that NK2 and HGF drive motogenic and anti-apoptotic signaling but only HGF drives cell proliferation by activating AKT-pathway signaling. These results have implications for the biological consequences of differential regulation of the two isoforms under pro-fibrotic conditions. PMID:27224506

  15. Secretory expression and characterization of a recombinant-deleted variant of human hepatocyte growth factor in Pichia pastoris

    PubMed Central

    Liu, Zhi-Min; Zhao, Hong-Liang; Xue, Chong; Deng, Bing-Bing; Zhang, Wei; Xiong, Xiang-Hua; Yang, Bing-Fen; Yao, Xue-Qin

    2005-01-01

    AIM: To study the secretory expression of human hepatocyte growth factor (hdHGF) gene in Pichia pastoris. METHODS: The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coli-yeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GS115 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Mut+ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor. RESULTS: The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g (DCW)/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L. CONCLUSION: The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes. PMID:16437654

  16. Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

    SciTech Connect

    Nagata, Takayuki; Murata, Kazuko; Murata, Ryo; Sun, Shu-lan; Saito, Yutaro; Yamaga, Shuhei; Tanaka, Nobuyuki; Tamai, Keiichi; Moriya, Kunihiko; Kasai, Noriyuki; Sugamura, Kazuo; Ishii, Naoto

    2014-01-10

    Highlights: •ESCRT-0 protein regulates the development of peripheral B-cells. •BCR expression on cell surface should be controlled by the endosomal-sorting system. •Hrs plays important roles in responsiveness to Ag stimulation in B lymphocytes. -- Abstract: Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs{sup flox/flox};mb1{sup cre/+}:Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

  17. Integrin-linked kinase (ILK) modulates wound healing through regulation of hepatocyte growth factor (HGF)

    SciTech Connect

    Serrano, Isabel; Diez-Marques, Maria L.; Rodriguez-Puyol, Manuel; Herrero-Fresneda, Inmaculada; Garcia del Moral, Raimundo; Dedhar, Shoukat; Ruiz-Torres, Maria P.; Rodriguez-Puyol, Diego

    2012-11-15

    Integrin-linked kinase (ILK) is an intracellular effector of cell-matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The present work analyzes the role of ILK in wound healing in adult animals using a conditional knock-out of the ILK gene generated with the tamoxifen-inducible Cre-lox system (CRE-LOX mice). Results show that ILK deficiency leads to retarded wound closure in skin. Intracellular mechanisms involved in this process were analyzed in cultured mouse embryonic fibroblast (MEF) isolated from CRE-LOX mice and revealed that wounding promotes rapid activation of phosphatidylinositol 3-kinase (PI3K) and ILK. Knockdown of ILK resulted in a retarded wound closure due to a decrease in cellular proliferation and loss of HGF protein expression during the healing process, in vitro and in vivo. Alterations in cell proliferation and wound closure in ILK-deficient MEF or mice could be rescued by exogenous administration of human HGF. These data demonstrate, for the first time, that the activation of PI3K and ILK after skin wounding are critical for HGF-dependent tissue repair and wound healing. -- Highlights: Black-Right-Pointing-Pointer ILK deletion results in decreased HGF expression and delayed scratch wound repair. Black-Right-Pointing-Pointer PI3K/ILK/AKT pathway signals through HGF to regulate wound healing. Black-Right-Pointing-Pointer An ILK-dependent increase in HGF expression is responsible for wound healing in vivo. Black-Right-Pointing-Pointer ILK-KO mice are used to confirm the requirement for ILK function in wound healing. Black-Right-Pointing-Pointer Human HGF treatment restores delayed wound closure in vitro and in vivo.

  18. Effect of size and conformation of the ligand on asialoglycoprotein receptor-mediated ligand internalization and degradation in rat hepatocytes

    SciTech Connect

    Chang, C.H.; Chang, T.M.

    1987-05-01

    The rates of internalization and degradation of /sup 125/-I-labeled desialylated cyanogen bromide fragment I of orosomucoid (AS-CNBr-I) and its reduced and carboxymethylated derivative (AS-RC-CNBr-I) were compared with those of /sup 125/I-labeled asialoorosomucoid (ASOR) in rat hepatocytes. At 30 nM the rates of internalization and degradation of /sup 125/I-AS-CNBr-I were greater than those of /sup 125/I-ASOR. /sup 125/I-AS-RC-CNBr-I also had a lower rate of internalization and degradation. In contrast to /sup 125/I-ASOR, when degradation was inhibited by 5 ..mu..M colchicine there was a significant intracellular accumulation of the smaller ligands. At 4/sup 0/C the hepatocytes were found to bind the fragmented ligands more than /sup 125/I-ASOR. Incubation of the cells with bound ligand at 37/sup 0/ indicated that diacytosis of /sup 125/I-ASOR was greater than the smaller ligands. Colchincine markedly enhanced diacytosis of /sup 125/I-ASOR. On the other hand, there were marked accumulation of the smaller ligands by colchicine. These results suggest that the rates of internalization, degradation and diacytosis of the ligand are affected by the size and conformation of the ligand through different rates of receptor binding and intracellular transport.

  19. Conformational Lability in Serine Protease Active Sites: Structures of Hepatocyte Growth Factor Activator (HGFA) Alone and with the Inhibitory Domain from HGFA Inhibitor-1B

    SciTech Connect

    Shia, Steven; Stamos, Jennifer; Kirchhofer, Daniel; Fan, Bin; Wu, Judy; Corpuz, Raquel T.; Santell, Lydia; Lazarus, Robert A.; Eigenbrot, Charles

    2010-07-20

    Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 {angstrom} resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 {angstrom} resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.

  20. Ex Vivo Stromal Cell-Derived Factor 1-Mediated Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells into Hepatocytes Is Enhanced by Chinese Medicine Yiguanjian Drug-Containing Serum

    PubMed Central

    Fu, Linlin; Pang, Bingyao; Zhu, Ying; Wang, Ling; Leng, Aijing; Chen, Hailong

    2016-01-01

    Yiguanjian is administered in traditional Chinese medicine for liver diseases and has been demonstrated to reduce liver fibrosis. This study investigated the effect of Yiguanjian drug-containing serum (YGJ) with Stromal Cell-Derived Factor 1 (SDF-1) and Hepatocyte Growth Factor (HGF) on the differentiation of murine bone-marrow-derived mesenchymal cells (BM-MSCs) into hepatocytes in vitro. Adherent MSCs were isolated from murine bone marrow. Differentiation was induced by 20 ng/mL HGF, 50 ng/mL SDF-1, and 20% Yiguanjian drug-containing serum for 7 to 28 days, and mature hepatocytes' marker albumin (ALB) and cholangiocytes' marker cytokeratin-18 (CK-18) were assessed by immunocytochemistry and western blot. BM-MSCs exhibited homogeneous spindle shape growth after subculture and stained positive for CD90 and negative for CD34. After induction with HGF + normal serum or YGJ for 14 days, HGF + SDF-1 + normal serum for 7 days, or HGF + SDF-1 + YGJ for 5 days, MSCs' morphology changed gradually and begun to resemble hepatocyte-like cells. Cultures supplemented with HGF + SDF-1 + YGJ contained significantly higher proportions of ALB and CK-18 positive cells than cultures supplemented with HGF + SDF-1 + normal serum at day 7. These observations corroborated the results of western blot. In conclusion, Yiguanjian drug-containing serum could facilitate the differentiation of murine BM-MSCs into hepatocytes in vitro and has a synergistic effect with SDF-1 and HGF. PMID:27190538

  1. Valproic acid overcomes transforming growth factor-β-mediated sorafenib resistance in hepatocellular carcinoma

    PubMed Central

    Matsuda, Yasunobu; Wakai, Toshifumi; Kubota, Masayuki; Osawa, Mami; Hirose, Yuki; Sakata, Jun; Kobayashi, Takashi; Fujimaki, Shun; Takamura, Masaaki; Yamagiwa, Satoshi; Aoyagi, Yutaka

    2014-01-01

    Sorafenib is a multi-kinase inhibitor approved for hepatocellular carcinoma, but rarely causes tumor regression in patients with chronic liver diseases. To investigate whether growth factor-mediated signaling is involved in sorafenib resistance, HepG2 and PLC/PRF/5 hepatoma cells were exposed to epidermal growth factor (EGF), hepatocyte growth factor (HGF) or transforming growth factor-β (TGF-β) prior to treatment with sorafenib. Furthermore, to identify an effective combination treatment with sorafenib, growth factor-sensitized cells were treated with sorafenib alone or in combination with celecoxib, lovastatin or valproic acid (VPA). Trypan blue staining and Annexin V assays showed that the cytotoxic effect of sorafenib was inhibited by 15-54% in cells sensitized to TGF-β (P<0.05). Western blotting analysis showed that TGF-β significantly activated extracellular signal-regulated kinase (ERK)-mediated AKT signaling, and sorafenib failed to suppress both ERK and AKT in TGF-β-sensitized cells. The decreased anti-tumor effect of sorafenib was rescued by chemical inhibition of ERK and AKT. When TGF-β-sensitized cells were treated with sorafenib plus VPA, the levels of phosphorylated ERK and AKT were considerably suppressed and the numbers of dead cells were increased by 3.7-5.7-fold compared with those exposed to sorafenib alone (P<0.05). Moreover, low dose sorafenib-induced cell migration was effectively suppressed by combination treatment with sorafenib and VPA. Collectively, TGF-β/ERK/AKT signaling might play a critical role in sorafenib resistance in hepatoma cells, and combination treatment with VPA may be effective against this drug resistance. PMID:24817927

  2. Growth Factor Mediated Signaling in Pancreatic Pathogenesis

    PubMed Central

    Nandy, Debashis; Mukhopadhyay, Debabrata

    2011-01-01

    Functionally, the pancreas consists of two types of tissues: exocrine and endocrine. Exocrine pancreatic disorders mainly involve acute and chronic pancreatitis. Acute pancreatitis typically is benign, while chronic pancreatitis is considered a risk factor for developing pancreatic cancer. Pancreatic carcinoma is the fourth leading cause of cancer related deaths worldwide. Most pancreatic cancers develop in the exocrine tissues. Endocrine pancreatic tumors are more uncommon, and typically are less aggressive than exocrine tumors. However, the endocrine pancreatic disorder, diabetes, is a dominant cause of morbidity and mortality. Importantly, different growth factors and their receptors play critical roles in pancreatic pathogenesis. Hence, an improved understanding of how various growth factors affect pancreatitis and pancreatic carcinoma is necessary to determine appropriate treatment. This chapter describes the role of different growth factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor (TGF) in various pancreatic pathophysiologies. Finally, the crosstalk between different growth factor axes and their respective signaling mechanisms, which are involved in pancreatitis and pancreatic carcinoma, are also discussed. PMID:24212642

  3. Effect of glucocorticoids, insulin and a growth promoting tripeptide on the biosynthesis of plasma proteins in serum-free hepatocyte cultures.

    PubMed

    Fouad, F M; Abd-El-Fattah, M; Scherer, R; Ruthenstroth-Bauer, G

    1981-01-01

    The effect of cortisol, dexamethasone, insulin and a liver cell growth promoting tripeptide on the secretion of plasma proteins into the medium of rat hepatocytes in monolayer cultures was studied. Cortisol and dexamethasone resulted in equal to or approximately 2.5-fold increase in the fibrinogen synthesis with general suppression of albumin and alpha-lipoprotein synthesis. On the other hand, insulin inhibited the biosynthesis of most plasma proteins except for the complement system and transferrin. Concentrations of alpha-lipoprotein, alpha-1-macroglobulin and haptoglobin were moderately elevated when the tripeptide Gly-His-Lys was applied in low concentration. PMID:7018103

  4. CARDIOVASCULAR MAGNETIC RESONANCE IMAGING IN DELIVERING AND EVALUATING THE EFFICACY OF HEPATOCYTE GROWTH FACTOR GENE IN CHRONIC INFARCT SCAR

    PubMed Central

    Saeed, Maythem; Saloner, David; Do, Loi; Wilson, Mark; Martin, Alastair

    2012-01-01

    Background In open-chest model of acute infarct, epicardial delivery of hepatocyte growth factor (HGF) gene improved LV function. This study was designed to test 1) the efficacy of HGF gene in infarct scar delivered under MR guidance and 2) the potential of multiple MR sequences in assessing the effects of pCK-HGF (treatment) and pCK-LacZ (control) genes on myocardial structure and function. Methods and Materials Swine (6 per group) were subjected to myocardial infarct, under X-ray fluoroscopy, developed LV remodelling at 5weeks. Multiple clinical MR imaging sequences were performed before delivery of gene (at 5 weeks after infarction) and 5 weeks after delivery of gene. Under MR-guidance, the active endovascular catheter was introduced into LV to transendocardially deliver 3.96×1011 viral copies of pCK-HGF or pCK-LacZ in the border and core of the infarct scar. Histological evaluation of the infarct scar was performed 5 weeks after delivery of gene. Results At 5weeks after infarction, there was no significant difference in measured cardiovascular MR parameters between the groups. PCK-HGF gene caused significant improvement in the following parameters (P<0.05 for these parameters): 3D strain (radial, circumferential, and longitudinal) , perfusion (maximum upslope, peak signal intensity, and time to peak) compared with control pCK-LacZ at 5 weeks after delivery of the genes. The ejection fraction was higher in pCK-HGF treated (43±1%) than pCK-LacZ control (37±1%, P<0.05). These changes are associated with a decrease in infarct scar size (11.3±2.0% in pCK-LacZ control and 6.7±1.3%, in pCK-HGF treated, P<0.01) and transmurality in 4 out of 5 infarct scar segments (P<0.05) on DE-MR imaging. Microscopic study confirmed the increase in capillary (P<0.05), and arteriole (P<0.05) density of infarct scar in pCK-HGF treated compared with pCK-LacZ control animals. Conclusions HGF gene delivered under MR-guidance into infarct scar ameliorated global function, 3D strain

  5. Mechanisms of cortisol action in fish hepatocytes.

    PubMed

    Faught, Erin; Vijayan, Mathilakath M

    2016-09-01

    Here we provide an overview of the mechanistic characterization of the hepatic action of cortisol during stress in fish. Cortisol is the main circulating glucocorticoid in fish and its action is mediated through its cytosolic receptor, the glucocorticoid receptor (GR), and regulates the expression of genes involved in growth, metabolism and immune function. When taken together, the data suggests that cortisol may be playing a key role in the energy substrate re-partitioning in hepatocytes to cope with stress. The proposed model is that cortisol upregulates pathways involved in energy substrate mobilization, including gluconeogenesis, while downregulating energy demanding pathways, including growth and immune function. Recent work also points to a role for cortisol in mediating rapid action that is non-genomic and includes modulation of secondary signalling cascades; however, the physiological relevance of these studies remains to be determined. Altogether, studies carried out in hepatocytes are bringing to fore the complex nature of the cortisol signalling pathways in the organismal stress response. The mode of actions and their physiological implications for stress coping awaits further study. PMID:27445122

  6. Estradiol-17beta protects against hypoxia-induced hepatocyte injury through ER-mediated upregulation of Bcl-2 as well as ER-independent antioxidant effects.

    PubMed

    Lee, Min Young; Jung, Sun Chul; Lee, Jang Hern; Han, Ho Jae

    2008-04-01

    Although many previous studies have suggested that estrogen functions as a cytoprotective agent under oxidative stress conditions, the underlying mechanism by which this effect is exerted remains to be elucidated. This study assessed the effects of estradiol-17beta (E(2)) (10(-8) M) on hypoxia-induced cell injury and its related signaling in primary cultured chicken hepatocytes. Hypoxic conditions were found to augment the level of DNA damage and to reduce cell viability and the level of [(3)H]-thymidine incorporation, and these phenomena were prevented through treatment with E(2). Hypoxia also increased caspase-3 expression, but showed no evidence of an influence on the expression of Bcl-2. However, E(2) induced an increase in the level of Bcl-2 expression under hypoxic conditions and reduced the level of caspase-3 expression. The effects of E(2) on Bcl-2 and caspase expression were blocked by ICI 182780 (E(2) receptor (ER) antagonist, 10(-7) M). In addition, hypoxia resulted in an increase in the intracellular reactive oxygen species (ROS) generated. These effects were blocked by E(2), but not by E(2)-BSA and ICI 182780. Hypoxia also activated p38 mitogen-activated protein kinase (MAPK), c-JUN N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and nuclear factor-kappaB (NF-kappaB). These effects were blocked by E(2), but not by ICI 182780. The inhibition of p38 MAPK and JNK/SAPK blocked NF-kappaB activation. In conclusion, E(2) was found to protect against hypoxia-induced cell injury in chicken hepatocytes through ER-mediated upregulation of Bcl-2 expression and through reducing the activity of ROS-dependent p38 MAPK, JNK/SAPK and NF-kappaB. PMID:18379592

  7. Hawthorn (Crataegus oxyacantha L.) bark extract regulates antioxidant response element (ARE)-mediated enzyme expression via Nrf2 pathway activation in normal hepatocyte cell line.

    PubMed

    Krajka-Kuźniak, Violetta; Paluszczak, Jarosław; Oszmiański, Jan; Baer-Dubowska, Wanda

    2014-04-01

    Hawthorn (Crataegus oxyacantha L.), a plant used in traditional medicine, is a rich source of procyanidins which have been reported to exhibit antioxidant and anti-carcinogenic activity. In this study, we assessed the effect of hawthorn bark extract (HBE) on Nrf2 pathway activation in THLE-2 and HepG2 cells. Treatment with 1.1 µg/mL, 5.5 µg/mL and 11 µg/mL of HBE resulted in the translocation of Nrf2 from the cytosol to the nucleus in both cell lines; however, the accumulation of phosphorylated Nrf2 was observed only in THLE-2. Accordingly, treatment of cells with HBE was associated with an increase in the mRNA and protein level of such Nrf2-dependent genes as glutathione S-transferases (GSTA, GSTP, GSTM, GSTT), NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) (0.2-1.1-fold change, p < 0.05), however, only in normal THLE-2 hepatocytes. The induction of NQO1 correlated with an increased level of p53 (0.21-0.42-fold change, p < 0.05). These effects may be related to induction of phosphorylation of upstream ERK and JNK kinases. Collectively, the results suggest that the Nrf2/ARE pathway may play an important role in the regulation of procyanidin-mediated antioxidant/detoxifying effects in hepatocytes, and this may explain the hepatoprotective and chemopreventive properties of these phytochemicals. PMID:23843400

  8. Ephedrae herba stimulates hepatocyte growth factor-induced MET endocytosis and downregulation via early/late endocytic pathways in gefitinib-resistant human lung cancer cells.

    PubMed

    Nishimura, Yukio; Hyuga, Sumiko; Takiguchi, Soichi; Hyuga, Masashi; Itoh, Kazuyuki; Hanawa, Toshihiko

    2016-05-01

    The MET tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), are known to be overexpressed in a variety of malignant tumor cells, and are implicated in the development of gefitinib-resistance in human non-small cell lung cancer (NSCLC) cells. Ephedrae herba was previously reported to prevent HGF-induced cancer cell motility by directly suppressing HGF/MET signaling through the inhibition of MET tyrosine kinase, and treatment with its extract also considerably reduced MET protein levels. To further investigate the mechanism underlying the Ephedrae herba-induced inhibition of MET phosphorylation as well as its degradation and subsequent disappearance, we examined the effect of Ephedrae herba on HGF-stimulated MET endocytosis and downregulation via early/late endocytic pathways in an NSCLC cell line. Using immunofluorescence microscopy, we found that pretreatment of cells with Ephedrae herba extract dramatically changed the intracellular distribution of plasma membrane-associated MET, and that the resultant MET staining was distributed throughout the cytoplasm. Pretreatment of the cells with Ephedrae herba extract also led to the rapid loss of MET and phosphorylated (p)-MET in HGF-stimulated cells. In contrast, inefficient endocytic delivery of MET and p-MET from early to late endosomes was observed in the absence of Ephedrae herba extract, since considerable amounts of the internalized MET accumulated in the early endosomes and were not delivered to lysosomes up to 1 h after HGF-stimulation. Furthermore, large amounts of MET and p-MET that had accumulated in late endosomes of Ephedrae herba-pretreated cells after HGF stimulation were observed along with bafilomycin A1. Therefore, we inferred that degradation of MET occurred in the late endosome/lysosome pathway. Moreover, western blot analysis revealed the accelerated degradation of MET and p-MET proceeds in cells pretreated with Ephedrae herba extract. Collectively, our results suggest that

  9. SPONTANEOUS REPOPULATION OF β-CATENIN NULL LIVERS WITH β-CATENIN POSITIVE HEPATOCYTES AFTER CHRONIC MURINE LIVER INJURY

    PubMed Central

    Thompson, Michael D.; Wickline, Emily D.; Bowen, William B.; Lu, Amy; Singh, Sucha; Misse, Amalea; Monga, Satdarshan P. S.

    2011-01-01

    Prolonged exposure of mice to diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) results in hepatobiliary injury, atypical ductular proliferation, oval cell appearance and limited fibrosis. Previously, we reported that short-term ingestion of DDC diet by hepatocyte-specific β-catenin conditional knockout (KO) mice, led to fewer A6-positive oval cells than wild-type (WT) littermates. To examine the role of β-catenin in chronic hepatic injury and repair, we exposed WT and KO mice to DDC for 80 and 150 days. Paradoxically, long-term DDC exposure led to significantly more A6-positive cells indicating greater atypical ductular proliferation in KO, which coincided with increased fibrosis and cholestasis. Surprisingly, at 80 and 150 days in KO, we observed a significant amelioration of hepatocyte injury. This coincided with extensive repopulation of β-catenin null livers with β-catenin-positive hepatocytes at 150 days, which was preceded by appearance of β-catenin-positive hepatocyte clusters at 80 days and a few β-catenin-positive hepatocytes at earlier times. Intriguingly, occasional β-catenin-positive hepatocytes that were negative for progenitor markers were also observed at baseline in the KO livers suggesting spontaneous escape from cre-mediated recombination. These cells with hepatocyte morphology expressed mature hepatocyte markers but lacked markers of hepatic progenitors. The gradual repopulation of KO livers with β-catenin-positive hepatocytes occurred only following DDC injury and coincided with a progressive loss of hepatic cre-recombinase expression. A few β-catenin-positive cholangiocytes were observed albeit only after long-term DDC-exposure and trailed the appearance of β-catenin-positive hepatocytes. In conclusion, in a chronic liver injury model, β-catenin-positive hepatocytes exhibit growth and survival advantages and repopulate KO livers eventually limiting hepatic injury and dysfunction despite increased fibrosis and

  10. Modulation of aflatoxin B1-mediated genotoxicity in primary cultures of human hepatocytes by diindolylmethane, curcumin, and xanthohumols.

    PubMed

    Gross-Steinmeyer, Kerstin; Stapleton, Patricia L; Tracy, Julia H; Bammler, Theo K; Strom, Stephen C; Buhler, Donald R; Eaton, David L

    2009-12-01

    This study employed cultured human primary hepatocytes to investigate the ability of the putative chemopreventive phytochemicals curcumin (CUR), 3,3'-diindolylmethane (DIM), isoxanthohumol (IXN), or 8-prenylnaringenin (8PN) to reduce DNA adduct formation of the hepatocarcinogen aflatoxin B1 (AFB). Following 48 h of pretreatment, DIM and 8PN significantly increased AFB-DNA adduct levels, whereas CUR and IXN had no effect. DIM greatly enhanced the transcriptional expression of cytochrome P450 (CYP) 1A1 and CYP1A2 mRNA. Glutathione S-transferase mRNAs were not increased by any of the treatments. In vitro enzyme activity assays demonstrated that 8PN and DIM, but not CUR or IXN, inhibited human CYP1A1, CYP1A2, and CYP3A4 activities. To distinguish between treatment effects on transcription versus direct effects on enzyme activity for DIM, we evaluated the effects of pretreatment alone (transcriptional activation) versus cotreatment alone (enzyme inhibition). The results demonstrated that effects on gene expression, but not catalytic activity, are responsible for the observed effects of DIM on AFB-DNA adduct formation. The increase in AFB-DNA damage following DIM treatment may be explained through its substantial induction of CYP1A2 and/or its downregulation of GSTM1, both of which were significant. The increase in DNA damage by DIM raises potential safety risks for dietary supplements of DIM and its precursor indole-3-carbinol. PMID:19770484

  11. Synergistic effects of tethered growth factors and adhesion ligands on DNA synthesis and function of primary hepatocytes cultured on soft synthetic hydrogels

    PubMed Central

    Mehta, Geeta; Williams, Courtney M.; Alvarez, Luis; Lesniewski, Martha; Kamm, Roger D.; Griffith, Linda G.

    2010-01-01

    The composition, presentation, and spatial orientation of extracellular matrix molecules and growth factors are key regulators of cell behavior. Here, we used self-assembling peptide nanofiber gels as a modular scaffold to investigate how fibronectin-derived adhesion ligands and different modes of epidermal growth factor (EGF) presentation synergistically regulate multiple facets of primary rat hepatocyte behavior in the context of a soft gel. In the presence of soluble EGF, inclusion of dimeric RGD and the heparin binding domain from fibronectin (HB) increased hepatocyte aggregation, spreading, and metabolic function compared to unmodified gels or gels modified with a single motif, but unlike rigid substrates, gels failed to induce DNA synthesis. Tethered EGF dramatically stimulated cell aggregation and spreading under all adhesive ligand conditions and also preserved metabolic function. Surprisingly, tethered EGF elicited DNA synthesis on gels with RGD and HB. Phenotypic differences between soluble and tethered EGF stimulation of cells on peptide gels are correlated with differences in expression and phosphorylation the EGF receptor and its heterodimerization partner ErbB2, and activation of the downstream signaling node ERK1/2. These modular matrices reveal new facets of hepatocellular biology in culture and may be more broadly useful in culture of other soft tissues. PMID:20304480

  12. Strategies for immortalization of primary hepatocytes

    PubMed Central

    Eva, Ramboer; Bram, De Craene; Joery, De Kock; Tamara, Vanhaecke; Geert, Berx; Vera, Rogiers; Mathieu, Vinken

    2014-01-01

    The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications. PMID:24911463

  13. Growth mediated feedback and the abrupt onset of antibiotic resistance

    NASA Astrophysics Data System (ADS)

    Barrett Deris, J.

    2010-03-01

    Recent results in our lab indicate that global gene expression will change in a growth-dependent manner for bacteria in sublethal antibiotic levels. We analyzed a system containing a constitutively expressed drug resistance gene and found that growth-mediated feedback provided a mechanism for bistable growth rates. That is, two identical cell-lines in the same antibiotic-infused media may respond with distinct growth rates. Our experimental work with cells carrying this resistance gene has shown that a rapid drop in growth occurs over a relatively small range of antibiotic. This result is consistent with a growth plateau arising in our analysis of the feedback mechanism. Furthermore, experiments have shown that a culture's degree of drug resistance depends on the initial growth conditions prior to exposure to high levels of antibiotics. This result is consistent with the predicted existence of a hysteretic regime near the growth plateau. The work reveals concrete mechanisms by which bacteria cope with high levels of antibiotics and illustrates the importance of considering growth-mediated feedback on gene circuits.

  14. l-carnitine protects human hepatocytes from oxidative stress-induced toxicity through Akt-mediated activation of Nrf2 signaling pathway.

    PubMed

    Li, Jinlian; Zhang, Yanli; Luan, Haiyun; Chen, Xuehong; Han, Yantao; Wang, Chunbo

    2016-05-01

    In our previous study, l-carnitine was shown to have cytoprotective effect against hydrogen peroxide (H2O2)-induced injury in human normal HL7702 hepatocytes. The aim of this study was to investigate whether the protective effect of l-carnitine was associated with the nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) pathway. Our results showed that pretreatment with l-carnitine augmented Nrf2 nuclear translocation, DNA binding activity and heme oxygenase-1 (HO-1) expression in H2O2-treated HL7702 cells, although l-carnitine treatment alone had no effect on them. Analysis using Nrf2 siRNA demonstrated that Nrf2 activation was involved in l-carnitine-induced HO-1 expression. In addition, l-carnitine-mediated protection against H2O2 toxicity was abrogated by Nrf2 siRNA, indicating the important role of Nrf2 in l-carnitine-induced cytoprotection. Further experiments revealed that l-carnitine pretreatment enhanced the phosphorylation of Akt in H2O2-treated cells. Blocking Akt pathway with inhibitor partly abrogated the protective effect of l-carnitine. Moreover, our finding demonstrated that the induction of Nrf2 translocation and HO-1 expression by l-carnitine directly correlated with the Akt pathway because Akt inhibitor showed inhibitory effects on the Nrf2 translocation and HO-1 expression. Altogether, these results demonstrate that l-carnitine protects HL7702 cells against H2O2-induced cell damage through Akt-mediated activation of Nrf2 signaling pathway. PMID:26889770

  15. The solution structure of the MANEC-type domain from hepatocyte growth factor activator inhibitor-1 reveals an unexpected PAN/apple domain-type fold.

    PubMed

    Hong, Zebin; Nowakowski, Michal; Spronk, Chris; Petersen, Steen V; Andreasen, Peter A; Koźmiński, Wiktor; Mulder, Frans A A; Jensen, Jan K

    2015-03-01

    A decade ago, motif at N-terminus with eight-cysteines (MANEC) was defined as a new protein domain family. This domain is found exclusively at the N-terminus of >400 multi-domain type-1 transmembrane proteins from animals. Despite the large number of MANEC-containing proteins, only one has been characterized at the protein level: hepatocyte growth factor activator inhibitor-1 (HAI-1). HAI-1 is an essential protein, as knockout mice die in utero due to placental defects. HAI-1 is an inhibitor of matriptase, hepsin and hepatocyte growth factor (HGF) activator, all serine proteases with important roles in epithelial development, cell growth and homoeostasis. Dysregulation of these proteases has been causatively implicated in pathological conditions such as skin diseases and cancer. Detailed functional understanding of HAI-1 and other MANEC-containing proteins is hampered by the lack of structural information on MANEC. Although many MANEC sequences exist, sequence-based database searches fail to predict structural homology. In the present paper, we present the NMR solution structure of the MANEC domain from HAI-1, the first three-dimensional (3D) structure from the MANEC domain family. Unexpectedly, MANEC is a new subclass of the PAN/apple domain family, with its own unifying features, such as two additional disulfide bonds, two extended loop regions and additional α-helical elements. As shown for other PAN/apple domain-containing proteins, we propose a similar active role of the MANEC domain in intramolecular and intermolecular interactions. The structure provides a tool for the further elucidation of HAI-1 function as well as a reference for the study of other MANEC-containing proteins. PMID:25510835

  16. Inhibition of hepatic organic anion-transporting polypeptide by RNA interference in sandwich-cultured human hepatocytes: an in vitro model to assess transporter-mediated drug-drug interactions.

    PubMed

    Liao, Mingxiang; Raczynski, Arek R; Chen, Michael; Chuang, Bei-Ching; Zhu, Qing; Shipman, Rob; Morrison, Jodi; Lee, David; Lee, Frank W; Balani, Suresh K; Xia, Cindy Q

    2010-09-01

    Organic anion-transporting polypeptides (OATPs), members of the SLCO/SLC21 family, mediate the transport of various endo- and xenobiotics. In human liver, OATP1B1, 1B3, and 2B1 are located at the basolateral membrane of hepatocytes and are involved in hepatic drug uptake and biliary elimination. Clinically significant drug-drug interactions (DDIs) mediated by hepatic OATPs have drawn great attention from clinical practitioners and researchers. However, there are considerable challenges to prospectively understanding the extent of OATP-mediated DDIs because of the lack of specific OATP inhibitors or substrates and the limitations of in vitro tools. In the present study, a novel RNA interference knockdown sandwich-cultured human hepatocyte model was developed and validated. Quantitative polymerase chain reaction, microarray and immunoblotting analyses, along with uptake assays, illustrated that the expression and transport activity of hepatic OATPs were reduced by small interfering (siRNA) efficiently and specifically in this model. Although OATP siRNA decreased only 20 to 30% of the total uptake of cerivastatin into human hepatocytes, it caused a 50% reduction in cerivastatin metabolism, which was observed by monitoring the formation of the two major metabolites of cerivastatin. The results suggest that coadministration of a drug that is a hepatic OATP inhibitor could significantly alter the pharmacokinetic profile of cerivastatin in clinical studies. Further studies with this novel model demonstrated that OATP and cytochrome P450 have a synergistic effect on cerivastatin-gemfibrozil interactions. The siRNA knockdown sandwich-cultured human hepatocytes may provide a new powerful model for evaluating DDIs. PMID:20516252

  17. Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: Multiple protective roles of cyclosporin A

    SciTech Connect

    Siu, W.P.; Pun, Pamela Boon Li; Latchoumycandane, Calivarathan; Boelsterli, Urs A.

    2008-03-15

    Diclofenac, a widely used nonsteroidal anti-inflammatory drug, has been associated with rare but severe cases of clinical hepatotoxicity. Diclofenac causes concentration-dependent cell death in human hepatocytes (after 24-48 h) by mitochondrial permeabilization via poorly defined mechanisms. To explore whether the cyclophilin D (CyD)-dependent mitochondrial permeability transition (mPT) and/or the mitochondrial outer membrane permeabilization (MOMP) was primarily involved in mediating cell death, we exposed immortalized human hepatocytes (HC-04) to apoptogenic concentrations of diclofenac (> 500 {mu}M) in the presence or absence of inhibitors of upstream mediators. The CyD inhibitor, cyclosporin A (CsA, 2 {mu}M) fully inhibited diclofenac-induced cell injury, suggesting that mPT was involved. However, CyD gene silencing using siRNA left the cells susceptible to diclofenac toxicity, and CsA still protected the CyD-negative cells from lethal injury. Diclofenac induced early (9 h) activation of Bax and Bak and caused mitochondrial translocation of Bax, indicating that MOMP was involved in cell death. Inhibition of Bax protein expression by using siRNA significantly protected HC-04 from diclofenac-induced cell injury. Diclofenac also induced early Bid activation (tBid formation, 6 h), which is an upstream mechanism that initiates Bax activation and mitochondrial translocation. Bid activation was sensitive to the Ca{sup 2+} chelator, BAPTA. In conclusion, we found that Bax/Bak-mediated MOMP is a key mechanism of diclofenac-induced lethal cell injury in human hepatocytes, and that CsA can prevent MOMP through inhibition of Bax activation. These data support our concept that the Ca{sup 2+}-Bid-Bax-MOMP axis is a critical pathway in diclofenac (metabolite)-induced hepatocyte injury.

  18. Interaction of ApoA-IV with NR4A1 and NR1D1 Represses G6Pase and PEPCK Transcription: Nuclear Receptor-Mediated Downregulation of Hepatic Gluconeogenesis in Mice and a Human Hepatocyte Cell Line

    PubMed Central

    Li, Xiaoming; Xu, Min; Wang, Fei; Ji, Yong; DavidsoN, W. Sean; Li, Zongfang; Tso, Patrick

    2015-01-01

    We have previously shown that the nuclear receptor, NR1D1, is a cofactor in ApoA-IV-mediated downregulation of gluconeogenesis. Nuclear receptor, NR4A1, is involved in the transcriptional regulation of various genes involved in inflammation, apoptosis, and glucose metabolism. We investigated whether NR4A1 influences the effect of ApoA-IV on hepatic glucose metabolism. Our in situ proximity ligation assays and coimmunoprecipitation experiments indicated that ApoA-IV colocalized with NR4A1 in human liver (HepG2) and kidney (HEK-293) cell lines. The chromatin immunoprecipitation experiments and luciferase reporter assays indicated that the ApoA-IV and NR4A1 colocalized at the RORα response element of the human G6Pase promoter, reducing its transcriptional activity. Our RNA interference experiments showed that knocking down the expression of NR4A1 in primary mouse hepatocytes treated with ApoA-IV increased the expression of NR1D1, G6Pase, and PEPCK, and that knocking down NR1D1 expression increased the level of NR4A1. We also found that ApoA-IV induced the expression of endogenous NR4A1 in both cultured primary mouse hepatocytes and in the mouse liver, and decreased glucose production in primary mouse hepatocytes. Our findings showed that ApoA-IV colocalizes with NR4A1, which suppresses G6Pase and PEPCK gene expression at the transcriptional level, reducing hepatic glucose output and lowering blood glucose. The ApoA-IV-induced increase in NR4A1 expression in hepatocytes mediates further repression of gluconeogenesis. Our findings suggest that NR1D1 and NR4A1 serve similar or complementary functions in the ApoA-IV-mediated regulation of gluconeogenesis. PMID:26556724

  19. Mediation Analysis in a Latent Growth Curve Modeling Framework

    ERIC Educational Resources Information Center

    von Soest, Tilmann; Hagtvet, Knut A.

    2011-01-01

    This article presents several longitudinal mediation models in the framework of latent growth curve modeling and provides a detailed account of how such models can be constructed. Logical and statistical challenges that might arise when such analyses are conducted are also discussed. Specifically, we discuss how the initial status (intercept) and…

  20. Epidermal growth factor (EGF)-stimulated inositol phosphate formation in hepatocytes is abolished by pertussis toxin and phorbol esters

    SciTech Connect

    Johnson, R.M.; Garrison, J.C.

    1987-05-01

    The EGF-stimulated rise in intracellular Ca/sup 2 +/ (Ca/sup 2 +/)/sub i/ and Ca/sup 2 +/-dependent protein phosphorylation events in isolated hepatocytes are blocked by pertussis toxin and phorbol ester pretreatment. The present study characterized the EGF-stimulated formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P/sub 3/) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P/sub 3/) in hepatocytes using HPLC methodology to separate the InsP/sub 3/ isomers. Both 66 nM EGF and 10 nM angiotensin II (ANG II) caused a rapid increase in the Ins(1,4,5)P/sub 3/ isomer although EGF-stimulated formation was smaller. At a concentration of ANG II (0.1 nM) which gave an equivalent rise in (Ca/sup 2 +/)/sub i/ as 66 nM EGF, the kinetics and magnitude of Ins(1,4,5)P/sub 3/ formation were similar. EGF or ANG II-stimulated formation of the Ins(1,3,4)P/sub 3/ isomer was more gradual and increased beyond the level of Ins(1,4,5)P/sub 3/ after 60 sec. The initial EGF and ANG II-stimulated increase in both InsP/sub 3/ isomers was not affected by removing external Ca/sup 2 +/ with a 10-fold excess of EGTA. Pretreatment of rats with pertussis toxin for 72 hrs blocked the ability of EGF to increase Ins(1,4,5)P/sub 3/ but did not affect the increase due to ANG II. Three main pretreatment of cells with 1 ..mu..g/ml phorbol 12-myristate-13-acetate (PMA) also inhibited the EGF-stimulated Ins(1,4,5)P/sub 3/ formation. PMA slightly attenuated Ins(1,4,5)P/sub 3/ formation stimulated by 0.1 nM ANG II but not enough to affect the Ca/sup 2 +/ signal. These data suggest that the signal transduction system used by EGF receptors to increase Ins (1,4,5)P/sub 3/ in hepatocytes is somehow different from that used by ANG II receptors.

  1. Human hepatocyte growth factor in alcoholic liver disease: a comparison with change in alpha-fetoprotein. Department of Veterans Affairs Cooperative Study Group 275.

    PubMed

    Mendenhall, C L; Roos, F; Moritz, T E; Roselle, G A; Chedid, A; Grossman, C J; Rouster, S D; Bennett, G L; Lake, J R

    1996-12-01

    To evaluate the hepatic regenerative response in patients with alcoholic liver disease, sera from 263 patients with severe alcoholic hepatitis and/or cirrhosis were analyzed for hepatocyte growth factor (HGF) and alpha-fetoprotein (AFP). HGF concentration was elevated above healthy controls in 95% of the patients (median level = 2.4 ng/ml), whereas AFP tended to be depressed below controls (median level = 4.1 ng/ml). Correlations with parameters of liver injury (i.e., ascites, encephalopathy, AST bilirubin, and protime) all showed a more significant correlation with HGF concentrations than those of AFP. Patients with HGF levels below the mean (4 ng/ml) exhibited significantly better survival (median survival = 35 months vs. 8.5 months for those with HGF > or = 4 ng/ml; p = 0.007). Serum HGF levels were associated with various specific histologic features of alcoholic hepatitis that included, but were not exclusively related to, necrosis. PMID:8986214

  2. Hepatocyte growth factor-induced up-regulation of Twist drives epithelial-mesenchymal transition in a canine mammary tumour cell line.

    PubMed

    Yoshida, Kota; Choisunirachon, Nan; Saito, Tomochika; Matsumoto, Kaori; Saeki, Kohei; Mochizuki, Manabu; Nishimura, Ryohei; Sasaki, Nobuo; Nakagawa, Takayuki

    2014-12-01

    Epithelial-mesenchymal transition (EMT) is a crucial step in tumour progression. However, the molecular mechanisms underlying EMT in canine tumours remain to be elucidated. In this study, the similarity or difference in the molecular mechanism of EMT in canine cells was evaluated and compared with that reported in human and mouse cells. We used eight cell lines derived from canine mammary cancers. Stimulation with hepatocyte growth factor (HGF) increased cell motility and changed EMT-related markers towards mesenchyme in CHMm cell line. These changes were accompanied by an increase in Twist expression and did not occur in CHMm transfected with Twist siRNA, indicating that Twist plays a key role in this phenomenon in CHMm. However, the down-regulation of E-cadherin was not observed by HGF stimulation. Further studies are required to elucidate the difference between human and canine Twist. PMID:25278141

  3. Three Dimensional Primary Hepatocyte Culture

    NASA Technical Reports Server (NTRS)

    Yoffe, Boris

    1998-01-01

    Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

  4. Death receptor and mitochondria-mediated hepatocyte apoptosis underlies liver dysfunction in rats exposed to organic pollutants from drinking water.

    PubMed

    Yang, Guanghong; Zhou, Zhiwei; Cen, Yanli; Gui, Xiaolin; Zeng, Qibing; Ao, Yunxia; Li, Qian; Wang, Shiran; Li, Jun; Zhang, Aihua

    2015-01-01

    Persistent organic pollutants in drinking water impose a substantial risk to the health of human beings, but the evidence for liver toxic effect and the underlying mechanism is scarce. This study aimed to examine the liver toxicity and elucidate the molecular mechanism of organic pollutants in drinking water in normal human liver cell line L02 cells and rats. The data showed that organic extraction from drinking water remarkably impaired rat liver function, evident from the increase in the serum level of alanine aminotransferase, aspartate aminotransferase, and cholinesterase, and decrease in the serum level of total protein and albumin. Organic extraction dose-dependently induced apoptotic cell death in rat liver and L02 cells. Administration of rats with organic extraction promoted death receptor signaling pathway through the increase in gene and protein expression level of Fas and FasL. Treatment of rats with organic extraction also induced mitochondria-mediated apoptosis via increasing the expression level of proapoptotic protein, Bax, but decreasing the expression level of antiapoptotic protein, Bcl-2, resulting in an upregulation of cytochrome c and activation of caspase cascade at both transcriptional and post-transcriptional levels. Moreover, organic extraction enhanced rat liver glutathione S-transferases activity and reactive oxygen species generation, and upregulated aryl hydrocarbon receptor and glutathione S-transferase A1 at both transcriptional and translational levels. Collectively, the results indicate that organic extraction from drinking water impairs liver function, with the involvement of death receptor and mitochondria-mediated apoptosis in rats. The results provide evidence and molecular mechanisms for organic pollutants in drinking water-induced liver dysfunction, which may help prevent and treat organic extraction-induced liver injury. PMID:26316710

  5. Death receptor and mitochondria-mediated hepatocyte apoptosis underlies liver dysfunction in rats exposed to organic pollutants from drinking water

    PubMed Central

    Yang, Guanghong; Zhou, Zhiwei; Cen, Yanli; Gui, Xiaolin; Zeng, Qibing; Ao, Yunxia; Li, Qian; Wang, Shiran; Li, Jun; Zhang, Aihua

    2015-01-01

    Persistent organic pollutants in drinking water impose a substantial risk to the health of human beings, but the evidence for liver toxic effect and the underlying mechanism is scarce. This study aimed to examine the liver toxicity and elucidate the molecular mechanism of organic pollutants in drinking water in normal human liver cell line L02 cells and rats. The data showed that organic extraction from drinking water remarkably impaired rat liver function, evident from the increase in the serum level of alanine aminotransferase, aspartate aminotransferase, and cholinesterase, and decrease in the serum level of total protein and albumin. Organic extraction dose-dependently induced apoptotic cell death in rat liver and L02 cells. Administration of rats with organic extraction promoted death receptor signaling pathway through the increase in gene and protein expression level of Fas and FasL. Treatment of rats with organic extraction also induced mitochondria-mediated apoptosis via increasing the expression level of proapoptotic protein, Bax, but decreasing the expression level of antiapoptotic protein, Bcl-2, resulting in an upregulation of cytochrome c and activation of caspase cascade at both transcriptional and post-transcriptional levels. Moreover, organic extraction enhanced rat liver glutathione S-transferases activity and reactive oxygen species generation, and upregulated aryl hydrocarbon receptor and glutathione S-transferase A1 at both transcriptional and translational levels. Collectively, the results indicate that organic extraction from drinking water impairs liver function, with the involvement of death receptor and mitochondria-mediated apoptosis in rats. The results provide evidence and molecular mechanisms for organic pollutants in drinking water-induced liver dysfunction, which may help prevent and treat organic extraction-induced liver injury. PMID:26316710

  6. TGF-β signaling deficient fibroblasts enhance Hepatocyte Growth Factor signaling in mammary carcinoma cells to promote scattering and invasion

    PubMed Central

    Cheng, Nikki; Chytil, Anna; Shyr, Yu; Joly, Alison; Moses, Harold L.

    2009-01-01

    Fibroblasts are major cellular components of the tumor microenvironment, regulating tumor cell behavior in part through secretion of extracellular matrix proteins, growth factors and angiogenic factors. In previous studies, conditional deletion of the type II TGF-β receptor in fibroblasts (Tgfbr2FspKO) was shown to promote mammary tumor metastasis in fibroblast: epithelial cell co-transplantation studies in mice, correlating with increased expression of HGF. Here, we advance our findings to show that Tgfbr2FspKO fibroblasts enhance HGF/c-Met and HGF/Ron signaling to promote scattering and invasion of mammary carcinoma cells. Blockade of c-Met and Ron by siRNA silencing and pharmacologic inhibitors significantly reduced mammary carcinoma cell scattering and invasion caused by Tgfbr2FspKO fibroblasts. Moreover, neutralizing antibodies to c-Met and Ron significantly inhibited HGF-induced cell scattering and invasion correlating with reduced Stat3 and p42/44MAPK phosphorylation. Investigation of the Stat3 and MAPK signaling pathways by pharmacologic inhibition and siRNA silencing revealed a cooperative interaction between the two pathways to regulate HGF- induced invasion, scattering and motility of mammary tumor cells. Furthermore, while c-Met was found to regulate both the Stat3 and MAPK signaling pathways, Ron was found to regulate Stat3, but not MAPK signaling in mammary carcinoma cells. These studies demonstrate a tumor suppressive role for TGF-β signaling in fibroblasts, in part by suppressing HGF signaling between mammary fibroblasts and epithelial cells. These studies characterize complex functional roles for HGF and TGF-β signaling in mediating tumor: stromal interactions during mammary tumor cell scattering and invasion, with important implications in the metastatic process. PMID:18922968

  7. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression

    PubMed Central

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A.; Choi, Hueng-Sik

    2016-01-01

    Background Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Results Activation of the hepatic CB1 receptor by arachidonyl-2’-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Conclusion Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion. PMID:27455076

  8. The homeodomain Pbx2-Prep1 complex modulates hepatocyte nuclear factor 1alpha-mediated activation of the UDP-glucuronosyltransferase 2B17 gene.

    PubMed

    Gregory, Philip A; Mackenzie, Peter I

    2002-07-01

    UDP glucuronosyltransferases (UGT) are expressed in a wide range of tissues in which their levels of expression and distribution are dependent on cell-type specific regulatory mechanisms. The presence of a hepatocyte nuclear factor (HNF) 1 binding site in the proximal promoters of several UGT2B genes has been shown to contribute to their expression in liver cells and possibly other HNF1-containing cell types. In some of these UGT2B genes, a putative pre-B cell homeobox (Pbx) transcription factor binding site is found directly adjacent to the functional HNF1 site. To determine whether this putative Pbx site contributes to the regulation of UGT2B expression, we chose the UGT2B17 gene and investigated the capacity of its Pbx site to bind specific transcription factors and alter promoter activity. The UGT2B17 Pbx site matches a consensus Pbx site known to bind members of the Pbx, Hox, Meis, and Prep1 families of homeodomain-containing proteins and has previously been shown to bind nuclear proteins in DNaseI footprint assays. In this study, we used gel shift and functional assays to show that a Pbx2-Prep1 heterodimer can bind to the UGT2B17 Pbx site and interfere with the binding of HNF1alpha to its site adjacent to the Pbx site. This interaction of Pbx2-Prep1 and HNF1alpha results in down-regulation of HNF1alpha-mediated activation of the UGT2B17 promoter. Modulation of transcription by restricting the binding of transcriptional effectors to their target site is a novel role for Pbx2-Prep1 complexes. PMID:12065766

  9. Ascorbic acid partly antagonizes resveratrol mediated heme oxygenase-1 but not paraoxonase-1 induction in cultured hepatocytes - role of the redox-regulated transcription factor Nrf2

    PubMed Central

    2011-01-01

    Background Both resveratrol and vitamin C (ascorbic acid) are frequently used in complementary and alternative medicine. However, little is known about the underlying mechanisms for potential health benefits of resveratrol and its interactions with ascorbic acid. Methods The antioxidant enzymes heme oxygenase-1 and paraoxonase-1 were analysed for their mRNA and protein levels in HUH7 liver cells treated with 10 and 25 μmol/l resveratrol in the absence and presence of 100 and 1000 μmol/l ascorbic acid. Additionally the transactivation of the transcription factor Nrf2 and paraoxonase-1 were determined by reporter gene assays. Results Here, we demonstrate that resveratrol induces the antioxidant enzymes heme oxygenase-1 and paraoxonase-1 in cultured hepatocytes. Heme oxygenase-1 induction by resveratrol was accompanied by an increase in Nrf2 transactivation. Resveratrol mediated Nrf2 transactivation as well as heme oxygenase-1 induction were partly antagonized by 1000 μmol/l ascorbic acid. Conclusions Unlike heme oxygenase-1 (which is highly regulated by Nrf2) paraoxonase-1 (which exhibits fewer ARE/Nrf2 binding sites in its promoter) induction by resveratrol was not counteracted by ascorbic acid. Addition of resveratrol to the cell culture medium produced relatively low levels of hydrogen peroxide which may be a positive hormetic redox-signal for Nrf2 dependent gene expression thereby driving heme oxygenase-1 induction. However, high concentrations of ascorbic acid manifold increased hydrogen peroxide production in the cell culture medium which may be a stress signal thereby disrupting the Nrf2 signalling pathway. PMID:21199573

  10. A food contaminant ochratoxin A suppresses pregnane X receptor (PXR)-mediated CYP3A4 induction in primary cultures of human hepatocytes.

    PubMed

    Doricakova, Aneta; Vrzal, Radim

    2015-11-01

    Ochratoxin A (OCHA) is a mycotoxin, which can be found in food such as coffee, wine, cereals, meat, nuts. Since it is absorbed via gastrointestinal tract, it is reasonable to anticipate that the liver will be the first organ to which OCHA comes into the contact before systemic circulation. Many xenobiotics are metabolically modified after the passage of the liver to biologically more active substances, sometimes with more harmful activity. Promoting own metabolism is often achieved via transcriptional regulation of biotransformation enzymes through ligand-activated transcription factors. Pregnane X receptor (PXR) belongs to such a group of regulators and it was demonstrated to be activated by many compounds of synthetic as well as natural origin. Our intention was to investigate if OCHA is capable of activating the PXR with consequent induction of PXR-regulated CYP3A4 gene. We found that OCHA does not activate PXR but displays antagonist-like behavior when combined with rifampicin (RIF) in gene reporter assay in human embryonal kidney cells (Hek293T). It was very weak inducer of CYP3A4 mRNA in primary cultures of human hepatocytes and it antagonized RIF-mediated CYP3A4 induction of mRNA as well as protein. In addition, it caused the decline of PXR protein as well as mRNA which was faster than that with actinomycin D, a transcription inhibitor. Since we found that OCHA induced the expression of miR-148a, which was described to regulate PXR expression, we conclude that antagonist-like behavior of OCHA is not due to the antagonism itself but due to the downregulation of PXR gene expression. Herein we provide important findings which bring a piece of puzzle into the understanding of mechanism of toxic action of ochratoxin A. PMID:26341324

  11. Continuous but not intermittent administration of growth hormone to hypophysectomized rats increases apolipoprotein-E secretion from cultured hepatocytes.

    PubMed

    Sjöberg, A; Oscarsson, J; Edén, S; Olofsson, S O

    1994-02-01

    Hypophysectomy of female rats has been shown to decrease the serum levels of apolipoprotein E (apoE). Continuous but not intermittent administration of GH to hypophysectomized (HX) rats increases these levels to those of normal rats, indicating that the sexually dimorphic secretion of GH is important in the regulation of apoE metabolism. In this study, these effects of GH were further investigated by studying the biosynthesis and secretion of apoE from isolated hepatocytes. Hepatocytes were isolated from HX rats as well as from HX rats that had received hormonal treatment with T4 and cortisol (C) or T4 and C together with GH given either as two daily sc injections (GH x 2) or as a continuous infusion (GHc). Hypophysectomy decreased by 47% the amount of apoE present in the culture medium after a 4-h incubation. Treatment of HX rats with T4 and C alone or in combination with GH x 2 did not influence the amount apoE present in the medium, whereas treatment with T4, C, and GHc increased the amount of apoE to that of normal controls. The different levels of apoE in the medium was not due to differences in the disappearance of apoE, indicating that it was caused by changes in the rate of apoE secretion. Consistent with this, hypophysectomy decreased the rate of intracellular accumulation of apoE measured by incubation of the cells with [35S]methionine for 0, 8, and 20 min. Treatment with T4, C, and GHc increased the rate of accumulation, but T4, C, and GH x 2 had no effect. The differences in the initial rate of intracellular accumulation of apoE were not due to variations in apoE messenger RNA pools or to differences in the degradation of apoE at a step early in the secretory pathway. These results indicate that the differences in the initial rate of accumulation of apoE results from differences in the translational rate. The major amount of apoE that was secreted to the medium appeared in the high-density lipoprotein fraction, whereas small amounts were present in the

  12. Hepatocyte growth factor-stimulated renal tubular mitogenesis: effects on expression of c-myc, c-fos, c-met, VEGF and the VHL tumour-suppressor and related genes.

    PubMed Central

    Clifford, S. C.; Czapla, K.; Richards, F. M.; O'Donoghue, D. J.; Maher, E. R.

    1998-01-01

    Hepatocyte growth factor (HGF/SF) is a potent renal proximal tubular cell (PTEC) mitogen involved in renal development. HGF/SF is the functional ligand for the c-met proto-oncogene, and germline c-met mutations are associated with familial papillary renal cell carcinoma. Somatic von Hippel-Lindau disease tumour-suppressor gene (VHL) mutations are frequently detected in sporadic clear cell renal cell carcinomas (RCC), and germline VHL mutations are the commonest cause of familial clear cell RCC. pVHL binds to the positive regulatory components of the trimeric elongin (SIII) complex (elongins B and C) and has been observed to deregulate expression of the vascular endothelial growth factor (VEGF) gene. HGF/SF has similarly been reported to up-regulate expression of the VEGF gene in non-renal experimental systems. To investigate the mechanism of HGF/SF action in PTECs and, specifically, to examine potential interactions between the HGF/c-met and the VHL-mediated pathways for renal tubular growth control, we have isolated untransformed PTECs from normal kidneys, developed conditions for their culture in vitro and used these cells to investigate changes in mRNA levels of the VHL, elongin A, B and C, VEGF, c-myc, c-fos and c-met genes after HGF/SF exposure. Significant elevations in the mRNA levels of VEGF, c-myc, c-fos, c-met and elongins A, B and C, but not VHL, were detected after HGF/SF stimulation of human PTECs (P < 0.02), with a consistent order of peak levels observed over successive replicates (c-fos at 1 h, VEGF at 2-4 h, c-myc, at 4 h, followed by c-met and all three elongin subunits at 8 h). This study highlights the spectrum of changes in gene expression observed in PTECs after HGF/SF stimulation and has identified possible candidate mediators of the HGF/SF-induced mitogenic response. Our evidence would suggest that the changes in PTEC VEGF expression induced by HGF/SF are mediated by a VHL-independent pathway. Images Figure 1 PMID:9652757

  13. Receptor-mediated endocytosis of insulin in lower vertebrates: internalization and intracellular processing of 125I-insulin in isolated hepatocytes of lamprey and frog.

    PubMed

    Lappova, Y L; Leibush, B N

    1995-10-01

    The binding of 125I-insulin to cellular insulin receptors and the internalization of insulin-receptor complexes have been studied in isolated hepatocytes of frog and lamprey. Two classes of binding sites (Kd 10(-9) and 10(-8) M) were found in cells of both species. The molecular weight of the insulin receptor alpha-subunit was 130 kDa in both species. Internalization of bound 125I-insulin in both species was found in the temperature range 0 to 20 degrees. Cells "loaded" with 125I-insulin were used to estimate the fate of the internalized ligand. Release of internalized ligand from frog cells increased at temperatures ranging from 0 to 20 degrees. At 0 degrees the degraded 125I-insulin was 5%, at 5 degrees 7%, and at 20 degrees 17% of total radioactivity accumulated in the medium. In lamprey hepatocytes there was neither radioactivity accumulation in the incubation medium nor release from cells at all temperatures studied. The intracellular degradation of internalized 125I-insulin in frog hepatocytes was much lower than that in lamprey cells. In frog hepatocytes the specific binding of 125I-insulin was increased twofold in the presence of the lysosomal inhibitor chloroquine. In contrast no increase was found in lamprey hepatocytes. In conclusion, the processing pathways of internalized insulin in the cells of ectothermal and endothermal vertebrates are generally similar but in ectothermal animals all events take place at lower temperatures and at lower rates. The peculiarities of insulin processing in lamprey hepatocytes most likely result from the transformation of hepatocytes during the nonfeeding prespawning period. PMID:8575649

  14. The discovery of Hepatocyte Growth Factor (HGF) and its significance for cell biology, life sciences and clinical medicine

    PubMed Central

    NAKAMURA, Toshikazu; MIZUNO, Shinya

    2010-01-01

    It has been more than 25 years since HGF was discovered as a mitogen of hepatocytes. HGF is produced by stromal cells, and stimulates epithelial cell proliferation, motility, morphogenesis and angiogenesis in various organs via tyrosine phosphorylation of its receptor, c-Met. In fetal stages, HGF-neutralization, or c-Met gene destruction, leads to hypoplasia of many organs, indicating that HGF signals are essential for organ development. Endogenous HGF is required for self-repair of injured livers, kidneys, lungs and so on. In addition, HGF exerts protective effects on epithelial and non-epithelial organs (including the heart and brain) via anti-apoptotic and anti-inflammatory signals. During organ diseases, plasma HGF levels significantly increased, while anti-HGF antibody infusion accelerated tissue destruction in rodents. Thus, endogenous HGF is required for minimization of diseases, while insufficient production of HGF leads to organ failure. This is the reason why HGF supplementation produces therapeutic outcomes under pathological conditions. Moreover, emerging studies delineated key roles of HGF during tumor metastasis, while HGF-antagonism leads to anti-tumor outcomes. Taken together, HGF-based molecules, including HGF-variants, HGF-fragments and c-Met-binders are available as regenerative or anti-tumor drugs. Molecular analysis of the HGF-c-Met system could provide bridges between basic biology and clinical medicine. PMID:20551596

  15. Penfluridol suppresses pancreatic tumor growth by autophagy-mediated apoptosis

    PubMed Central

    Ranjan, Alok; Srivastava, Sanjay K.

    2016-01-01

    Pancreatic tumors exhibit enhanced autophagy as compared to any other cancer, making it resistant to chemotherapy. We evaluated the effect of penfluridol against pancreatic cancer. Penfluridol treatment induced apoptosis and inhibited the growth of Panc-1, BxPC-3 and AsPC-1, pancreatic cancer cells with IC50 ranging between 6–7 μM after 24 h of treatment. Significant autophagy was induced by penfluridol treatment in pancreatic cancer cells. Punctate LC3B and autophagosomes staining confirmed autophagy. Inhibiting autophagy by chloroquine, bafilomycin, 3-methyladenine or LC3BsiRNA, significantly blocked penfluridol-induced apoptosis, suggesting that autophagy lead to apoptosis in our model. Penfluridol treatment suppressed the growth of BxPC-3 tumor xenografts by 48% as compared to 17% when treated in combination with chloroquine. Similarly, penfluridol suppressed the growth of AsPC-1 tumors by 40% versus 16% when given in combination with chloroquine. TUNEL staining and caspase-3 cleavage revealed less apoptosis in the tumors from mice treated with penfluridol and chloroquine as compared to penfluridol alone. Penfluridol treatment also suppressed the growth of orthotopically implanted Panc-1 tumors by 80% by inducing autophagy-mediated apoptosis in the tumors. These studies established that penfluridol inhibits pancreatic tumor growth by autophagy-mediated apoptosis. Since penfluridol is already in clinic, positive findings from our study will accelerate its clinical development. PMID:27189859

  16. Hepatocyte Growth Factor and MET Support Mouse Enteric Nervous System Development, the Peristaltic Response, and Intestinal Epithelial Proliferation in Response to Injury

    PubMed Central

    Avetisyan, Marina; Wang, Hongtao; Schill, Ellen Merrick; Bery, Saya; Grider, John R.; Hassell, John A.; Stappenbeck, Thaddeus

    2015-01-01

    Factors providing trophic support to diverse enteric neuron subtypes remain poorly understood. We tested the hypothesis that hepatocyte growth factor (HGF) and the HGF receptor MET might support some types of enteric neurons. HGF and MET are expressed in fetal and adult enteric nervous system. In vitro, HGF increased enteric neuron differentiation and neurite length, but only if vanishingly small amounts (1 pg/ml) of glial cell line-derived neurotrophic factor were included in culture media. HGF effects were blocked by phosphatidylinositol-3 kinase inhibitor and by MET-blocking antibody. Both of these inhibitors and MEK inhibition reduced neurite length. In adult mice, MET was restricted to a subset of calcitonin gene-related peptide-immunoreactive (IR) myenteric plexus neurons thought to be intrinsic primary afferent neurons (IPANs). Conditional MET kinase domain inactivation (Metfl/fl; Wnt1Cre+) caused a dramatic loss of myenteric plexus MET-IR neurites and 1–1′-dioctodecyl-3,3,3′,3′-tetramethylindocarbocyamine perchlorate (DiI) labeling suggested reduced MET-IR neurite length. In vitro, Metfl/fl; Wnt1Cre+ mouse bowel had markedly reduced peristalsis in response to mucosal deformation, but normal response to radial muscle stretch. However, whole-bowel transit, small-bowel transit, and colonic-bead expulsion were normal in Metfl/fl; Wnt1Cre+ mice. Finally, Metfl/fl; Wnt1Cre+ mice had more bowel injury and reduced epithelial cell proliferation compared with WT animals after dextran sodium sulfate treatment. These results suggest that HGF/MET signaling is important for development and function of a subset IPANs and that these cells regulate intestinal motility and epithelial cell proliferation in response to bowel injury. SIGNIFICANCE STATEMENT The enteric nervous system has many neuronal subtypes that coordinate and control intestinal activity. Trophic factors that support these neuron types and enhance neurite growth after fetal development are not well

  17. Phosphorylation of Adaptor Protein Containing Pleckstrin Homology Domain, Phosphotyrosine Binding Domain, and Leucine Zipper Motif 1 (APPL1) at Ser430 Mediates Endoplasmic Reticulum (ER) Stress-induced Insulin Resistance in Hepatocytes*

    PubMed Central

    Liu, Meilian; Zhou, Lijun; Wei, Li; Villarreal, Ricardo; Yang, Xin; Hu, Derong; Riojas, Ramon A.; Holmes, Bekke M.; Langlais, Paul R.; Lee, Hakjoo; Dong, Lily Q.

    2012-01-01

    APPL1 is an adaptor protein that plays a critical role in regulating adiponectin and insulin signaling. However, how APPL1 is regulated under normal and pathological conditions remains largely unknown. In this study, we show that APPL1 undergoes phosphorylation at Ser430 and that this phosphorylation is enhanced in the liver of obese mice displaying insulin resistance. In cultured mouse hepatocytes, APPL1 phosphorylation at Ser430 is stimulated by phorbol 12-myristate 13-acetate, an activator of classic PKC isoforms, and by the endoplasmic reticulum (ER) stress inducer, thapsigargin. Overexpression of wild-type but not dominant negative PKCα increases APPL1 phosphorylation at Ser430 in mouse hepatocytes. In addition, suppressing PKCα expression by shRNA in hepatocytes reduces ER stress-induced APPL1 phosphorylation at Ser430 as well as the inhibitory effect of ER stress on insulin-stimulated Akt phosphorylation. Consistent with a negative regulatory role of APPL1 phosphorylation at Ser430 in insulin signaling, overexpression of APPL1S430D but not APPL1S430A impairs the potentiating effect of APPL1 on insulin-stimulated Akt phosphorylation at Thr308. Taken together, our results identify APPL1 as a novel target in ER stress-induced insulin resistance and PKCα as the kinase mediating ER stress-induced phosphorylation of APPL1 at Ser430. PMID:22685300

  18. Twin-mediated crystal growth: an enigma resolved

    PubMed Central

    Shahani, Ashwin J.; Gulsoy, E. Begum; Poulsen, Stefan O.; Xiao, Xianghui; Voorhees, Peter W.

    2016-01-01

    During crystal growth, faceted interfaces may be perturbed by defects, leading to a rich variety of polycrystalline growth forms. One such defect is the coherent Σ3 {111} twin boundary, which is widely known to catalyze crystal growth. These defects have a profound effect on the properties of many materials: for example, electron-hole recombination rates strongly depend on the character of the twin boundaries in polycrystalline Si photovoltaic cells. However, the morphology of the twinned interface during growth has long been a mystery due to the lack of four-dimensional (i.e., space and time resolved) experiments. Many controversial mechanisms have been proposed for this process, most of which lack experimental verification. Here, we probe the real-time interfacial dynamics of polycrystalline Si particles growing from an Al-Si-Cu liquid via synchrotron-based X-ray tomography. Our novel analysis of the time evolution of the interfacial normals allows us to quantify unambiguously the habit plane and grain boundary orientations during growth. This, when combined with direct measurements of the interfacial morphology provide the first confirmation of twin-mediated growth, proposed over 50 years ago. Using the insights provided by these experiments, we have developed a unified picture of the phenomena responsible for the dynamics of faceted Si growth. PMID:27346073

  19. Twin-mediated crystal growth: an enigma resolved

    NASA Astrophysics Data System (ADS)

    Shahani, Ashwin J.; Gulsoy, E. Begum; Poulsen, Stefan O.; Xiao, Xianghui; Voorhees, Peter W.

    2016-06-01

    During crystal growth, faceted interfaces may be perturbed by defects, leading to a rich variety of polycrystalline growth forms. One such defect is the coherent Σ3 {111} twin boundary, which is widely known to catalyze crystal growth. These defects have a profound effect on the properties of many materials: for example, electron-hole recombination rates strongly depend on the character of the twin boundaries in polycrystalline Si photovoltaic cells. However, the morphology of the twinned interface during growth has long been a mystery due to the lack of four-dimensional (i.e., space and time resolved) experiments. Many controversial mechanisms have been proposed for this process, most of which lack experimental verification. Here, we probe the real-time interfacial dynamics of polycrystalline Si particles growing from an Al-Si-Cu liquid via synchrotron-based X-ray tomography. Our novel analysis of the time evolution of the interfacial normals allows us to quantify unambiguously the habit plane and grain boundary orientations during growth. This, when combined with direct measurements of the interfacial morphology provide the first confirmation of twin-mediated growth, proposed over 50 years ago. Using the insights provided by these experiments, we have developed a unified picture of the phenomena responsible for the dynamics of faceted Si growth.

  20. Twin-mediated crystal growth: an enigma resolved.

    PubMed

    Shahani, Ashwin J; Gulsoy, E Begum; Poulsen, Stefan O; Xiao, Xianghui; Voorhees, Peter W

    2016-01-01

    During crystal growth, faceted interfaces may be perturbed by defects, leading to a rich variety of polycrystalline growth forms. One such defect is the coherent Σ3 {111} twin boundary, which is widely known to catalyze crystal growth. These defects have a profound effect on the properties of many materials: for example, electron-hole recombination rates strongly depend on the character of the twin boundaries in polycrystalline Si photovoltaic cells. However, the morphology of the twinned interface during growth has long been a mystery due to the lack of four-dimensional (i.e., space and time resolved) experiments. Many controversial mechanisms have been proposed for this process, most of which lack experimental verification. Here, we probe the real-time interfacial dynamics of polycrystalline Si particles growing from an Al-Si-Cu liquid via synchrotron-based X-ray tomography. Our novel analysis of the time evolution of the interfacial normals allows us to quantify unambiguously the habit plane and grain boundary orientations during growth. This, when combined with direct measurements of the interfacial morphology provide the first confirmation of twin-mediated growth, proposed over 50 years ago. Using the insights provided by these experiments, we have developed a unified picture of the phenomena responsible for the dynamics of faceted Si growth. PMID:27346073

  1. Hepatic Stellate Cell–Targeted Delivery of Hepatocyte Growth Factor Transgene via Bile Duct Infusion Enhances Its Expression at Fibrotic Foci to Regress Dimethylnitrosamine-Induced Liver Fibrosis

    PubMed Central

    Narmada, Balakrishnan Chakrapani; Kang, Yuzhan; Venkatraman, Lakshmi; Peng, Qiwen; Sakban, Rashidah Binte; Nugraha, Bramasta; Jiang, Xuan; Bunte, Ralph M.; So, Peter T.C.; Tucker-Kellogg, Lisa

    2013-01-01

    Abstract Liver fibrosis generates fibrotic foci with abundant activated hepatic stellate cells and excessive collagen deposition juxtaposed with healthy regions. Targeted delivery of antifibrotic therapeutics to hepatic stellate cells (HSCs) might improve treatment outcomes and reduce adverse effects on healthy tissue. We delivered the hepatocyte growth factor (HGF) gene specifically to activated hepatic stellate cells in fibrotic liver using vitamin A–coupled liposomes by retrograde intrabiliary infusion to bypass capillarized hepatic sinusoids. The antifibrotic effects of DsRed2-HGF vector encapsulated within vitamin A–coupled liposomes were validated by decreases in fibrotic markers in vitro. Fibrotic cultures transfected with the targeted transgene showed a significant decrease in fibrotic markers such as transforming growth factor-β1. In rats, dimethylnitrosamine-induced liver fibrosis is manifested by an increase in collagen deposition and severe defenestration of sinusoidal endothelial cells. The HSC-targeted transgene, administered via retrograde intrabiliary infusion in fibrotic rats, successfully reduced liver fibrosis markers alpha-smooth muscle actin and collagen, accompanied by an increase in the expression of DsRed2-HGF near the fibrotic foci. Thus, targeted delivery of HGF gene to hepatic stellate cells increased the transgene expression at the fibrotic foci and strongly enhanced its antifibrotic effects. PMID:23527815

  2. Transcriptional regulation of the hepatocyte growth factor gene by the nuclear receptors chicken ovalbumin upstream promoter transcription factor and estrogen receptor.

    PubMed

    Jiang, J G; Bell, A; Liu, Y; Zarnegar, R

    1997-02-14

    Hepatocyte growth factor (HGF) is a multifunctional cytokine that controls the growth and differentiation of various tissues. Previously, we described the existence of a negative cis-acting regulatory element(s) within the -1- to -0.7-kilobase pair (kb) portion of the 5'-flanking region of the mouse HGF promoter. In the present study, we show that the repressor element is located at position -872 to -860 base pairs and comprises an imperfect estrogen-responsive element 5'-AGGTCAGAAAGACCA-3'. We demonstrate that chicken ovalbumin upstream promoter transcription factor (COUP-TF), a nuclear orphan receptor belonging to the steroid/thyroid hormone receptor superfamily, through binding to this site effectively silences the transcriptional activity of the HGF promoter. We show that estrogen receptor, on the other hand, relieves the repressive action of COUP-TF, resulting in the induction of the HGF promoter. Using mice transgenic for either 2.7 or 0.7 kb of the HGF promoter region linked to the chloramphenicol acetyltransferase reporter gene, we found that injection of estradiol stimulates HGF promoter activity in tissues such as the mammary gland and ovary of mice harboring 2.7 but not 0.7 kb of the mouse HGF promoter region. Potential involvement of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors in the regulation of HGF gene expression is also discussed. PMID:9020096

  3. Expression of hepatocyte growth factor and c-Met is characteristic of α-fetoprotein-producing colorectal adenocarcinoma: A report of three cases

    PubMed Central

    LI, JUN; LIU, YUE; XU, JING-HONG; XU, ZHENG-PING; ZHENG, SHU; DING, KE-FENG

    2016-01-01

    α-fetoprotein (AFP)-producing colorectal adenocarcinoma is rare and typically not well recognized. In the present study, 3 cases of AFP-producing colorectal cancer are described. All 3 of these cases demonstrated increased levels of blood AFP associated with disease progression. Only case 2 exhibited classical histological hepatoid features. Following immunohistochemical tissue staining, all 3 cases were observed to be positive for AFP expression. In addition, the expression of hepatocyte growth factor (HGF), c-Met receptor and the transcription factor c-Myc were identified to be associated with the expression of AFP. The 3 cases demonstrated resistance to multiple drugs, including epidermal growth factor receptor inhibitors, despite the presence of wild-type Kirsten rat sarcoma viral oncogene homolog (K-RAS; codons 12 and 13), neuroblastoma-RAS (codons 12 and 13) and B-Raf proto-oncogene, serine/threonine kinase (V600E). We propose that hepatoid histological features or a positive AFP finding by immunohistochemistry are sufficient for a diagnosis of AFP-producing colorectal adenocarcinoma. Furthermore, we speculates that autocrine HGF/c-Met activation may be capable of inducing the dedifferentiation of common adenocarcinoma cells, reverting them to a cancer stem cell state and producing AFP or hepatoid differentiation. Consequently, therapy targeted to the HGF/c-Met signaling pathway may potentially be effective for the treatment of AFP-producing colorectal adenocarcinoma. PMID:26870275

  4. BAG-1 enhances cell-cell adhesion, reduces proliferation and induces chaperone-independent suppression of hepatocyte growth factor-induced epidermal keratinocyte migration

    SciTech Connect

    Hinitt, C.A.M.; Wood, J.; Lee, S.S.; Williams, A.C.; Howarth, J.L.; Glover, C.P.; Uney, J.B.; Hague, A.

    2010-08-01

    Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF) in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell-cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell-cell adhesion and suggest an important function in epidermal cohesion.

  5. Trans-Ethnic Meta-Analysis Identifies Common and Rare Variants Associated with Hepatocyte Growth Factor Levels in the Multi-Ethnic Study of Atherosclerosis (MESA)

    PubMed Central

    Larson, Nicholas B.; Berardi, Cecilia; Decker, Paul A.; Wassel, Christina L.; Kirsch, Phillip S.; Pankow, James S.; Sale, Michele M.; de Andrade, Mariza; Sicotte, Hugues; Tang, Weihong; Hanson, Naomi Q.; Tsai, Michael Y.; Taylor, Kent D.; Bielinski, Suzette J.

    2015-01-01

    Summary Hepatocyte growth factor (HGF) is a mesenchyme-derived pleiotropic factor that regulates cell growth, motility, mitogenesis, and morphogenesis in a variety of cells, and increased serum levels of HGF have been linked to a number of clinical and subclinical cardiovascular disease phenotypes. However, little is currently known regarding what genetic factors influence HGF levels, despite evidence of substantial genetic contributions to HGF variation. Based upon ethnicity-stratified single-variant association analysis and trans-ethnic meta-analysis of 6201 participants of the Multi-Ethnic Study of Atherosclerosis (MESA), we discovered five statistically significant common and low-frequency variants: HGF missense polymorphism rs5745687 (p.E299K) as well as four variants (rs16844364, rs4690098, rs114303452, rs3748034) within or in proximity to HGFAC. We also identified two significant ethnicity-specific gene-level associations (A1BG in African Americans; FASN in Chinese Americans) based upon low-frequency/rare variants, while meta-analysis of gene-level results identified a significant association for HGFAC. However, identified single-variant associations explained modest proportions of the total trait variation and were not significantly associated with coronary artery calcium or coronary heart disease. Our findings indicate genetic factors influencing circulating HGF levels may be complex and ethnically diverse. PMID:25998175

  6. Peroxisome proliferator-activated receptor alpha acts as a mediator of endoplasmic reticulum stress-induced hepatocyte apoptosis in acute liver failure

    PubMed Central

    Zhang, Li; Ren, Feng; Zhang, Xiangying; Wang, Xinxin; Shi, Hongbo; Zhou, Li; Zheng, Sujun; Chen, Yu; Chen, Dexi; Li, Liying; Duan, Zhongping

    2016-01-01

    ABSTRACT Peroxisome proliferator-activated receptor α (PPARα) is a key regulator to ameliorate liver injury in cases of acute liver failure (ALF). However, its regulatory mechanisms remain largely undetermined. Endoplasmic reticulum stress (ER stress) plays an important role in a number of liver diseases. This study aimed to investigate whether PPARα activation inhibits ER stress-induced hepatocyte apoptosis, thereby protecting against ALF. In a murine model of D-galactosamine (D-GalN)- and lipopolysaccharide (LPS)-induced ALF, Wy-14643 was administered to activate PPARα, and 4-phenylbutyric acid (4-PBA) was administered to attenuate ER stress. PPARα activation ameliorated liver injury, because pre-administration of its specific inducer, Wy-14643, reduced the serum aminotransferase levels and preserved liver architecture compared with that of controls. The protective effect of PPARα activation resulted from the suppression of ER stress-induced hepatocyte apoptosis. Indeed, (1) PPARα activation decreased the expression of glucose-regulated protein 78 (Grp78), Grp94 and C/EBP-homologous protein (CHOP) in vivo; (2) the liver protection by 4-PBA resulted from the induction of PPARα expression, as 4-PBA pre-treatment promoted upregulation of PPARα, and inhibition of PPARα by small interfering RNA (siRNA) treatment reversed liver protection and increased hepatocyte apoptosis; (3) in vitro PPARα activation by Wy-14643 decreased hepatocyte apoptosis induced by severe ER stress, and PPARα inhibition by siRNA treatment decreased the hepatocyte survival induced by mild ER stress. Here, we demonstrate that PPARα activation contributes to liver protection and decreases hepatocyte apoptosis in ALF, particularly through regulating ER stress. Therefore, targeting PPARα could be a potential therapeutic strategy to ameliorate ALF. PMID:27482818

  7. Peroxisome proliferator-activated receptor alpha acts as a mediator of endoplasmic reticulum stress-induced hepatocyte apoptosis in acute liver failure.

    PubMed

    Zhang, Li; Ren, Feng; Zhang, Xiangying; Wang, Xinxin; Shi, Hongbo; Zhou, Li; Zheng, Sujun; Chen, Yu; Chen, Dexi; Li, Liying; Zhao, Caiyan; Duan, Zhongping

    2016-07-01

    Peroxisome proliferator-activated receptor α (PPARα) is a key regulator to ameliorate liver injury in cases of acute liver failure (ALF). However, its regulatory mechanisms remain largely undetermined. Endoplasmic reticulum stress (ER stress) plays an important role in a number of liver diseases. This study aimed to investigate whether PPARα activation inhibits ER stress-induced hepatocyte apoptosis, thereby protecting against ALF. In a murine model of D-galactosamine (D-GalN)- and lipopolysaccharide (LPS)-induced ALF, Wy-14643 was administered to activate PPARα, and 4-phenylbutyric acid (4-PBA) was administered to attenuate ER stress. PPARα activation ameliorated liver injury, because pre-administration of its specific inducer, Wy-14643, reduced the serum aminotransferase levels and preserved liver architecture compared with that of controls. The protective effect of PPARα activation resulted from the suppression of ER stress-induced hepatocyte apoptosis. Indeed, (1) PPARα activation decreased the expression of glucose-regulated protein 78 (Grp78), Grp94 and C/EBP-homologous protein (CHOP) in vivo; (2) the liver protection by 4-PBA resulted from the induction of PPARα expression, as 4-PBA pre-treatment promoted upregulation of PPARα, and inhibition of PPARα by small interfering RNA (siRNA) treatment reversed liver protection and increased hepatocyte apoptosis; (3) in vitro PPARα activation by Wy-14643 decreased hepatocyte apoptosis induced by severe ER stress, and PPARα inhibition by siRNA treatment decreased the hepatocyte survival induced by mild ER stress. Here, we demonstrate that PPARα activation contributes to liver protection and decreases hepatocyte apoptosis in ALF, particularly through regulating ER stress. Therefore, targeting PPARα could be a potential therapeutic strategy to ameliorate ALF. PMID:27482818

  8. Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: Comparisons with AHR1-mediated reporter gene activity and in ovo toxicity

    SciTech Connect

    Manning, Gillian E.; Mundy, Lukas J.; Crump, Doug; Jones, Stephanie P.; Chiu, Suzanne; Klein, Jeff; Konstantinov, Alex; Potter, Dave; Kennedy, Sean W.

    2013-01-01

    Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, in combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species. -- Highlights: ► The chicken isn't the most sensitive species to CYP1A induction by PCB 105 and 118. ► The relative potency of PCBs differs between avian species. ► EROD activity was correlated with luciferase activity from the LRG assay. ► EROD activity was a better predictor of toxicity than CYP

  9. Nature and mechanisms of hepatocyte apoptosis induced by d-galactosamine/lipopolysaccharide challenge in mice

    PubMed Central

    WU, YI-HANG; HU, SHAO-QING; LIU, JUN; CAO, HONG-CUI; XU, WEI; LI, YONG-JUN; LI, LAN-JUAN

    2014-01-01

    Apoptosis plays a role in the normal development of liver. However, overactivation thereof may lead to hepatocellular damage. The aim of this study was to assess d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-induced hepatocyte apoptotic changes in mice and clarify the mechanisms involved in this process. DNA ladder detection was employed to determine the induction condition of hepatic apoptosis. An initial test indicated that typical hepatocyte apoptosis was observed at 6–10 h after the intraperitoneal injection of d-GalN (700 mg/kg) and LPS (10 μg/kg). Subsequently, we evaluated hepatocyte apoptosis at 8 h after administering d-GalN/LPS by histopathological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) detection, flow cytometry and electron microscopy analysis. To clarify the apoptosis-related gene expression, the expression levels of tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), caspase-3, and Fas/Fas ligand (FasL) were determined by serum enzyme immunoassay, immunohistochemistry and western blot analysis. Strong apoptotic positive signals following d-GalN/LPS injection were observed from the results of the serum analysis, histopathological and immunohistochemical analyses, DNA ladder detection, TUNEL detection, flow cytometry and electron microscopy analysis. Additionally, apoptotic hepatocytes were mainly at the late stage of cell apoptosis. The expression of TNF-α, TGF-β1, caspase-3 and Fas/FasL was significantly increased. In conclusion, this study evaluated the d-GalN/LPS-induced hepatocyte apoptotic changes and clarified the apoptosis-related gene expression in mice. The hepatocyte apoptosis induced by d-GalN/LPS may be mainly regulated by the death receptor pathway. TGF-β signaling pathway may also play a vital role in this process of hepatocyte apoptosis. PMID:24714963

  10. Immobilization of tripeptide growth factor glycyl-L-histidyl-L-lysine on poly(vinylalcohol)-quarternized stilbazole (PVA-SbQ) and its use as a ligand for hepatocyte attachment.

    PubMed

    Kawase, M; Miura, N; Kurikawa, N; Masuda, K; Higashiyama, S; Yagi, K; Mizoguchi, T

    1999-09-01

    A tripeptide growth factor, glycyl-L-histidyl-L-lysine (GHK), was immobilized on the surface of poly(vinylalcohol)-quarternized stilbazole (PVA-SbQ) gel. The photoreactive substance, 4-(3-trifluoromethylazirino)benzoyl-N-hydroxysuccinimide (TDBA-OSu), was employed to link the gel and ligand GHK. The density of immobilized GHK was 70 nmol/cm2. Isolated rat hepatocytes were inoculated on the GHK-immobilized PVA-SbQ gel and cultured for 5 d. About 24 h after inoculation, hepatocytes started to aggregate and formed multicellular spheroids while almost no cells attached to GHK-non-immobilized PVA-SbQ gel. The formed spheroids attached firmly to the surface of PVA-SbQ gel for 5 d. GHK was, thus, shown to be an effective ligand for hepatocyte attachment. Dodecamethylenediamine was used to extend the length between the gel surface and GHK. Extension of the length significantly increased the number of attached hepatocytes. PMID:10513632

  11. A serum component mediates food restriction-induced growth attenuation.

    PubMed

    Pando, Rakefet; Shtaif, Biana; Phillip, Moshe; Gat-Yablonski, Galia

    2014-03-01

    Proper nutrition in terms of calories and essential food components is required to maximize longitudinal growth in children. Our previous study showed that prepubertal male rats subjected to 10 days of 40% food restriction (RES) exhibited a dramatic reduction in weight and epiphyseal growth plate height, as well as changes in gene expression and microRNAs (miRNAs) in the epiphyseal growth plate. These findings reversed rapidly after renewal of the regular food supply (catch-up [CU]). To further elucidate the mechanisms underlying the nutrition-growth association, serum collected from the RES and CU rats and control rats fed ad libitum (AL) was added to the culture medium of the chondrocyte cell line ATDC5 (instead of fetal calf serum). Serum from the RES group induced a reduction in cell viability (25%, P < .05) concomitant with an increase in cell differentiation compared with that for the AL group serum. The most interesting observation, in our opinion, was the significant reduction in the expression of specific miRNAs, including the chondro-specific miR-140. These effects were not observed for serum from refed (CU) rats. Serum levels of IGF-I, leptin, and fibroblast growth factor 21 were reduced by food restriction. The addition of IGF-I and leptin to the culture increased cell viability, whereas fibroblast growth factor 21 reduced it, suggesting the involvement of IGF-I, leptin, and possibly other still unidentified serum factors in chondrocyte cell growth. In conclusion, specific miRNAs respond to nutritional cues, and these effects are mediated by serum-borne factors. These results may promote the development of superior interventions for children with malnutrition and growth abnormalities. PMID:24456162

  12. Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: Potential for gene therapy of hemophilia B

    SciTech Connect

    Thompson, A.R. Puget Sound Blood Center, Seattle, WA ); Darlington, G. ); Armentano, D.; Woo, S.L.C.

    1990-08-01

    Hemophilia B (Christmas disease) is a chromosome X-linked blood clotting disorder which results when factor IX is deficient or functionally defective. The enzyme is synthesized in the liver, and the existence of animal models for this genetic disease will permit the development of somatic gene therapy protocols aimed at transfer of the functional gene into the liver. The authors report the construction of an N2-based recombinant retroviral vector, NCMVFIX, for efficient transfer and expression of human factor IX cDNA in primary rabbit hepatocytes. In this construct the human cytomegalovirus immediate early promoter directs the expression of factor IX. Hepatocytes were isolated from 3-week-old New Zealand White rabbits, infected with the recombinant virus, and analyzed for secretion of active factor IX. The infected rabbit hepatocytes produced human factor IX that is indistinguishable from enzyme derived from normal human plasma. The recombinant protein is sufficiently {gamma}-carboxylated and is functionally active in clotting assays. These results establish the feasibility of using infected hepatocytes for the expression of this protein and are a step toward the goal of correcting hemophilia B by hepatic gene transfer.

  13. Environmental pollutants parathion, paraquat and bisphenol A show distinct effects towards nuclear receptors-mediated induction of xenobiotics-metabolizing cytochromes P450 in human hepatocytes.

    PubMed

    Vrzal, Radim; Zenata, Ondrej; Doricakova, Aneta; Dvorak, Zdenek

    2015-10-01

    Environmental pollutants parathion, bisphenol A and paraquat were not systematically studied towards the effects on the expression of phase I xenobiotics-metabolizing cytochromes P450 (CYPs). We monitored their effects on the expression of selected CYPs in primary cultures of human hepatocytes. Moreover, we investigated their effects on the receptors regulating these CYPs, particularly arylhydrocarbon receptor (AhR), pregnane X receptor (PXR) and glucocorticoid receptor (GR) by gene reporter assays. We found that parathion and bisphenol A are the activators of AhR. Moreover, they are the inducers of CYP1A1 mRNA in hepatoma cells HepG2 as well as in human hepatocytes by AhR-dependent mechanism via formation of AhR-DNA-binding complex, as revealed by gel shift assay. All three compounds possessed anti-glucocorticoid action as revealed by GR-dependent gene reporter assay and a decline in tyrosine aminotransferase (TAT) gene expression in human hepatocytes. Moreover, parathion and bisphenol A are the activators of PXR and inducers of CYP3A4 mRNA and protein in the primary cultures of human hepatocytes. In conclusion, the studied compounds displayed distinct activities towards nuclear receptors involved in many biological processes and these findings may help us to better understand their adverse actions in pathological states followed after their exposure. PMID:26196221

  14. The hepatocyte growth factor antagonist NK4 inhibits indoleamine-2,3-dioxygenase expression via the c-Met-phosphatidylinositol 3-kinase-AKT signaling pathway.

    PubMed

    Wang, Dongdong; Saga, Yasushi; Sato, Naoto; Nakamura, Toshikazu; Takikawa, Osamu; Mizukami, Hiroaki; Matsubara, Shigeki; Fujiwara, Hiroyuki

    2016-06-01

    Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme involved in tumor malignancy. However, the regulatory mechanism underlying its involvement remains largely uncharacterized. The present study aimed to investigate the hypothesis that NK4, an antagonist of hepatocyte growth factor (HGF), can regulate IDO and to characterize the signaling mechanism involved. Following successful transfection of the human ovarian cancer cell line SKOV-3 (which constitutively expresses IDO) with an NK4 expression vector, we observed that NK4 expression suppressed IDO expression; furthermore, NK4 expression did not suppress cancer cell growth in vitro [in the absence of natural killer (NK) cells], but did influence tumor growth in vivo. In addition, NK4 enhanced the sensitivity of cancer cells to NK cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. In an effort to clarify the mechanisms by which NK4 interacts with IDO, we performed investigations utilizing various biochemical inhibitors. The results of these investigations were as follows. First, c-Met (a receptor of HGF) tyrosine kinase inhibitor PHA-665752, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 both suppress IDO expression. Second, enhanced expression of PTEN (a known tumor suppressor) via negative regulation within a PI3K-AKT pathway, inhibits IDO expression. Conversely, neither the MEK1/2 inhibitor U0126 nor the STAT3 inhibitor WP1066 affects IDO expression. These results suggest that NK4 inhibits IDO expression via a c-Met-PI3K-AKT signaling pathway. PMID:27082119

  15. The hepatocyte growth factor antagonist NK4 inhibits indoleamine-2,3-dioxygenase expression via the c-Met-phosphatidylinositol 3-kinase-AKT signaling pathway

    PubMed Central

    WANG, DONGDONG; SAGA, YASUSHI; SATO, NAOTO; NAKAMURA, TOSHIKAZU; TAKIKAWA, OSAMU; MIZUKAMI, HIROAKI; MATSUBARA, SHIGEKI; FUJIWARA, HIROYUKI

    2016-01-01

    Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme involved in tumor malignancy. However, the regulatory mechanism underlying its involvement remains largely uncharacterized. The present study aimed to investigate the hypothesis that NK4, an antagonist of hepatocyte growth factor (HGF), can regulate IDO and to characterize the signaling mechanism involved. Following successful transfection of the human ovarian cancer cell line SKOV-3 (which constitutively expresses IDO) with an NK4 expression vector, we observed that NK4 expression suppressed IDO expression; furthermore, NK4 expression did not suppress cancer cell growth in vitro [in the absence of natural killer (NK) cells], but did influence tumor growth in vivo. In addition, NK4 enhanced the sensitivity of cancer cells to NK cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. In an effort to clarify the mechanisms by which NK4 interacts with IDO, we performed investigations utilizing various biochemical inhibitors. The results of these investigations were as follows. First, c-Met (a receptor of HGF) tyrosine kinase inhibitor PHA-665752, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 both suppress IDO expression. Second, enhanced expression of PTEN (a known tumor suppressor) via negative regulation within a PI3K-AKT pathway, inhibits IDO expression. Conversely, neither the MEK1/2 inhibitor U0126 nor the STAT3 inhibitor WP1066 affects IDO expression. These results suggest that NK4 inhibits IDO expression via a c-Met-PI3K-AKT signaling pathway. PMID:27082119

  16. Liver tumors escape negative control of proliferation via PI3K/Akt-mediated block of C/EBPα growth inhibitory activity

    PubMed Central

    Wang, Guo-Li; Iakova, Polina; Wilde, Margie; Awad, Samir; Timchenko, Nikolai A.

    2004-01-01

    Liver tumor cells arise from normal hepatocytes that escape negative control of proliferation. The transcription factor C/EBPα maintains quiescence of hepatocytes through two pathways: inhibition of cdks and repression of E2F. Nevertheless, liver tumors and cultured hepatoma cell lines proliferate in the presence of C/EBPα. In this paper, we present evidence that the activation of the PI3K/Akt pathway in liver tumor cells blocks the growth inhibitory activity of C/EBPα through the PP2A-mediated dephosphorylation of C/EBPα on Ser 193, leading to a failure of C/EBPα to interact with and inhibit cdks and E2F. Mutation of Ser 193 to Ala also abolishes the ability of C/EBPα to cause growth arrest because of a lack of interactions with cdk2 and E2F–Rb complexes. These data provide a molecular basis for the development of liver tumors in which the activation of PI3K/Akt pathway neutralizes C/EBPα growth inhibitory activity. PMID:15107404

  17. A cluster region of AP-1 responsive elements is required for transcriptional activity of mouse ODC gene by hepatocyte growth factor.

    PubMed

    Bianchi, Laura; Tacchini, Lorenza; Matteucci, Emanuela; Desiderio, Maria Alfonsina

    2002-05-01

    Ornithine decarboxylase (ODC) activity is regulated by a variety of mechanisms including transcription, translation, and RNA and protein half-life. Since in mouse B16-F1 melanoma cells an early and remarkable (about 6-fold) increase in steady state mRNA levels was observed after hepatocyte growth factor (HGF) treatment, we investigated the transcriptional regulation of mouse ODC promoter. Transient transfection of various ODC-luciferase promoter constructs into the B16-Fl cells in combination with electrophoretic mobility shift assays identified the HGF-responsive element as a cluster of three AP-1 binding sites (-1660 to -1572). Even if each site differs from the canonical TPA responsive element for one nucleotide, only the first two AP-1 consensus sequences seemed to be functional since allowed DNA-binding activity of nuclear proteins after HGF treatment. Comparison of the results of transfection assays with the pOD2.5-luc (2.5 kb gene fragment) and with the construct deprived of the AP-1 cluster pOD-B-luc showed that this 50 bp region was required for ODC transactivating activity in response to HGF. Since in B16-F1 cells HGF increased AP-1 activity and the mRNA expression of various AP-1 subunits, we may conclude that HGF-induced transcription of mouse ODC was largely due to triggering of AP-1 pathway. PMID:12054494

  18. Phase 1/2 open-label dose-escalation study of plasmid DNA expressing two isoforms of hepatocyte growth factor in patients with painful diabetic peripheral neuropathy.

    PubMed

    Ajroud-Driss, Senda; Christiansen, Mark; Allen, Jeffrey A; Kessler, John A

    2013-06-01

    This study aimed to evaluate the safety and preliminary efficacy of intramuscular injections of plasmid DNA (VM202) expressing two isoforms of hepatocyte growth factor (HGF) in subjects with painful diabetic peripheral neuropathy (PDPN). Twelve patients in three cohorts (4, 8, and 16 mg) received two sets of VM202 injections separated by two weeks. Safety and tolerability were evaluated and the visual analog scale (VAS), the short form McGill questionnaire (SF-MPQ), and the brief pain inventory for patients with diabetic peripheral neuropathy (BPI-DPN) measured pain level throughout 12 months after treatment. No serious adverse events (AEs) were observed. The mean VAS was reduced from baseline by 47.2% (P = 0.002) at 6 months and by 44.1% (P = 0.005) at 12 months after treatment. The VAS scores for the 4, 8, and 16 mg dose cohorts at 6 months follow-up decreased in a dose-responsive manner, by 21% (P = 0.971), 53% (P = 0.014), and 62% (P = 0.001), respectively. The results with the BPI-DPN and SF-MPQ showed patterns similar to the VAS scores. In conclusion, VM202 treatment appeared to be safe, well tolerated, and sufficient to provide long term symptomatic relief and improvement in the quality of life in patients with PDPN. PMID:23609019

  19. Phase 1/2 Open-label Dose-escalation Study of Plasmid DNA Expressing Two Isoforms of Hepatocyte Growth Factor in Patients With Painful Diabetic Peripheral Neuropathy

    PubMed Central

    Ajroud-Driss, Senda; Christiansen, Mark; Allen, Jeffrey A; Kessler, John A

    2013-01-01

    This study aimed to evaluate the safety and preliminary efficacy of intramuscular injections of plasmid DNA (VM202) expressing two isoforms of hepatocyte growth factor (HGF) in subjects with painful diabetic peripheral neuropathy (PDPN). Twelve patients in three cohorts (4, 8, and 16 mg) received two sets of VM202 injections separated by two weeks. Safety and tolerability were evaluated and the visual analog scale (VAS), the short form McGill questionnaire (SF-MPQ), and the brief pain inventory for patients with diabetic peripheral neuropathy (BPI-DPN) measured pain level throughout 12 months after treatment. No serious adverse events (AEs) were observed. The mean VAS was reduced from baseline by 47.2% (P = 0.002) at 6 months and by 44.1% (P = 0.005) at 12 months after treatment. The VAS scores for the 4, 8, and 16 mg dose cohorts at 6 months follow-up decreased in a dose–responsive manner, by 21% (P = 0.971), 53% (P = 0.014), and 62% (P = 0.001), respectively. The results with the BPI-DPN and SF-MPQ showed patterns similar to the VAS scores. In conclusion, VM202 treatment appeared to be safe, well tolerated, and sufficient to provide long term symptomatic relief and improvement in the quality of life in patients with PDPN. PMID:23609019

  20. Safety of a non-viral plasmid-encoding dual isoforms of hepatocyte growth factor in critical limb ischemia patients: a phase I study.

    PubMed

    Henry, T D; Hirsch, A T; Goldman, J; Wang, Y L; Lips, D L; McMillan, W D; Duval, S; Biggs, T A; Keo, H H

    2011-08-01

    We aimed to evaluate in a phase I dose-escalation study, the safety of intramuscular injections of a novel non-viral plasmid DNA expressing two isoforms of human hepatocyte growth factor (HGF) (VM202) in patients with critical limb ischemia (CLI). In total, 12 patients with CLI and unsuitable for revascularization were consecutively assigned to increasing doses (2 to 16 mg) of VM202 administered into the ischemic calf muscle at days 1 and 15. Patients were evaluated for safety and tolerability, changes in ankle- and toe brachial index (ABI and TBI), and pain severity score using a visual analog scale (VAS) throughout a 12-month follow-up period. Median age was 72 years and 53% of the patients were male. VM202 was safe and well tolerated with no death during the 12-month follow-up. Median ABI and TBI significantly increased from 0.35 to 0.52 (P=0.005) and from 0.15 to 0.24 (P=0.01) at 12 months follow-up. Median VAS decreased from 57.5 to 16.0 mm at 6 months follow-up (P=0.03). In this first human clinical trial, VM202, which expresses two isoforms of human HGF, appear to be safe and well tolerated with encouraging clinical results and thus supports the performance of a phase II randomized controlled trial. PMID:21430785

  1. Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors.

    PubMed

    Yu, Xiuju; He, Xiaoyan; Jiang, Junbing; He, Junping; Fan, Ruiwen; Wang, Haidong; Geng, Jianjun; Dong, Changsheng

    2015-09-01

    Hepatocyte growth factor (HGF)/c-Met signaling has been considered as a key pathway in both melanocyte development and melanogenesis. To understand better the expression patterns and tissue distribution characterization of HGF and its receptor c-Met in skin of white versus brown alpaca (Vicugna pacos), we detected the tissue distribution of HGF and c-Met using immunohistochemistry and analyzed the expression patterns by using Western blot and quantitative real time PCR (qPCR). Immunohistochemistry analysis demonstrated that HGF staining robustly increased in the dermal papilla and mesenchymal cells of white alpaca skin compared with that of brown. However, c-Met staining showed strongly positive result, particularly inhair matrix and root sheath in brown alpaca skin. Western blot and qPCR results suggested that HGF and c-Met were expressed at significantly high levels in white and brown alpaca skins, respectively, and protein and transcripts possessed the same expression pattern in white and brown alpaca skins. The results suggested that HGF/c-Met signaling functions in alpaca coat color formation offer essential theoretical basis for further exploration of the role of HGF/c-Met signaling in pigment formation. PMID:26099836

  2. Sustained functional improvement by hepatocyte growth factor-like small molecule BB3 after focal cerebral ischemia in rats and mice

    PubMed Central

    Chaparro, Rafael E; Izutsu, Miwa; Sasaki, Toshihiro; Sheng, Huaxin; Zheng, Yi; Sadeghian, Homa; Qin, Tao; von Bornstadt, Daniel; Herisson, Fanny; Duan, Bin; Li, Jing-Song; Jiang, Kai; Pearlstein, Molly; Pearlstein, Robert D; Smith, David E; Goldberg, Itzhak D; Ayata, Cenk; Warner, David S

    2015-01-01

    Hepatocyte growth factor (HGF), efficacious in preclinical models of acute central nervous system injury, is burdened by administration of full-length proteins. A multiinstitutional consortium investigated the efficacy of BB3, a small molecule with HGF-like activity that crosses the blood–brain barrier in rodent focal ischemic stroke using Stroke Therapy Academic Industry Roundtable (STAIR) and Good Laboratory Practice guidelines. In rats, BB3, begun 6 hours after temporary middle cerebral artery occlusion (tMCAO) reperfusion, or permanent middle cerebral artery occlusion (pMCAO) onset, and continued for 14 days consistently improved long-term neurologic function independent of sex, age, or laboratory. BB3 had little effect on cerebral infarct size and no effect on blood pressure. BB3 increased HGF receptor c-Met phosphorylation and synaptophysin expression in penumbral tissue consistent with a neurorestorative mechanism from HGF-like activity. In mouse tMCAO, BB3 starting 10 minutes after reperfusion and continued for 14 days improved neurologic function that persisted for 8 weeks in some, but not all measures. Study in animals with comorbidities and those exposed to common stroke drugs are the next steps to complete preclinical assessment. These data, generated in independent, masked, and rigorously controlled settings, are the first to suggest that the HGF pathway can potentially be harnessed by BB3 for neurologic benefit after ischemic stroke. PMID:25712497

  3. Hepatocyte growth factor inhibits apoptosis by the profibrotic factor angiotensin II via extracellular signal-regulated kinase 1/2 in endothelial cells and tissue explants.

    PubMed

    Lee, Young H; Marquez, Ana P; Mungunsukh, Ognoon; Day, Regina M

    2010-12-01

    Hepatocyte growth factor (HGF), an endogenous tissue repair factor, attenuates apoptosis in many primary cell types, but the mechanism is not completely understood. Our laboratory demonstrated that angiotensin (Ang) II activates the intrinsic apoptotic pathway in primary endothelial cells (ECs) via reduction of the antiapoptotic protein Bcl-x(L). Ang II decreased Bcl-x(L) mRNA half-life by reducing its binding to nucleolin, a protein that normally binds a 3' AU-rich region and stabilizes Bcl-x(L) mRNA. We hypothesized HGF may block apoptosis induced by Ang II. We used primary EC and ex vivo cultures of rat lung tissue to investigate HGF inhibition of Ang II-induced apoptosis. Our data indicated HGF abrogated Ang II-induced apoptosis by inhibiting cytochrome c release, caspase-3 activation, and DNA fragmentation. RNA-immunoprecipitation experiments demonstrated that HGF stabilized Bcl-x(L) mRNA by increasing nucleolin binding to the 3'-untranslated region that was associated with cytoplasmic localization of nucleolin. Cytoplasmic localization of nucleolin and Bcl-x(L) mRNA stabilization required HGF activation of extracellular signal-regulated kinase (ERK)1/2, but not phosphatidylinositol 3-kinase. HGF also blocked Ang II-induced caspase-3 activation and lactate dehydrogenase release in tissue explants in an ERK-dependent manner. PMID:20926686

  4. Hepatocyte Growth Factor Effects on Mesenchymal Stem Cells Derived from Human Arteries: A Novel Strategy to Accelerate Vascular Ulcer Wound Healing

    PubMed Central

    Valente, Sabrina; Pasanisi, Emanuela; Ricci, Francesca; Stella, Andrea

    2016-01-01

    Vascular ulcers are a serious complication of peripheral vascular disease, especially in diabetics. Several approaches to treat the wounds are proposed but they show poor outcomes and require long healing times. Hepatocyte Growth Factor/Scatter Factor (HGF/SF) is a pleiotropic cytokine exerting many biological activities through the c-Met receptor. This study was aimed at verifying whether HGF/SF influences proliferation, migration, and angiogenesis on mesenchymal stem cells isolated from human arteries (hVW-MSCs). hVW-MSCs were exposed to NIBSC HGF/SF (2.5, 5, 10, and 70 ng/mL) from 6 hrs to 7 days. HGF and c-MET mRNA and protein expression, cell proliferation (Alamar Blue and Ki–67 assay), migration (scratch and transwell assays), and angiogenesis (Matrigel) were investigated. hVW-MSCs displayed stemness features and expressed HGF and c-MET. HGF/SF did not increase hVW-MSC proliferation, whereas it enhanced the cell migration, the formation of capillary-like structures, and the expression of angiogenic markers (vWF, CD31, and KDR). The HGF/SF effects on hVW-MSC migration and angiogenic potential are of great interest to accelerate wound healing process. Local delivery of HGF/SF could therefore improve the healing of unresponsive vascular ulcers. PMID:26788066

  5. M10, a caspase cleavage product of the hepatocyte growth factor receptor, interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo.

    PubMed

    Atanelishvili, Ilia; Shirai, Yuichiro; Akter, Tanjina; Buckner, Taylor; Noguchi, Atsushi; Silver, Richard M; Bogatkevich, Galina S

    2016-04-01

    Hepatocyte growth factor receptor, also known as cellular mesenchymal-epithelial transition factor (c-MET, MET), is an important antifibrotic molecule that protects various tissues, including lung, from injury and fibrosis. The intracellular cytoplasmic tail of MET contains a caspase-3 recognition motif "DEVD-T" that on cleavage by caspase-3 generates a 10-amino acid peptide, TRPASFWETS, designated as "M10". M10 contains at its N-terminus the uncharged amino acid proline (P) directly after a cationic amino acid arginine (R) which favors the transport of the peptide through membranes. M10, when added to cell culture medium, remains in the cytoplasm and nuclei of cells for up to 24 hours. M10 effectively decreases collagen in both scleroderma and TGFβ-stimulated normal lung and skin fibroblasts. M10 interacts with the Mad Homology 2 domain of Smad2 and inhibits TGFβ-induced Smad2 phosphorylation, suggesting that the antifibrotic effects of M10 are mediated in part by counteracting Smad-dependent fibrogenic pathways. In the bleomycin murine model of pulmonary fibrosis, M10 noticeably reduced lung inflammation and fibrosis. Ashcroft fibrosis scores and lung collagen content were significantly lower in bleomycin-treated mice receiving M10 as compared with bleomycin-treated mice receiving scrambled peptide. We conclude that M10 peptide interacts with Smad2 and demonstrates strong antifibrotic effects in vitro and in vivo in an animal model of lung fibrosis and should be considered as a potential therapeutic agent for systemic sclerosis and other fibrosing diseases. PMID:26772959

  6. D1398G Variant of MET Is Associated with Impaired Signaling of Hepatocyte Growth Factor in Alveolar Epithelial Cells and Lung Fibroblasts.

    PubMed

    Atanelishvili, Ilia; Shirai, Yuichiro; Akter, Tanjina; Noguchi, Atsushi; Ash, Kurt T; Misra, Suniti; Ghatak, Sibnath; Silver, Richard M; Bogatkevich, Galina S

    2016-01-01

    Pulmonary fibrosis represents the terminal stage of a diverse group of lung diseases including scleroderma associated interstitial lung disease. The molecular mechanisms underlying the pathogenesis of lung fibrosis are not well understood and there is a great need for more effective treatment for this lethal disease. We recently discovered a small fragment of hepatocyte growth factor (HGF) receptor MET as a peptide designated "M10," with strong antifibrotic properties. Furthermore, we showed that aspartic acid at position 1398 of MET is essential for M10 generation. The current study was undertaken to investigate the D1398G variant of MET in which aspartic acid at position 1398 was mutated to glycine resulting in loss of M10. We demonstrate that lung fibroblasts, A549, and primary alveolar epithelial cells (AEC) expressing D1398G MET exhibit reduced auto-phosphorylation on tyrosine residues and reduced activation of Ras and MAPK. HGF treatment of scleroderma lung fibroblasts as well as HGF treatment of TGFβ-treated normal lung fibroblasts transfected with wild type MET is associated with decreased collagen, connective tissue growth factor (CTGF, CCN2) and smooth muscle α-actin (SMA). However, HGF has no such effects in cells transfected with MET D1398G. Cisplatin- and FasL-induced apoptosis is significantly reduced in AEC transfected with MET wild type, but not in AEC transfected with MET D1398G. We conclude that the D1398G variant of MET is associated with compromised phosphorylation and impaired HGF signaling in lung fibroblasts and AEC, two cell types implicated in the pathogenesis of pulmonary fibrosis associated with scleroderma. Ongoing studies will explore the frequency of this variant and its relationship to pulmonary outcomes in scleroderma patients. PMID:27584154

  7. Melatonin Mediates Monochromatic Light-induced Insulin-like Growth Factor 1 Secretion of Chick Liver: Involvement of Membrane Receptors.

    PubMed

    Li, Suqi; Cao, Jing; Wang, Zixu; Dong, Yulan; Wang, Wenli; Chen, Yaoxing

    2016-07-01

    Monochromatic lights influenced the proliferation and differentiation of skeletal satellite cells in broilers by the enhancement of insulin-like growth factor 1 (IGF-1) secretion. However, whether melatonin (MEL)-mediated monochromatic lights influenced the IGF-1 secretion remains unclear. Newly hatched broilers, including intact, sham operation and pinealectomy groups, were exposed to blue (BL), green (GL), red (RL) and white light (WL) from a light-emitting diode system for 14 days. The results showed that GL effectively promoted the secretion of MEL and IGF-1, the expression of proliferating cell nuclear antigen and MEL receptor subtypes Mel1a, Mel1b and Mel1c in the liver compared to BL and RL in vivo. Moreover, those was a positive correlation between MEL and IGF-1 (r = 0.834). After pinealectomy, however, these parameters declined, and there were no differences between GL and other monochromatic light treatments. In vitro, exogenous MEL increased hepatocyte proliferation and IGF-1 secretion. Meanwhile, the MEL enhancements were suppressed by prazosin (selective Mel1c antagonist), followed by luzindole (nonselective Mel1a/Mel1b antagonist), but not suppressed by 4-phenyl-2-propionamideotetralin (selective Mel1b antagonist). These findings demonstrated that MEL mediated the monochromatic light-induced secretion of IGF-1 in chicks' livers by Mel1c and that Mel1a may be involved in this process. PMID:27128575

  8. Humanized anti-hepatocyte growth factor (HGF) antibody suppresses innate irinotecan (CPT-11) resistance induced by fibroblast-derived HGF

    PubMed Central

    Kim, BoRa; Park, Byung Hee; Shin, Kum-Joo; Song, Seong-Won; Kim, Jung Ju; Kim, Hwan-Mook; Lee, Sang-Jin; Oh, Seung Hyun

    2015-01-01

    The growth factors derived from the microenvironment create an environment conducive to tumor growth and survival. HGF deprivation using neutralizing antibody enhanced chemosensitivity in colorectal cancer cells (CRC). We determined secreted HGF in fibroblast conditioned medium (CM). Combination treatment of anti-HGF antibody and irinotecan (CPT-11) directly enhanced CPT-11 sensitivity in CRC. We generated xenograft in NOD/SCID mice inoculating HCT-116 human colorectal cancer cells subcutaneously with or without fibroblast. We found that the combination of CPT-11 and anti-HGF antibody induced marked suppression of tumor development. These results suggest that HGF produced by fibroblast induce CPT-11 resistance, and that anti-HGF antibody abrogate such resistance in vivo. fibroblast-derived HGF is important determinant of chemoresistance. Anti-HGF monoclonal antibody treatment confirmed the importance of this growth factor for chemoresistance in CRC. These results present new options toward the early diagnosis of chemoresistance and suggest novel combinations of chemotherapy and anti-HGF agents to prevent or significantly delay the onset of therapy resistance. PMID:26090722

  9. A simple model for dislocation emission mediated dynamic nanovoid growth

    NASA Astrophysics Data System (ADS)

    Wilkerson, Justin; Ramesh, K. T.

    2015-06-01

    Failure of ductile metals has long been attributed to the microscopic processes of void nucleation, growth, and finally coalescence leading to fracture. Our traditional view of void nucleation is associated with interface debonding at second-phase particles. However, much of this understanding has been gleaned from observations of quasi-static fracture surfaces. Under more extreme dynamic loading conditions second-phase particles may not necessarily be the dominant source of void nucleating material defects, and a few key experimental observations of laser spall surfaces seem to support this assertion. Here, we motivate an alternative mechanism to the traditional view, namely shock-induced vacancy generation and clustering followed by nanovoid growth mediated by dislocation emission. This mechanism only becomes active at very large stresses, and thus it is desirable to establish a closed-form criterion for the macroscopic stress required to activate dislocation emission in porous materials. Following an approach similar to Lubarda and co-workers, we make use of stability arguments applied to the analytic solutions of the elastic interactions of dislocations and voids to derive the desired criterion. We then propose a dynamic nanovoid growth law that is motivated by the kinetics of dislocation emission. The resulting failure model is validated against a number of molecular dynamics simulations with favorable agreement. Lastly, we make use of our simple model to predict some interesting anomalous behaviors associated with high surface energies and nonlinear elasticity.

  10. Elevated hepatocyte growth factor expression as an autocrine c-Met activation mechanism in acquired resistance to sorafenib in hepatocellular carcinoma cells.

    PubMed

    Firtina Karagonlar, Zeynep; Koc, Dogukan; Iscan, Evin; Erdal, Esra; Atabey, Neşe

    2016-04-01

    Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the third leading cause of cancer-related deaths worldwide. Limitations in HCC treatment result due to poor prognosis and resistance against traditional radiotherapy and chemotherapies. The multikinase inhibitor sorafenib is the only FDA approved drug available for advanced HCC patients, and development of second-line treatment options for patients who cannot tolerate or develop resistance to sorafenib is an urgent medical need. In this study, we established sorafenib-resistant cells from Huh7 and Mahlavu cell lines by long-term sorafenib exposure. Sorafenib-resistant HCC cells acquired spindle-shape morphology, upregulated mesenchymal markers, and showed significant increase in both migration and invasion abilities compared to their parental counterparts. Moreover, after long-term sorafenib treatment, HCC cells showed induction of hepatocyte growth factor (HGF) synthesis and secretion along with increased levels of c-Met kinase and its active phosphorylated form, indicating autocrine activation of HGF/c-Met signaling. Importantly, the combined treatment of the resistant cells with c-Met kinase inhibitor SU11274 and HGF neutralizing antibody significantly reversed the increased invasion ability of the cells. The combined treatment also significantly augmented sorafenib-induced apoptosis, suggesting restoration of sorafenib sensitivity. These results describe, for the first time, compensatory upregulation of HGF synthesis leading to autocrine activation of HGF/c-Met signaling as a novel cellular strategy in the acquisition of sorafenib resistance. Therefore, we suggest that combinatorial therapeutic strategies with HGF and c-Met inhibitors comprise promising candidates for overcoming sorafenib resistance. PMID:26790028

  11. Detection of hepatocyte growth factor (HGF) ligand-c-MET receptor activation in formalin-fixed paraffin embedded specimens by a novel proximity assay.

    PubMed

    Dua, Rajiv; Zhang, Jianhuan; Parry, Gordon; Penuel, Elicia

    2011-01-01

    Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffin embedded (FFPE) tumor specimens. FFPE is a standard method of specimen preparation used in the histological analysis of solid tumors. Due to variability in FFPE preparations and the labile nature of protein phosphorylation, measurements of phospho-proteins are unreliable and create ambiguities in clinical interpretation. Here, we describe an alternative, novel approach to measure receptor activation by detecting and quantifying ligand-receptor complexes in FFPE specimens. We used hepatocyte growth factor (HGF)-c-MET as our model ligand-receptor system. HGF is the only known ligand of the c-MET tyrosine kinase receptor and HGF binding triggers c-MET phosphorylation. Novel antibody proximity-based assays were developed and used to detect and quantify total c-MET, total HGF, and HGF-c-MET ligand-receptor interactions in FFPE cell line and tumor tissue. In glioma cells, autocrine activation of c-MET by HGF-c-MET increased basal levels of c-MET phosphorylation at tyrosine (Tyr) 1003. Furthermore, HGF-c-MET activation in glioma cell lines was verified by Surface Protein-Protein Interaction by Crosslinking ELISA (SPPICE) assay in corresponding soluble cell lysates. Finally, we profiled levels ofc-MET, HGF, and HGF-c-MET complexes in FFPE specimens of human Non-Small Cell Lung Cancer (NSCLC), Gastric Cancer, Head and Neck Squamous Cell, and Head and Neck Non-Squamous Cell carcinomas. This report describes a novel approach for the detection and quantification of ligand-receptor interactions that can be widely applied to measure receptor activation in FFPE preclinical models and archived FFPE human tissue specimens. PMID:21283737

  12. Detection of Hepatocyte Growth Factor (HGF) Ligand-c-MET Receptor Activation in Formalin-Fixed Paraffin Embedded Specimens by a Novel Proximity Assay

    PubMed Central

    Dua, Rajiv; Zhang, Jianhuan; Parry, Gordon; Penuel, Elicia

    2011-01-01

    Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffin embedded (FFPE) tumor specimens. FFPE is a standard method of specimen preparation used in the histological analysis of solid tumors. Due to variability in FFPE preparations and the labile nature of protein phosphorylation, measurements of phospho-proteins are unreliable and create ambiguities in clinical interpretation. Here, we describe an alternative, novel approach to measure receptor activation by detecting and quantifying ligand-receptor complexes in FFPE specimens. We used hepatocyte growth factor (HGF)-c-MET as our model ligand-receptor system. HGF is the only known ligand of the c-MET tyrosine kinase receptor and HGF binding triggers c-MET phosphorylation. Novel antibody proximity-based assays were developed and used to detect and quantify total c-MET, total HGF, and HGF-c-MET ligand-receptor interactions in FFPE cell line and tumor tissue. In glioma cells, autocrine activation of c-MET by HGF-c-MET increased basal levels of c-MET phosphorylation at tyrosine (Tyr) 1003. Furthermore, HGF-c-MET activation in glioma cell lines was verified by Surface Protein-Protein Interaction by Crosslinking ELISA (SPPICE) assay in corresponding soluble cell lysates. Finally, we profiled levels of c-MET, HGF, and HGF-c-MET complexes in FFPE specimens of human Non-Small Cell Lung Cancer (NSCLC), Gastric Cancer, Head and Neck Squamous Cell, and Head and Neck Non-Squamous Cell carcinomas. This report describes a novel approach for the detection and quantification of ligand-receptor interactions that can be widely applied to measure receptor activation in FFPE preclinical models and archived FFPE human tissue specimens. PMID:21283737

  13. Increased miR-16 expression induced by hepatitis C virus infection promotes liver fibrosis through downregulation of hepatocyte growth factor and Smad7.

    PubMed

    Zhu, Bin; Wei, Xiao-Xia; Wang, Tian-Bao; Zhou, Yan-Cai; Liu, A-Min; Zhang, Guang-Wen

    2015-08-01

    Hepatitis C virus (HCV) is involved in the initiation and progression of liver fibrosis by regulating genes encoding host proteins. However, the underlying mechanism of HCV-induced liver fibrosis is still to be determined. Reverse transcription polymerase chain reaction (RT-PCR) and western blot were performed to investigate the effect of HCV infection on the expression of the cellular microRNA miR-16 and its target genes encoding hepatocyte growth factor (HGF) and Smad7 in patients infected with HCV and in a liver cell line, QSG-7701, transfected with Ad-HCV, a recombinant adenovirus construct for expression of the HCV core protein. Regulation of HGF and Smad7 expression by miR-16 was assessed using luciferase reporter construct assays and miR-16 mimic transfection. Interferon-α (IFN-α) was used to verify the alteration of gene expression induced by HCV in QSG-7701 cells. Here, we found that miR-16 levels were increased in patients with HCV infection and were correlated with HGF and Smad7 expression levels in patients with HCV infection. Furthermore, HGF and Smad7 were predicted by bioinformatics analysis to be targets of miR-16. Upregulation of miR-16 and decreased HGF and Smad7 expression were still shown in QSG-7701 cells infected with Ad-HCV. Additionally, interferon-α (IFN-α) could reverse the changes in gene expression induced by HCV infection. These results suggest that the upregulation of miR-16 expression induced by HCV infection is a novel mechanism that contributes to downregulation of HGF and Smad7 in the development of liver fibrosis. PMID:26071245

  14. Study of surfactant mediated growth of Ni/V superlattices

    SciTech Connect

    Amir, S. M.; Gupta, Mukul; Potdar, Satish; Gupta, Ajay; Stahn, Jochen

    2013-07-14

    The Ni/V multilayers are useful as soft x-ray mirrors, polarizers, and phase retarders. For these applications, it is necessary that the interfaces roughness and interdiffusion must be as small as possible. The V-on-Ni and Ni-on-V interfaces are asymmetric due to the difference in the surface free energy of Ni and V. In this work, we report Ag surfactant mediated growth of Ni/V superlattices prepared using ion beam sputter deposition technique. These superlattices were studied using x-ray and neutron scattering techniques. It was found that when added in an optimum amount, Ag surfactant results in reduced interface roughness and interdiffusion across the interfaces. Obtained results can be understood with the surfactant floating-off mechanism leading to a balance in the surface free energy of Ni and V.

  15. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth.

    PubMed

    Martin, Claire; Lafosse, Jean-Michel; Malavaud, Bernard; Cuvillier, Olivier

    2010-01-01

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK. PMID:19932089

  16. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

    SciTech Connect

    Martin, Claire; Lafosse, Jean-Michel; Malavaud, Bernard; Cuvillier, Olivier

    2010-01-01

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

  17. Accelerated adhesion of grafted skins by laser-induced stress wave-based gene transfer of hepatocyte growth factor

    NASA Astrophysics Data System (ADS)

    Aizawa, Kazuya; Sato, Shunichi; Saitoh, Daizoh; Tsuda, Hitoshi; Ashida, Hiroshi; Obara, Minoru

    2009-02-01

    In our previous study, we delivered plasmid DNA coding for human hepatocyto growth factor (hHGF) to rat skin grafts based on laser-induced stress wave (LISW), by which production of CD31-positive cells in the grafted skins was found to be enhanced, suggesting improved angiogenesis. In this study, we validated the efficacy of this method to accelerate adhesion of grafted skins; reperfusion and reepithelialization in the grafted skins were examined. As a graft, dorsal skin of a rat was exsected and its subcutaneous fat was removed. Plasmid DNA expression vector for hHGF was injected into the graft; on its back surface a laser target with a transparent sheet for plasma confinement was placed, and irradiated with three nanosecond laser pulses at a laser fluence of 1.2 J/cm2 (532 nm; spot diameter, 3 mm) to generate LISWs. After the application of LISWs, the graft was transplanted onto its donor site. We evaluated blood flow by laser Doppler imaging and analyzed reepithelialization based on immunohistochemistry as a function of postgrafting time. It was found that both reperfusion and reepithelialization were significantly enhanced for the grafts with gene transfection than for normal grafts; reepithelialization was completed within 7 days after transplantation with the transfected grafts. These findings demonstrate that adhesion of grafted skins can be accelerated by delivering HGF gene to the grafts based on LISWs.

  18. Highly efficient expression of functional recombinant human keratinocyte growth factor 1 and its protective effects on hepatocytes.

    PubMed

    Xue, Ping; Zhu, Xiaojing; Shi, Junqing; Fu, Hongqi; Zhang, Jian; Liu, Min; Jiang, Chao; Li, Xiaokun

    2014-05-01

    Three forms of recombinant human keratinocyte growth factor 1 (rhKGF1) with or without the native signal peptide or a 23-amino acid truncation were expressed in Spodoptera frugiperda 9 (Sf9) cells by designing with insect codon usage. Immunoblotting demonstrated that these rhKGF1 proteins were recognized by a human anti-KGF1 antibody. The multiplicity of infection and timing of harvest had a significant effect on protein yield, protein quality, and cytotoxicity. Our results indicated that the native signal peptide directed KGF1 secretion from insect cells, reaching a maximum at 60 h postinfection. Although secretion of rhKGF1194 was less efficient than that of rhKGF1163 and rhKGF1140, protein secretion is an attractive pathway for simple purification of biologically active rhKGF1 at a high yield. Moreover, the sizes of rhKGF1194 and rhKGF1163 were similar (20 kDa), suggesting that the signal peptide may be recognized and removed in Sf9 cells. A 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay was used to analyze the biological function of rhKGF1, indicating that the three forms of rhKGF1 had a similar mitogenic function in BaF3 cells. Furthermore, to elucidate the effect of rhKGF1 on cytoprotection of liver cells, we used KGF1 pretreatment of an acute liver injury model. The results indicated that rhKGF1 prevented necrosis and apoptosis of CCl4-treated HL7702 cells in vitro and in vivo. These results suggest that KGF1 may be a candidate therapeutic drug for acute liver injury. PMID:24463717

  19. Enhanced expression of hepatocyte-specific microRNAs in valproic acid mediated hepatic trans-differentiation of human umbilical cord derived mesenchymal stem cells.

    PubMed

    Raut, Akshata; Khanna, Aparna

    2016-05-01

    MicroRNAs (miRNAs) play an important role in the control of cell fate determination during differentiation. In this study, we analyzed the expression pattern of microRNAs (miRNAs) during hepatic trans-differentiation. The protocol employed the use of histone deacetylase inhibitor (HDACI), valproic acid (VPA) to induce hepatic trans-differentiation of human umbilical cord Wharton's jelly derived mesenchymal stem cells (hUC-MSCs). The differentiated hepatocyte like cells (HLCs) from hUC-MSCs shared typical characteristics with mature hepatocytes, including morphology, expression of hepatocyte -specific genes at the molecular and cellular level. Moreover, the functionality of HLCs was confirmed through various liver function tests such as periodic acid-Schiff (PAS) stain for glycogen accumulation, enzyme-linked immunosorbent assay (ELISA) for synthesis of albumin and release of urea. The aim of the present work was to examine the effect of VPA treatment on miRNA expression during hepatic trans-differentiation. The analysis at miRNA level showed that there was a significant increase in expression of miRNAs involved in hepatic differentiation, due to VPA pre-treatment during differentiation. The study, thus demonstrated that improved expression of hepatocyte-specific miRNAs, miR-23b cluster (miR-27b-3p, miR-24-1-5p and miR-23b-3p), miR-30a-5p, miR-26a-5p, miR-148a-3p, miR-192-5p, miR-122-5p due to VPA pre-treatment contributed to a more efficient hepatic trans-differentiation from hUC-MSCs. The putative targets of these upregulated miRNAs were predicted using Bioinformatics analysis. Finally, miR-122-5p, highly upregulated miRNA during hepatic differentiation, was selected for target verification studies. Thus, this study also provides the basis for the function of miR-122-5p during hepatic differentiation of hUC-MSCs. PMID:27001466

  20. Axl as a mediator of cellular growth and survival

    PubMed Central

    Axelrod, Haley; Pienta, Kenneth J.

    2014-01-01

    The control of cellular growth and proliferation is key to the maintenance of homeostasis. Survival, proliferation, and arrest are regulated, in part, by Growth Arrest Specific 6 (Gas6) through binding to members of the TAM receptor tyrosine kinase family. Activation of the TAM receptors leads to downstream signaling through common kinases, but the exact mechanism within each cellular context varies and remains to be completely elucidated. Deregulation of the TAM family, due to its central role in mediating cellular proliferation, has been implicated in multiple diseases. Axl was cloned as the first TAM receptor in a search for genes involved in the progression of chronic to acute-phase leukemia, and has since been established as playing a critical role in the progression of cancer. The oncogenic nature of Axl is demonstrated through its activation of signaling pathways involved in proliferation, migration, inhibition of apoptosis, and therapeutic resistance. Despite its recent discovery, significant progress has been made in the development of effective clinical therapeutics targeting Axl. In order to accurately define the role of Axl in normal and diseased processes, it must be analyzed in a cell type-specific context. PMID:25344858

  1. Syntrophic growth via quinone-mediated interspecies electron transfer

    PubMed Central

    Smith, Jessica A.; Nevin, Kelly P.; Lovley, Derek R.

    2015-01-01

    The mechanisms by which microbial species exchange electrons are of interest because interspecies electron transfer can expand the metabolic capabilities of microbial communities. Previous studies with the humic substance analog anthraquinone-2,6-disulfonate (AQDS) suggested that quinone-mediated interspecies electron transfer (QUIET) is feasible, but it was not determined if sufficient energy is available from QUIET to support the growth of both species. Furthermore, there have been no previous studies on the mechanisms for the oxidation of anthrahydroquinone-2,6-disulfonate (AHQDS). A co-culture of Geobacter metallireducens and G. sulfurreducens metabolized ethanol with the reduction of fumarate much faster in the presence of AQDS, and there was an increase in cell protein. G. sulfurreducens was more abundant, consistent with G. sulfurreducens obtaining electrons from acetate that G. metallireducens produced from ethanol, as well as from AHQDS. Co-cultures initiated with a citrate synthase-deficient strain of G. sulfurreducens that was unable to use acetate as an electron donor also metabolized ethanol with the reduction of fumarate and cell growth, but acetate accumulated over time. G. sulfurreducens and G. metallireducens were equally abundant in these co-cultures reflecting the inability of the citrate synthase-deficient strain of G. sulfurreducens to metabolize acetate. Evaluation of the mechanisms by which G. sulfurreducens accepts electrons from AHQDS demonstrated that a strain deficient in outer-surface c-type cytochromes that are required for AQDS reduction was as effective at QUIET as the wild-type strain. Deletion of additional genes previously implicated in extracellular electron transfer also had no impact on QUIET. These results demonstrate that QUIET can yield sufficient energy to support the growth of both syntrophic partners, but that the mechanisms by which electrons are derived from extracellular hydroquinones require further investigation. PMID

  2. Source-Related Effects of Wastewater on Transcription Factor (AhR, CAR and PXR)-Mediated Induction of Gene Expression in Cultured Rat Hepatocytes and Their Association with the Prevalence of Antimicrobial-Resistant Escherichia coli.

    PubMed

    Guruge, Keerthi S; Yamanaka, Noriko; Sonobe, Miyuki; Fujizono, Wataru; Yoshioka, Miyako; Akiba, Masato; Yamamoto, Takehisa; Joshua, Derrick I; Balakrishna, Keshava; Yamashita, Nobuyoshi; Kannan, Kurunthachalam; Tsutsui, Toshiyuki

    2015-01-01

    Extracts of wastewater collected from 4 sewage treatment plants (STPs) receiving effluents from different sources in South India were investigated for their levels of transcription factor-mediated gene induction in primary cultured rat hepatocytes. In addition, the relation between gene induction levels and the prevalence of antimicrobial-resistant Escherichia coli (E. coli) in wastewater was examined. STP-3, which treats only hospital wastewater, exhibited significantly greater induction potency of all 6 drug metabolizing cytochrome P450 (CYP) genes examined, CYP1A1, 1A2, 1B1, 2B15, 3A1, and 3A2, whereas the wastewater at STP-1, which exclusively receives domestic sewage, showed significantly diminished levels of induction of 3 CYP genes when compared to the levels of CYP induction at STP-2, which receives mixed wastewater. Samples collected during the monsoon season showed a significantly altered gene induction capacity compared to that of samples from the pre-monsoon period. The data suggest that the toxicity of wastewater in STPs was not significantly diminished during the treatment process. The chemical-gene interaction data predicted that a vast number of chemicals present in the wastewater would stimulate the genes studied in the rat hepatocytes. The multivariable logistic regression analysis demonstrated that the prevalence of isolates resistant to cefotaxime, imipenem and streptomycin was significantly correlated with the levels of induction of at least three CYP-isozymes in STP wastewater. In addition, the resistance of isolates in treatment plants was not altered by the treatment steps, whereas the sampling season did have an impact on the resistance to specific antimicrobials. The identification of receptor-mediated gene regulation capacities offers important data not limited to the (synergistic) physiological role of chemicals in biological systems but may provide new insight into the link between the effects of known/unknown drugs and prevalence of

  3. Source-Related Effects of Wastewater on Transcription Factor (AhR, CAR and PXR)-Mediated Induction of Gene Expression in Cultured Rat Hepatocytes and Their Association with the Prevalence of Antimicrobial-Resistant Escherichia coli

    PubMed Central

    Guruge, Keerthi S.; Yamanaka, Noriko; Sonobe, Miyuki; Fujizono, Wataru; Yoshioka, Miyako; Akiba, Masato; Yamamoto, Takehisa; Joshua, Derrick I.; Balakrishna, Keshava; Yamashita, Nobuyoshi; Kannan, Kurunthachalam; Tsutsui, Toshiyuki

    2015-01-01

    Extracts of wastewater collected from 4 sewage treatment plants (STPs) receiving effluents from different sources in South India were investigated for their levels of transcription factor-mediated gene induction in primary cultured rat hepatocytes. In addition, the relation between gene induction levels and the prevalence of antimicrobial-resistant Escherichia coli (E. coli) in wastewater was examined. STP-3, which treats only hospital wastewater, exhibited significantly greater induction potency of all 6 drug metabolizing cytochrome P450 (CYP) genes examined, CYP1A1, 1A2, 1B1, 2B15, 3A1, and 3A2, whereas the wastewater at STP-1, which exclusively receives domestic sewage, showed significantly diminished levels of induction of 3 CYP genes when compared to the levels of CYP induction at STP-2, which receives mixed wastewater. Samples collected during the monsoon season showed a significantly altered gene induction capacity compared to that of samples from the pre-monsoon period. The data suggest that the toxicity of wastewater in STPs was not significantly diminished during the treatment process. The chemical-gene interaction data predicted that a vast number of chemicals present in the wastewater would stimulate the genes studied in the rat hepatocytes. The multivariable logistic regression analysis demonstrated that the prevalence of isolates resistant to cefotaxime, imipenem and streptomycin was significantly correlated with the levels of induction of at least three CYP-isozymes in STP wastewater. In addition, the resistance of isolates in treatment plants was not altered by the treatment steps, whereas the sampling season did have an impact on the resistance to specific antimicrobials. The identification of receptor-mediated gene regulation capacities offers important data not limited to the (synergistic) physiological role of chemicals in biological systems but may provide new insight into the link between the effects of known/unknown drugs and prevalence of

  4. Nonylphenol-mediated CYP induction is PXR-dependent: The use of humanized mice and human hepatocytes suggests that hPXR is less sensitive than mouse PXR to nonylphenol treatment

    SciTech Connect

    Mota, Linda C.; Barfield, Christina; Hernandez, Juan P.; Baldwin, William S.

    2011-05-01

    Nonylphenol (NP), a by-product of alkylphenol ethoxylates, is a pervasive surfactant that activates the xenosensing nuclear receptor, the pregnane X-receptor (PXR) in transactivation assays in vitro. We are interested in determining if NP activates PXR in vivo, determining if hPXR and mPXR act similarly, and investigating the role of PXR in protecting individuals from NP. Wild-type (WT), PXR-null, and humanized PXR (hPXR) mice were treated with NP at 0, 50 or 75 mg/kg/day for one week, and cytochrome P450 (CYP) induction, liver histopathology, and serum NP concentrations were examined. WT mice treated with NP showed induction of Cyp2b, and male-specific induction of Cyp2c and Cyp3a. CYPs were not induced in PXR-null mice, demonstrating that PXR is necessary for NP-mediated CYP induction. CAR-mediated CYP induction was not observed in the PXR-null mice despite previous data demonstrating that NP is also a CAR activator. hPXR mice only showed moderate Cyp induction, suggesting that hPXR is not as sensitive to NP as mPXR in vivo. NP-mediated Cyp3a induction from three human hepatocyte donors was not significant, confirming that hPXR is not very sensitive to NP-mediated CYP induction. Lastly, mice with PXR (mPXR and hPXR) showed lower NP serum concentrations than PXR-null mice treated with NP suggesting that PXR plays a role in decreasing liver toxicity by basally regulating phase I-III detoxification enzymes that promote the metabolism and elimination of NP. In summary, PXR is required for NP-mediated CYP-induction, mPXR mediates greater CYP induction than hPXR in vivo, and the presence of PXR, especially mPXR, is associated with altered histopathology and increased clearance of NP.

  5. Nonylphenol-mediated CYP induction is PXR-dependent: The use of humanized mice and human hepatocytes suggests that hPXR is less sensitive than mouse PXR to nonylphenol treatment

    PubMed Central

    Mota, Linda C; Barfield, Christina; Hernandez, Juan P; Baldwin, William S.

    2011-01-01

    Nonylphenol (NP), a by-product of alkylphenol ethoxylates, is a pervasive surfactant that activates the xenosensing nuclear receptor, the pregnane X-receptor (PXR) in transactivation assays in vitro. We are interested in determining if NP activates PXR in vivo, determining if hPXR and mPXR act similarly, and investigating the role of PXR in protecting individuals from NP. Wild-type (WT), PXR-null, and humanized PXR (hPXR) mice were treated with NP at 0, 50 or 75 mg/kg/day for one week, and cytochrome P450 (CYP) induction, liver histopathology, and serum NP concentrations were examined. WT mice treated with NP showed induction of Cyp2b, and male-specific induction of Cyp2c and Cyp3a. CYPs were not induced in PXR-null mice, demonstrating that PXR is necessary for NP-mediated CYP induction. CAR-mediated CYP induction was not observed in the PXR-null mice despite previous data demonstrating NP is also a CAR activator. hPXR mice only showed moderate Cyp induction, suggesting that hPXR is not as sensitive to NP as mPXR in vivo. NP-mediated Cyp3a induction from three human hepatocyte donors was not significant, confirming that hPXR is not very sensitive to NP-mediated CYP induction. Lastly, mice with PXR (mPXR and hPXR) showed lower NP serum concentrations than PXR-null mice treated with NP suggesting that PXR plays a role in decreasing liver toxicity by basally regulating Phase I-III detoxification enzymes that promote the metabolism and elimination of NP. In summary, PXR is required for NP-mediated CYP-induction, and mPXR mediates greater CYP induction than hPXR in vivo, and the presence of PXR, especially mPXR, is associated with altered histopathology and increased clearance of NP. PMID:21376070

  6. Mechanisms of lysophosphatidylcholine-induced hepatocyte lipoapoptosis

    PubMed Central

    Kakisaka, Keisuke; Cazanave, Sophie C.; Fingas, Christian D.; Guicciardi, Maria E.; Bronk, Steven F.; Werneburg, Nathan W.; Mott, Justin L.

    2012-01-01

    Isolated hepatocytes undergo lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH2-terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC. PMID:21995961

  7. Hepatocytes as Immunological Agents.

    PubMed

    Crispe, Ian N

    2016-01-01

    Hepatocytes are targeted for infection by a number of major human pathogens, including hepatitis B virus, hepatitis C virus, and malaria. However, hepatocytes are also immunological agents in their own right. In systemic immunity, they are central in the acute-phase response, which floods the circulation with defensive proteins during diverse stresses, including ischemia, physical trauma, and sepsis. Hepatocytes express a variety of innate immune receptors and, when challenged with pathogen- or damage-associated molecular patterns, can deliver cell-autonomous innate immune responses that may result in host defense or in immunopathology. Important human pathogens have evolved mechanisms to subvert these responses. Finally, hepatocytes talk directly to T cells, resulting in a bias toward immune tolerance. PMID:26685314

  8. MR Assessment of Myocardial Perfusion, Viability, and Function after Intramyocardial Transfer of VM202, a New Plasmid Human Hepatocyte Growth Factor in Ischemic Swine Myocardium1

    PubMed Central

    Saeed, Maythem; Martin, Alastair; Ursell, Phillip; Do, Loi; Bucknor, Matt; Higgins, Charles B.; Saloner, David

    2008-01-01

    Purpose: VM202, a newly constructed plasmid human hepatocyte growth factor, was transferred intramyocardially after infarction for the purpose of evaluating this strategy as a therapeutic approach for protection from left ventricular (LV) remodeling. Materials and Methods: The institutional animal care and use committee approved this study. Pigs underwent coronary artery occlusion and reperfusion and served as either control (n = 8) or VM202-treated (n = 8) animals. VM202 was transferred intramyocardially into four infarcted and four periinfarcted sites. Cardiac magnetic resonance (MR) imaging (cine, perfusion, delayed enhancement) was performed in acute (3 days) and chronic (50 days ± 3 [standard error of the mean]) infarction. Histopathologic findings were used to characterize and quantify neovascularization. The t test was utilized to compare treated and control groups and to assess changes over time. Results: In acute infarction, MR imaging estimates of function, perfusion, and viability showed no difference between the groups. In chronic infarction, however, VM202 increased maximum signal intensity and upslope at first-pass perfusion imaging and reduced infarct size at perfusion and delayed-enhancement imaging. These changes were associated with a decrease in end-diastolic (2.15 mL/kg ± 0.12 to 1.73 mL/kg ± 0.10, P < .01) and end-systolic (1.33 mL/kg ± 0.07 to 0.92 mL/kg ± 0.08, P < .001) volumes and an increase in ejection fraction (38.2% ± 1.3 to 47.0% ± 1.8, P < .001). In contrast, LV function deteriorated further in control animals. Compared with control animals, VM202-treated animals revealed peninsulas and/or islands of viable myocardium in infarcted and periinfarcted regions and greater number of capillaries (218 per square millimeter ± 19 vs 119 per square millimeter ± 17, P < .05) and arterioles (21 per square millimeter ± 4 vs 3 per square millimeter ± 1, P < .001). Conclusion: Intramyocardial transfer of VM202 improved myocardial

  9. Usefulness of the Hepatocyte Growth Factor as a Predictor of Mortality in Patients Hospitalized With Acute Heart Failure Regardless of Ejection Fraction.

    PubMed

    Pérez-Calvo, Juan-Ignacio; Morales-Rull, José-Luis; Gimeno-Orna, José-Antonio; Lasierra-Díaz, Pilar; Josa-Laorden, Claudia; Puente-Lanzarote, Juan-José; Bettencourt, Paulo; Pascual-Figal, Domingo A

    2016-08-15

    Hepatocyte growth factor (HGF) plays a role in the improvement of cardiac function and remodeling. Their serum levels are strongly related with mortality in chronic systolic heart failure (HF). The aim of this study was to study prognostic value of HGF in acute HF, interaction with ejection fraction, renal function, and natriuretic peptides. We included 373 patients (age 76 ± 10 years, left ventricular ejection fraction [LVEF] 46 ± 14%, 48% men) consecutively admitted for acute HF. Blood samples were obtained at admission. All patients were followed up until death or close of study (>1 year, median 371 days). HGF concentrations were determined using a commercial enzyme-linked immunosorbent assay (human HGF immunoassay). The predictive power of HGF was estimated by Cox regression with calculation of Harrell C-statistic. HGF had a median of 1,942 pg/ml (interquartile rank 1,354). According to HGF quartiles, mortality rates (per 1,000 patients/year) were 98, 183, 375, and 393, respectively (p <0.001). In Cox regression analysis, HGF (hazard ratio1SD = 1.5, 95% confidence interval 1.1 to 2.1, p = 0.002) and N-terminal pro b-type natriuretic peptide (NT-proBNP; hazard ratio1SD = 1.8, 95% confidence interval 1.2 to 2.6, p = 0.002) were independent predictors of mortality. Interaction between HGF and LVEF, origin, and renal function was nonsignificant. The addition of HGF improved the predictive ability of the models (C-statistic 0.768 vs 0.741, p = 0.016). HGF showed a complementary value over NT-proBNP (p = 0.001): mortality rate was 490 with both above the median versus 72 with both below. In conclusion, in patients with acute HF, serum HGF concentrations are elevated and identify patients at higher risk of mortality, regardless of LVEF, ischemic origin, or renal function. HGF had independent and additive information over NT-proBNP. PMID:27338207

  10. Hepatocyte Growth Factor Activator Inhibitor-1 Is Induced by Bone Morphogenetic Proteins and Regulates Proliferation and Cell Fate of Neural Progenitor Cells

    PubMed Central

    Koivuniemi, Raili; Mäkelä, Johanna; Hokkanen, Marie-Estelle; Bruelle, Céline; Ho, Tho Huu; Ola, Roxana; Korhonen, Laura; Schröder, Jim; Kataoka, Hiroaki; Lindholm, Dan

    2013-01-01

    Background Neural progenitor cells (NPCs) in the developing neuroepithelium are regulated by intrinsic and extrinsic factors. There is evidence that NPCs form a self-supporting niche for cell maintenance and proliferation. However, molecular interactions and cell-cell contacts and the microenvironment within the neuroepithelium are largely unknown. We hypothesized that cellular proteases especially those associated with the cell surface of NPCs play a role in regulation of progenitor cells in the brain. Methodology/Principal Findings In this work, we show that NPCs, isolated from striatal anlage of developing rat brain, express hepatocyte growth factor activator inhibitor-1 and -2 (HAI-1 and HAI-2) that are cell surface-linked serine protease inhibitors. In addition, radial glia cells derived from mouse embryonic stem cells also express HAI-1 and HAI-2. To study the functional significance of HAI-1 and HAI-2 in progenitor cells, we modulated their levels using expression plasmids or silencing RNA (siRNA) transfected into the NPCs. Data showed that overexpression of HAI-1 or HAI-2 decreased cell proliferation of cultured NPCs, whilst their siRNAs had opposite effects. HAI-1 also influenced NPC differentiation by increasing the number of glial fibrillary acidic protein (GFAP) expressing cells in the culture. Expression of HAI-1 in vivo decreased cell proliferation in developing neuroepithelium in E15 old animals and promoted astrocyte cell differentiation in neonatal animals. Studying the regulation of HAI-1, we observed that Bone morphogenetic protein-2 (BMP-2) and BMP-4 increased HAI-1 levels in the NPCs. Experiments using HAI-1-siRNA showed that these BMPs act on the NPCs partly in a HAI-1-dependent manner. Conclusions This study shows that the cell-surface serine protease inhibitors, HAI-1 and HAI-2 influence proliferation and cell fate of NPCs and their expression levels are linked to BMP signaling. Modulation of the levels and actions of HAI-1 in NPCs may be of

  11. Mesenchymal stem cells overexpressing hepatocyte growth factor (HGF) inhibit collagen deposit and improve bladder function in rat model of bladder outlet obstruction.

    PubMed

    Song, Yun Seob; Lee, Hong Jun; Doo, Seung Hwan; Lee, Sun Ju; Lim, Inja; Chang, Kyu-Tae; Kim, Seung U

    2012-01-01

    Bladder outlet obstruction (BOO) caused by collagen deposit is one of the most common problems in elderly male. This study was performed to examine the capability of human mesenchymal stem cells (MSCs) overexpressing hepatocyte growth factor (HGF) to inhibit collagen deposition in rat model of bladder outlet obstruction (BOO). HGF is known for its antifibrotic effect and the most promising agent for treating bladder fibrosis. BM3.B10 stable immortalized human MSC line (B10) was transduced to encode human HGF with a retroviral vector was prepared (B10.HGF). Two weeks after the onset of BOO, B10, and B10.HGF cells were injected into the rat's bladder wall. After 4 weeks, bladder tissues were harvested and Masson's trichrome staining was performed. Transgene expression in HGF-expressing B10 cells was demonstrated by reverse transcriptase polymerase chain reaction and immunohistochemical staining, and the high levels of HGF secreted by B10.HGF cells was confirmed by ELISA. The mean bladder weight in BOO rats was 5.8 times of the normal controls, while in animals grafted with B10.HGF cells, the weight was down to four times of the control [90.2 ± 1.6 (control), 89.9 ± 2.8 (sham), 527.9 ± 150.9 (BOO), 447.7 ± 41.0 (BOO + B10), and 362.7 ± 113.2 (BOO + B10.HGF)]. The mean percentage of collagen area increased in BOO rats, while in the animals transplanted with B10.HGF cells, the collagen area decreased to the normal control level [12.2 ± 1.3, (control), 12.8 ± 1.1 (sham), 26.6 ± 2.7 (BOO), 19.9 ± 6.0 (BOO + B10), and 13.3 ± 2.1 (BOO + B10.HGF)]. The expression of collagen and TGF-b protein increased after BOO, while the expression of HGF and c-met protein increased in the group with B10.HGF transplantation after BOO. Intercontraction interval decreased after BOO, but it recovered after B10.HGF transplantation. Maximal voiding pressure (MVP) increased after BOO, and it recovered to levels of the normal control after transplantation of B10.HGF cells. Residual

  12. Chemokine Receptors, CXCR1 and CXCR2, Differentially Regulate Exosome Release in Hepatocytes

    PubMed Central

    Nojima, Hiroyuki; Konishi, Takanori; Freeman, Christopher M.; Schuster, Rebecca M.; Japtok, Lukasz; Kleuser, Burkhard; Edwards, Michael J.; Gulbins, Erich; Lentsch, Alex B.

    2016-01-01

    Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect. PMID:27551720

  13. Chemokine Receptors, CXCR1 and CXCR2, Differentially Regulate Exosome Release in Hepatocytes.

    PubMed

    Nojima, Hiroyuki; Konishi, Takanori; Freeman, Christopher M; Schuster, Rebecca M; Japtok, Lukasz; Kleuser, Burkhard; Edwards, Michael J; Gulbins, Erich; Lentsch, Alex B

    2016-01-01

    Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect. PMID:27551720

  14. Metabolism-mediated drug interaction potential of HS-23, a new herbal drug for the treatment of sepsis in human hepatocytes and liver microsomes.

    PubMed

    Jeong, Hyeon-Uk; Lee, Ji Young; Kwon, Soon-Sang; Kim, Ju Hyun; Kim, Young-Mok; Hong, Sung-Woon; Yeon, Sung Hum; Lee, Sun-Mee; Cho, Yong-Yeon; Lee, Hye Suk

    2015-02-01

    HS-23, an extract of the dried flower buds of Lonicera japonica, is a new botanical drug currently being evaluated in a phase I clinical study in Korea for the treatment of sepsis. The in vitro induction and inhibition potentials of HS-23 on the drug-metabolizing enzymes using human hepatocytes and liver microsomes were assessed to evaluate herb-drug interaction according to botanical drug guideline and drug interaction guidance of FDA. HS-23 slightly inhibited CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 enzyme activities in human liver microsomes with IC50 values of 80.6, 160.7, 169.5, 85.4, and 76.6 μg/mL, respectively. HS-23 showed negligible inhibition of CYP1A2, CYP2C8, CYP2D6, UGT1A1, UGT1A4, UGT1A9, and UGT2B7 activities in human liver microsomes. Based on these results, HS-23 may not inhibit the metabolism of CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP3A4-catalyzed drugs in humans. HS-23 did not affect the mRNA expression of CYP1A2, CYP2B6, and CYP3A4 after 48 h treatment at three concentrations (0.5, 5, and 50 μg/mL) in three independent human hepatocytes, indicating that HS-23 has no effect on herb-drug interactions that up- or down-regulate CYP1A2, CYP2B6, and CYP3A4. These results indicate that the administration of HS-23 in human may not cause clinically relevant inhibition and induction of these cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes and HS-23 may be promising therapeutic agent for treatment of sepsis. PMID:25052959

  15. The Zinc Transporter Zip14 Influences c-Met Phosphorylation and Hepatocyte Proliferation During Liver Regeneration in Mice

    PubMed Central

    AYDEMIR, TOLUNAY BEKER; SITREN, HARRY S.; COUSINS, ROBERT J.

    2013-01-01

    BACKGROUND & AIMS Zinc homeostasis in cells is maintained through tight regulation of zinc influx, efflux, and distribution to intracellular organelles by zinc transporters. The Zrt-Irt-like protein (ZIP) transporters facilitate zinc influx to the cytosol. Expression of the ZIP family member Zip14 can be induced by inflammatory cytokines, which also initiate liver regeneration. Hepatocyte proliferation is required for liver regeneration. Zinc regulates cell proliferation, tissue growth, and many mitogenic signaling pathways; we investigated its role in hepatocytes. METHODS Wild-type and Zip14−/− mice that underwent partial hepatectomy (70% of liver removed) were used as models of liver regeneration. We also analyzed AML12 hepatocytes that overexpressed Zip14. Proliferation was assessed with proliferating cell nuclear antigen, CD1, and Ki67 markers and along with assays of zinc content was related to protein tyrosine phosphatase 1B (PTP1B) and extracellular signal–regulated kinase 1/2 signaling. RESULTS Zip14 was up-regulated and hepatic zinc content increased during liver regeneration. Increased hepatic zinc inhibited activity of the phosphatase PTP1B and increased phosphorylation of c-Met, which promoted hepatocyte proliferation. AML12 cells that overexpressed Zip14 increased in zinc content and proliferation; PTP1B was inhibited and phosphorylation of c-Met increased. The increases in hepatic levels of zinc and hepatocyte proliferation that occurred following partial hepatectomy were not observed in Zip14−/− mice. CONCLUSIONS The transporter Zip14 mediates hepatic uptake of zinc during liver regeneration and for hepatocyte proliferation. These findings indicate that zinc transporter activity regulates liver tissue growth by sequestering zinc. Reagents that regulate ZIP14 activity might be developed as therapeutics to promote liver regeneration in patients with chronic liver disease. PMID:22374166

  16. The potential of induced pluripotent stem cell derived hepatocytes.

    PubMed

    Hannoun, Zara; Steichen, Clara; Dianat, Noushin; Weber, Anne; Dubart-Kupperschmitt, Anne

    2016-07-01

    Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers. PMID:26916529

  17. Epidermal growth factor (EGF) stimulated Ca/sup 2 +/ mobilization in hepatocytes is abolished by phorbol esters, pertussis toxin and partial hepatectomy

    SciTech Connect

    Johnson, R.M.; Garrison, J.C.

    1986-05-01

    EGF has been demonstrated to increase free intracellular Ca/sup 2 +/ levels in isolated hepatocytes putatively by generation of the second messenger inositol trisphosphate (IP/sub 3/). Pretreatment of cells with phorbol 12-myristate 13-acetate (PMA) inhibited the EGF (66 nM) stimulated Ca/sup 2 +/ response as measured by quin2. Inhibition by PMA was maximal within 3 min and was concentration dependent (IC/sub 50/ = 13.5 nM). Four other active phorbol ester analogues blocked the Ca/sup 2 +/ response while inactive analogues did not. EGF was unable to increase intracellular Ca/sup 2 +/ levels in hepatocytes isolated from rats treated with pertussis toxin for 72 hrs. Neither PMA nor toxin pretreatment was able to inhibit the Ca/sup 2 +/ response to angiotensin II (Ang II). In hepatocytes isolated 24 hrs after partial hepatectomy, the Ca/sup 2 +/ response to EGF (as measured by phosphorylase activity, EC/sub 50/ = 5 nM) was completely abolished and remained attenuated for 7 days post-hepatectomy. The Ca/sup 2 +/ response to Ang II in this model system was also blunted but required 3 days for development of the full effect and within 7 days full activity is nearly restored. The results suggest that fundamental differences exist in the transduction mechanisms used by these two Ca/sup 2 +/-linked hormones to mobilize intracellular Ca/sup 2 +/ (and putatively increase IP/sub 3/ formation).

  18. SiO2 nanoparticle-induced impairment of mitochondrial energy metabolism in hepatocytes directly and through a Kupffer cell-mediated pathway in vitro

    PubMed Central

    Xue, Yang; Chen, Qingqing; Ding, Tingting; Sun, Jiao

    2014-01-01

    The liver has been shown to be a primary target organ for SiO2 nanoparticles in vivo, and may be highly susceptible to damage by these nanoparticles. However, until now, research focusing on the potential toxic effects of SiO2 nanoparticles on mitochondria-associated energy metabolism in hepatocytes has been lacking. In this work, SiO2 nanoparticles 20 nm in diameter were evaluated for their ability to induce dysfunction of mitochondrial energy metabolism. First, a buffalo rat liver (BRL) cell line was directly exposed to SiO2 nanoparticles, which induced cytotoxicity and mitochondrial damage accompanied by decreases in mitochondrial dehydrogenase activity, mitochondrial membrane potential, enzymatic expression in the Krebs cycle, and activity of the mitochondrial respiratory chain complexes I, III and IV. Second, the role of rat-derived Kupffer cells was evaluated. The supernatants from Kupffer cells treated with SiO2 nanoparticles were transferred to stimulate BRL cells. We observed that SiO2 nanoparticles had the ability to activate Kupffer cells, leading to release of tumor necrosis factor-α, nitric oxide, and reactive oxygen species from these cells and subsequently to inhibition of mitochondrial respiratory chain complex I activity in BRL cells. PMID:24959077

  19. Liver irradiation: a potential preparative regimen for hepatocyte transplantation.

    PubMed

    Guha, C; Parashar, B; Deb, N J; Sharma, A; Gorla, G R; Alfieri, A; Roy-Chowdhury, N; Roy-Chowdhury, J; Vikram, B

    2001-02-01

    Advances in the understanding of hepatocyte engraftment and repopulation of the host liver have already led to the use of hepatocyte transplantation (HT) with some success in the treatment of inherited and acquired liver diseases. Wider application of HT is severely limited by the unavailability of large number of transplantable hepatocytes and difficulties associated with transplanting an adequate number of cells for achieving therapeutically satisfactory levels of metabolic correction. Therefore, there is a need for preparative regimens that provide a growth advantage to the transplanted (healthy) hepatocytes over the host's own (diseased) hepatocytes so that the former can repopulate the host liver. We have recently shown that when the liver of recipient rats was subjected to radiotherapy and partial hepatectomy before HT, the transplanted hepatocytes engrafted in and massively repopulated the liver, and also ameliorated the adverse clinical and histopathological changes associated with hepatic irradiation. This protocol was then used as a preparative regimen for transplanting normal hepatocytes into jaundice mutant rats (Gunn strain), which lack hepatic bilirubin-uridinediphosphoglucuronate glucuronosyltransferase and is a model of Crigler-Najjar syndrome Type I. The results showed long-term correction of the metabolic abnormality, suggesting that the transplanted hepatocytes repopulated an irradiated liver and were metabolically functional. This strategy could be useful in the treatment of various genetic, metabolic, or malignant diseases of the liver. PMID:11173140

  20. Isolated rat hepatocytes can signal to other hepatocytes and bile duct cells by release of nucleotides.

    PubMed Central

    Schlosser, S F; Burgstahler, A D; Nathanson, M H

    1996-01-01

    Intercellular communication among certain cell types can occur via ATP secretion, which leads to stimulation of nucleotide receptors on target cells. In epithelial cells, however, intercellular communication is thought to occur instead via gap junctions. Here we examined whether one epithelial cell type, hepatocytes, can also communicate via nucleotide secretion. The effects on cytosolic Ca2+ ([Ca2+]i) of mechanical stimulation, including microinjection, were examined in isolated rat hepatocytes and in isolated bile duct units using confocal fluorescence video microscopy. Mechanical stimulation of a single hepatocyte evoked an increase in [Ca2+]i in the stimulated cell plus an unexpected [Ca2+]i rise in neighboring noncontacting hepatocytes. Perifusion with ATP before mechanical stimulation suppressed the [Ca2+]i increase, but pretreatment with phenylephrine did not. The P2 receptor antagonist suramin inhibited these intercellular [Ca2+]i signals. The ATP/ADPase apyrase reversibly inhibited the [Ca2+]i rise induced by mechanical stimulation, and did not block vasopressin-induced [Ca2+]i signals. Mechanical stimulation of hepatocytes also induced a [Ca2+]i increase in cocultured isolated bile duct units, and this [Ca2+]i increase was inhibited by apyrase as well. Finally, this form of [Ca2+]i signaling could be elicited in the presence of propidium iodide without nuclear labeling by that dye, indicating that this phenomenon does not depend on disruption of the stimulated cell. Thus, mechanical stimulation of isolated hepatocytes, including by microinjection, can evoke [Ca2+]i signals in the stimulated cell as well as in neighboring noncontacting hepatocytes and bile duct epithelia. This signaling is mediated by release of ATP or other nucleotides into the extracellular space. This is an important technical consideration given the widespread use of microinjection techniques for examining mechanisms of signal transduction. Moreover, the evidence provided suggests a

  1. Autocrine production of TGF-{beta} confers resistance to apoptosis after an epithelial-mesenchymal transition process in hepatocytes: Role of EGF receptor ligands

    SciTech Connect

    Castillo, Gaelle del; Murillo, Miguel M.; Bertran, Esther; Sanchez, Aranzazu; Fabregat, Isabel . E-mail: ifabregat@iro.es

    2006-09-10

    Transforming growth factor-beta (TGF-{beta}) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives, concomitant with changes in phenotype, reminiscent of an epithelial-mesenchymal transition (EMT). We have previously suggested that EMT might confer cell resistance to apoptosis (Valdes et al., Mol. Cancer Res., 1: 68-78, 2002). However, the molecular mechanisms responsible for this resistance are not explored yet. In this work, we have isolated and subcultured the population of hepatocytes that suffered the EMT process and are resistant to apoptosis (TGF-{beta}-treated fetal hepatocytes: T{beta}T-FH). We prove that they secrete mitogenic and survival factors, as analyzed by the proliferative and survival capacity of conditioned medium. Inhibition of the epidermal growth factor receptor (EGFR) sensitizes T{beta}T-FH to die after serum withdrawal. T{beta}T-FH expresses high levels of transforming growth factor-alpha (TGF-{alpha}) and heparin-binding EGF-like growth factor (HB-EGF) and shows constitutive activation of the EGFR pathway. A blocking anti-TGF-{alpha} antibody restores the capacity of cells to die. TGF-{beta}, which is expressed by T{beta}T-FH, mediates up-regulation of TGF-{alpha} and HB-EGF expression in those cells. In summary, results suggest that an autocrine loop of TGF-{beta} confers resistance to apoptosis after an EMT process in hepatocytes, through the increase in the expression of EGFR ligands.

  2. STAT1 and STAT3 do not participate in FGF-mediated growth arrest in chondrocytes.

    PubMed

    Krejci, Pavel; Salazar, Lisa; Goodridge, Helen S; Kashiwada, Tamara A; Schibler, Matthew J; Jelinkova, Petra; Thompson, Leslie Michels; Wilcox, William R

    2008-02-01

    Activating mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human skeletal dysplasias as a result of attenuation of cartilage growth. It is believed that FGFR3 inhibits chondrocyte proliferation via activation of signal transducers and activators of transcription (STAT) proteins, although the exact mechanism of both STAT activation and STAT-mediated inhibition of chondrocyte growth is unclear. We show that FGFR3 interacts with STAT1 in cells and is capable of activating phosphorylation of STAT1 in a kinase assay, thus potentially serving as a STAT1 kinase in chondrocytes. However, as demonstrated by western blotting with phosphorylation-specific antibodies, imaging of STAT nuclear translocation, STAT transcription factor assays and STAT luciferase reporter assays, FGF does not activate STAT1 or STAT3 in RCS chondrocytes, which nevertheless respond to a FGF stimulus with potent growth arrest. Moreover, addition of active STAT1 and STAT3 to the FGF signal, by means of cytokine treatment, SRC-mediated STAT activation or expression of constitutively active STAT mutants does not sensitize RCS chondrocytes to FGF-mediated growth arrest. Since FGF-mediated growth arrest is rescued by siRNA-mediated downregulation of the MAP kinase ERK1/2 but not STAT1 or STAT3, our data support a model whereby the ERK arm but not STAT arm of FGF signaling in chondrocytes accounts for the growth arrest phenotype. PMID:18198189

  3. Underpotential deposition-mediated layer-by-layer growth of thin films

    SciTech Connect

    Wang, Jia Xu; Adzic, Radoslav R.

    2015-05-19

    A method of depositing contiguous, conformal submonolayer-to-multilayer thin films with atomic-level control is described. The process involves the use of underpotential deposition of a first element to mediate the growth of a second material by overpotential deposition. Deposition occurs between a potential positive to the bulk deposition potential for the mediating element where a full monolayer of mediating element forms, and a potential which is less than, or only slightly greater than, the bulk deposition potential of the material to be deposited. By cycling the applied voltage between the bulk deposition potential for the mediating element and the material to be deposited, repeated desorption/adsorption of the mediating element during each potential cycle can be used to precisely control film growth on a layer-by-layer basis. This process is especially suitable for the formation of a catalytically active layer on core-shell particles for use in energy conversion devices such as fuel cells.

  4. USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

    PubMed Central

    Jaworski, Jakub; de la Vega, Michelle; Fletcher, Sarah J.; McFarlane, Cheryl; Greene, Michelle K.; Smyth, Andrew W.; Van Schaeybroeck, Sandra; Johnston, James A.; Scott, Christopher J.; Rappoport, Joshua Z.; Burrows, James F.

    2014-01-01

    Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of ‘CaaX’ motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis. PMID:25026282

  5. Hormonal regulation of hepatocyte tight junctional permeability

    SciTech Connect

    Lowe, P.J.; Miyai, K.; Steinbach, J.H.; Hardison, W.G.M. Univ. of California, San Diego )

    1988-10-01

    The authors have investigated the effects of hormones on the permeability of the hepatocyte tight junction to two probes, ({sup 14}C)sucrose and horseradish peroxidase, using one-pass perfused rat livers. Using a single injection of horseradish peroxidase the authors have demonstrated that this probe can enter bile by two pathways that are kinetically distinct, a fast pathway, which corresponds to the passage of the probe through the hepatocyte tight junctions, and a slow pathway, which corresponds to the transcytotic entry into bile. The passage of horseradish peroxidase through the hepatocyte tight junctions was confirmed by electron microscopic histochemistry. Vasopressin, epinephrine, and angiotensin II, hormones that act in the hepatocyte through the intracellular mediators calcium, the inositol polyphosphates, and diacylglycerol, increased the bile-to-perfusion fluid ratio of ({sup 14}C)sucrose and the rapid entry of horseradish peroxidase into bile, indicating that the permeability of the tight junctions to these probes was increased. The effect of these hormones was dose dependent and in the cases of angiotensin II and epinephrine was inhibited by the specific inhibitors (Sar{sup 1},Thr{sup 8})angiotensin II and prazosin, respectively. Dibutyryl adenosine 3{prime},5{prime}-cyclic monophosphate did not affect the ({sup 14}C)sucrose bile-to-perfusion fluid ratio or the fast entry of horseradish peroxidase into bile. These results suggest that the hepatocyte tight junction can no longer be considered a static system of pores separating blood from bile. It is rather a dynamic barrier potentially capable of influencing the composition of the bile.

  6. Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1: FUNCTIONAL INTERACTIONS BETWEEN THE KUNITZ-TYPE INHIBITOR DOMAIN-1 AND THE NEIGHBORING POLYCYSTIC KIDNEY DISEASE-LIKE DOMAIN.

    PubMed

    Hong, Zebin; De Meulemeester, Laura; Jacobi, Annemarie; Pedersen, Jan Skov; Morth, J Preben; Andreasen, Peter A; Jensen, Jan K

    2016-07-01

    Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain). PMID:27189939

  7. Industry growth, work role characteristics, and job satisfaction: a cross-level mediation model.

    PubMed

    Ford, Michael T; Wooldridge, Jessica D

    2012-10-01

    The associations between industry revenue growth, individual work role characteristics, and job satisfaction were examined in this cross-level mediation analysis. Work roles were expected to be more autonomous, involve greater skill variety, and offer more opportunities for growth and development for workers in growing industries than for workers in declining industries. Supervisor support was also hypothesized to be stronger for workers in high-growth industries. Results from a nationally representative (U.S.) sample of service industry workers, using multilevel modeling, supported these propositions and suggest that job enrichment mediates relations between industry growth and job satisfaction. Associations between industry growth and autonomy were also stronger among workers in occupations that are less normatively autonomous, suggesting that industry growth fosters a weakening, and industry decline a strengthening, of traditional differences in autonomy across work roles. These results contribute to a multilevel perspective on organizational environments, individual work roles, and worker attitudes and well-being. PMID:22888860

  8. Death Receptor 5 Signaling Promotes Hepatocyte Lipoapoptosis*

    PubMed Central

    Cazanave, Sophie C.; Mott, Justin L.; Bronk, Steven F.; Werneburg, Nathan W.; Fingas, Christian D.; Meng, X. Wei; Finnberg, Niklas; El-Deiry, Wafik S.; Kaufmann, Scott H.; Gores, Gregory J.

    2011-01-01

    Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR−/−) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor. PMID:21941003

  9. HDM2 promotes WIP1-mediated medulloblastoma growth

    PubMed Central

    Buss, Meghan C.; Read, Tracy-Ann; Schniederjan, Matthew J.; Gandhi, Khanjan; Castellino, Robert C.

    2012-01-01

    Medulloblastoma is the most common malignant childhood brain tumor. The protein phosphatase and oncogene WIP1 is over-expressed or amplified in a significant number of primary human medulloblastomas and cell lines. In the present study, we examine an important mechanism by which WIP1 promotes medulloblastoma growth using in vitro and in vivo models. Human cell lines and intracerebellar xenografted animal models were used to study the role of WIP1 and the major TP53 regulator, HDM2, in medulloblastoma growth. Stable expression of WIP1 enhances growth of TP53 wild-type medulloblastoma cells, compared with cells with stable expression of an empty-vector or mutant WIP1. In an animal model, WIP1 enhances proliferation and reduces the survival of immunodeficient mice bearing intracerebellar xenografted human medulloblastoma cells. Cells with increased WIP1 expression also exhibit increased expression of HDM2. HDM2 knockdown or treatment with the HDM2 inhibitor Nutlin-3a, the active enantomer of Nutlin-3, specifically inhibits the growth of medulloblastoma cells with increased WIP1 expression. Nutlin-3a does not affect growth of medulloblastoma cells with stable expression of an empty vector or of mutant WIP1. Knockdown of WIP1 or treatment with the WIP1 inhibitor CCT007093 results in increased phosphorylation of known WIP1 targets, reduced HDM2 expression, and reduced growth specifically in WIP1 wild-type and high-expressing medulloblastoma cells. Combined WIP1 and HDM2 inhibition is more effective than WIP1 inhibition alone in blocking growth of WIP1 high-expressing medulloblastoma cells. Our preclinical study supports a role for therapies that target WIP1 and HDM2 in the treatment of medulloblastoma. PMID:22379189

  10. Metabolism of lipoproteins by human fetal hepatocytes

    SciTech Connect

    Carr, B.R.

    1987-12-01

    The rate of clearance of lipoproteins from plasma appears to play a role in the development of atherogenesis. The liver may account for as much as two thirds of the removal of low-density lipoprotein and one third of the clearance of high-density lipoprotein in certain animal species and humans, mainly by receptor-mediated pathways. The purpose of the present investigation was to determine if human fetal hepatocytes maintained in vitro take up and degrade lipoproteins. We first determined that the maximal binding capacity of iodine 125-iodo-LDL was approximately 300 ng of low-density lipoprotein protein/mg of membrane protein and an apparent dissociation constant of approximately 60 micrograms low-density lipoprotein protein/ml in membranes prepared from human fetal liver. We found that the maximal uptake of (/sup 125/I)iodo-LDL and (/sup 125/I)iodo-HDL by fetal hepatocytes occurred after 12 hours of incubation. Low-density lipoprotein uptake preceded the appearance of degradation products by 4 hours, and thereafter the degradation of low-density lipoprotein increased linearly for at least 24 hours. In contrast, high-density lipoprotein was not degraded to any extent by fetal hepatocytes. (/sup 125/I)Iodo-LDL uptake and degradation were inhibited more than 75% by preincubation with low-density lipoprotein but not significantly by high-density lipoprotein, whereas (/sup 125/I)iodo-HDL uptake was inhibited 70% by preincubation with high-density lipoprotein but not by low-density lipoprotein. In summary, human fetal hepatocytes take up and degrade low-density lipoprotein by a receptor-mediated process similar to that described for human extrahepatic tissues.

  11. Light-Mediated Hormonal Regulation of Plant Growth and Development.

    PubMed

    de Wit, Mieke; Galvão, Vinicius Costa; Fankhauser, Christian

    2016-04-29

    Light is crucial for plant life, and perception of the light environment dictates plant growth, morphology, and developmental changes. Such adjustments in growth and development in response to light conditions are often established through changes in hormone levels and signaling. This review discusses examples of light-regulated processes throughout a plant's life cycle for which it is known how light signals lead to hormonal regulation. Light acts as an important developmental switch in germination, photomorphogenesis, and transition to flowering, and light cues are essential to ensure light capture through architectural changes during phototropism and the shade avoidance response. In describing well-established links between light perception and hormonal changes, we aim to give insight into the mechanisms that enable plants to thrive in variable light environments. PMID:26905653

  12. Regulation of gene expression mediating indeterminate muscle growth in teleosts.

    PubMed

    Ahammad, A K Shakur; Asaduzzaman, Md; Asakawa, Shuichi; Watabe, Shugo; Kinoshita, Shigeharu

    2015-08-01

    Teleosts are unique among vertebrates due to their indeterminate muscle growth, i.e., continued production of neonatal muscle fibers until death. However, the molecular mechanism(s) underlying this property is unknown. Here, we focused on the torafugu (Takifugu rubripes) myosin heavy chain gene, MYHM2528-1, which is specifically expressed in neonatal muscle fibers produced by indeterminate muscle growth. We examined the flanking region of MYHM2528-1 through an in vivo reporter assay using zebrafish (Danio rerio) and identified a 2100 bp 5'-flanking sequence that contained sufficient promoter activity to allow specific gene expression. The effects of enhanced promoter activity were observed at the outer region of the fast muscle and the dorsal edge of slow muscle in zebrafish larvae. At the juvenile stage, the promoter was specifically activated in small diameter muscle fibers scattered throughout fast muscle and in slow muscle near the septum separating slow and fast muscles. This spatio-temporal promoter activity overlapped with known myogenic zones involved in teleost indeterminate muscle growth. A deletion mutant analysis revealed that the -2100 to -600 bp 5'flanking sequence of MYHM2528-1 is essential for promoter activity. This region contains putative binding sites for several representative myogenesis-related transcription factors and nuclear factor of activated T-cell (NFAT), a transcription activator involved in regeneration of mammalian adult skeletal muscle. A significant reduction in the promoter activity of the MYHM2528-1 deletion constructs was observed in accordance with a reduction in the number of these binding sites, suggesting the involvement of specific transcription factors in indeterminate muscle growth. PMID:25842264

  13. Ethanol-induced impairments in receptor-mediated endocytosis of asialoorosomucoid in isolated rat hepatocytes: Time course of impairments and recovery after ethanol withdrawal

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J.

    1989-04-01

    Chronic ethanol administration markedly impairs the process of receptor-mediated endocytosis (RME) of a representative asialoglycoprotein, asialoorosomucoid (ASOR), by the liver. In this study, we further characterized these impairments by identifying the time of onset for ethanol-induced changes in RME as well as establishing the time course for recovery to normal endocytotic values after ethanol withdrawal. Ethanol administration for 3 days did not alter any aspect of endocytosis examined in this study. After feeding ethanol to rats for 7 days, however, significant decreases in amounts of ligand bound, internalized, and degraded were apparent. These impairments persisted throughout the 5-week feeding study although the effects were somewhat attenuated with more prolonged ethanol feeding. In addition, an accumulation of intracellular receptors was observed in ethanol-fed animals relative to controls after 7 days of ethanol feeding. In all cases, recovery of endocytotic values to control levels was partially completed after 2 to 3 days of refeeding control diet and was fully completed after 7 days of refeeding. These results indicate that ethanol feeding for as little as 7 days profoundly impairs the process of RME by the liver. These impairments can be reversed after refeeding control diet for 7 days.

  14. Pathogen Virulence Impedes Mutualist-Mediated Enhancement of Host Juvenile Growth via Inhibition of Protein Digestion

    PubMed Central

    Erkosar, Berra; Storelli, Gilles; Mitchell, Mélanie; Bozonnet, Loan; Bozonnet, Noémie; Leulier, François

    2015-01-01

    Summary The microbial environment impacts many aspects of metazoan physiology through largely undefined molecular mechanisms. The commensal strain Lactobacillus plantarumWJL (LpWJL) sustains Drosophila hormonal signals that coordinate systemic growth and maturation of the fly. Here we examine the underlying mechanisms driving these processes and show that LpWJL promotes intestinal peptidase expression, leading to increased intestinal proteolytic activity, enhanced dietary protein digestion, and increased host amino acid levels. LpWJL-mediated peptidase upregulation is partly driven by the peptidoglycan recognition and signaling cascade PGRP-LE/Imd/Relish. Additionally, this mutualist-mediated physiological benefit is antagonized upon pathogen infection. Pathogen virulence selectively impedes LpWJL-mediated intestinal peptidase activity enhancement and juvenile growth promotion but does not alter growth of germ-free animals. Our study reveals the adaptability of host physiology to the microbial environment, whereby upon acute infection the host switches to pathogen-mediated host immune defense at the expense of mutualist-mediated growth promotion. PMID:26439865

  15. Interactions between macrophage/Kupffer cells and hepatocytes in surgical sepsis

    SciTech Connect

    West, M.A.

    1988-01-01

    Experiments were performed to investigate the role of Kupffer cell/macrophage interactions with hepatocytes in modulating liver function during infections using direct in vitro cocultivation of rat macrophages or Kupffer cells with rat hepatocytes. Protein synthesis was assayed as a sensitive indicator of integrated hepatocellular function by measuring {sup 3}H-leucine incorporation into hepatocyte protein. Septic stimuli such as lipoploysaccharide and killed bacteria were added to cocultures of hepatocytes and macrophages or Kupffer cells and the responses compared to hepatocytes alone. Information about the types of proteins synthesized by hepatocytes under various culture conditions was determined using polyacrylamide gel electrophoresis and autoradiography. These experiments showed that septic stimuli alter the amount and type of protein synthesized by hepatocytes and had no direct effect on hepatocytes in the absence of macrophages or Kupffer cells. The mediator(s) appears to be a heat labile, soluble monokine(s) which is distinct from interleukin-1 or tumor necrosis factor. The important role of Kupffer cells/macrophages in mediating alterations in hepatocellular function in sepsis may ultimately improve patient care.

  16. Autophagy is required for IL-2-mediated fibroblast growth

    SciTech Connect

    Kang, Rui; Tang, Daolin; Lotze, Michael T.; Zeh III, Herbert J.

    2013-02-15

    Autophagy is an evolutionarily conserved pathway responsible for delivery of cytoplasmic material into the lysosomal degradation pathway to enable vesicular exocytosis. Interleukin (IL)-2 is produced by T-cells and its activity is important for immunoregulation. Fibroblasts are an immune competent cell type, playing a critical role in wound healing, chronic inflammation, and tumor development. Although autophagy plays an important role in each of these processes, whether it regulates IL-2 activity in fibroblasts is unknown. Here, we show that autophagy is required for IL-2-induced cell growth in fibroblasts. IL-2 significantly induced autophagy in mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts. Autophagy inhibitors (e.g., 3-methylamphetamine and bafilomycin A1) or knockdown of ATG5 and beclin 1 blocked clinical grade IL-2-induced autophagy. Moreover, IL-2 induced HMGB1 cytoplasmic translocation in MEFs and promoted interaction between HMGB1 and beclin1, which is required for autophagy induction. Pharmacological and genetic inhibition of autophagy inhibited IL-2-induced cell proliferation and enhanced IL-2-induced apoptosis. These findings suggest that autophagy is an important pro-survival regulator for IL-2-induced cell growth in fibroblasts.

  17. Dynamic designing of microstructures by chemical gradient-mediated growth

    PubMed Central

    Shim, Tae Soup; Yang, Seung-Man; Kim, Shin-Hyun

    2015-01-01

    Shape is one of the most important determinants of the properties of microstructures. Despite of a recent progress on microfabrication techniques, production of three-dimensional micro-objects are yet to be fully achieved. Nature uses reaction–diffusion process during bottom-up self-assembly to create functional shapes and patterns with high complexity. Here we report a method to produce polymeric microstructures by using a dynamic reaction–diffusion process during top-down photolithography, providing unprecedented control over shape and composition. In radical polymerization, oxygen inhibits reaction, and therefore diffusion of oxygen significantly alters spatial distribution of growth rate. Therefore, growth pathways of the microstructures can be controlled by engineering a concentration gradient of oxygen. Moreover, stepwise control of chemical gradients enables the creation of highly complex microstructures. The ease of use and high controllability of this technology provide new opportunities for microfabrication and for fundamental studies on the relationships between shape and function for the materials. PMID:25766762

  18. [Receptor-mediated endocytosis in the cells of cold-blooded animals. II. The fate of internalized 125I-insulin in the isolated hepatocytes of the lamprey and the frog].

    PubMed

    Lappova, Iu L; Leĭbush, B N

    1994-01-01

    The 125I-insulin outflow from isolated hepatocytes of the frog and lamprey "loaded" with the labeled hormone has been studied. It is shown that the ligand outflow from the frog cells increased with the increase in the incubation temperature from 0 up to 20 degrees C. The curves of the rest cell radioactivity were reciprocal to those of the radioactivity accumulated in the medium at the corresponding temperatures. At 0.5 and 20 degrees C the degraded 125I-insulin made 5.7 and 17% of the whole hormone accumulated in the medium. In the lamprey hepatocytes, neither accumulation in the incubation medium nor outflow of the radioactivity from cell was seen at all temperatures studied. The intracellular degradation of 125I-insulin in the frog hepatocytes was no more than 7% of the internalized ligand, compared to about 25% in the lamprey cells. The specific binding of 125I-insulin was twice increased in the presence of lysosomal inhibitor chloroquin; contrary to this, no increase was found in the lamprey hepatocytes. The results of experiments on the frog hepatocytes lead us to a conclusion that the processing pathway of internalized insulin in cold-blooded vertebrate cells is similar mainly to that in cells of warm-blooded species, but takes place at lower temperatures and with slower rates. The peculiarities of processing in the lamprey hepatocytes (extralysosomal ligand degradation, the inability to release the internalized ligand and its degradation products) are dependent on a deep transformation of hepatocytes during prespawning migration period. PMID:7701627

  19. INHIBITION OF INTERCELLULAR COMMUNICATION BETWEEN MOUSE HEPATOCYTES BY TUMOR PROMOTERS

    EPA Science Inventory

    Tumor promoters can inhibit gap junction-mediated intercellular communication in cultured cells. The authors evaluated the effects of tumor promoters on intercellular communication between B6C3F1 mouse hepatocytes in primary culture. Intercellular communication between donor and ...

  20. Investigation of Mediational Processes Using Parallel Process Latent Growth Curve Modeling.

    ERIC Educational Resources Information Center

    Cheong, JeeWon; MacKinnon, David P.; Khoo, Siek Toon

    2003-01-01

    Investigated a method to evaluate mediational processes using latent growth curve modeling and tested it with empirical data from a longitudinal steroid use prevention program focusing on 1,506 high school football players over 4 years. Findings suggest the usefulness of the approach. (SLD)

  1. Reactive oxygen species mediate growth and death in submerged plants

    PubMed Central

    Steffens, Bianka; Steffen-Heins, Anja; Sauter, Margret

    2013-01-01

    Aquatic and semi-aquatic plants are well adapted to survive partial or complete submergence which is commonly accompanied by oxygen deprivation. The gaseous hormone ethylene controls a number of adaptive responses to submergence including adventitious root growth and aerenchyma formation. Reactive oxygen species (ROS) act as signaling intermediates in ethylene-controlled submergence adaptation and possibly also independent of ethylene. ROS levels are controlled by synthesis, enzymatic metabolism, and non-enzymatic scavenging. While the actors are by and large known, we still have to learn about altered ROS at the subcellular level and how they are brought about, and the signaling cascades that trigger a specific response. This review briefly summarizes our knowledge on the contribution of ROS to submergence adaptation and describes spectrophotometrical, histochemical, and live cell imaging detection methods that have been used to study changes in ROS abundance. Electron paramagnetic resonance (EPR) spectroscopy is introduced as a method that allows identification and quantification of specific ROS in cell compartments. The use of advanced technologies such as EPR spectroscopy will be necessary to untangle the intricate and partially interwoven signaling networks of ethylene and ROS. PMID:23761805

  2. 17α-Ethinylestradiol (EE2) effect on global gene expression in primary rainbow trout (Oncorhynchus mykiss) hepatocytes.

    PubMed

    Hultman, Maria T; Song, You; Tollefsen, Knut Erik

    2015-12-01

    The potential impact of endocrine disrupting chemicals (EDCs) in the aquatic environment has driven the development of screening assays to evaluate the estrogenic properties of chemicals and their effects on aquatic organisms such as fish. However, obtaining full concentration-response relationships in animal (in vivo) exposure studies are laborious, costly and unethical, hence a need for developing feasible alternative (non-animal) methods. Use of in vitro bioassays such as primary fish hepatocytes, which retain many of the native properties of the liver, has been proposed for in vitro screening of estrogen receptor (ER) agonists and antagonists. The aim of present study was to characterize the molecular mode of action (MoA) of the ER agonist 17α-ethinylestradiol (EE2) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes. A custom designed salmonid 60,000-feature (60k) oligonucleotide microarray was used to characterize the potential MoAs after 48h exposure to EE2. The microarray analysis revealed several concentration-dependent gene expression alterations including classical estrogen sensitive biomarker gene expression (e.g. estrogen receptor α, vitellogenin, zona radiata). Gene Ontology (GO) analysis displayed transcriptional changes suggesting interference of cellular growth, fatty acid and lipid metabolism potentially mediated through the estrogen receptor (ER), which were proposed to be associated with modulation of genes involved in endocrine function and reproduction. Pathway analysis supported the identified GOs and revealed modulation of additional genes associated with apoptosis and cholesterol biosynthesis. Differentially expressed genes (DEGs) related to impaired lipid metabolism (e.g. peroxisome proliferator-activated receptor α and γ), growth (e.g. insulin growth factor protein 1), phase I and II biotransformation (e.g. cytochrome P450 1A, sulfotransferase, UDP-glucuronosyltransferase and glutathione S-transferase) provided additional

  3. Anion-Mediated End-Shape Control in Seed-Mediated Growth of Gold Nanorods.

    PubMed

    Kim, Tae Youl; Kim, Joo-Hyung; Kim, Minsoo P; Yi, Gi-Ra

    2016-06-01

    End-shape-controlled gold nanorods (GNRs) were synthesized at room-temperature by a seeded-growth method, in which hexadecyltrimethylammonium bromide (CTAB) was used as a stabilizer and capping agent. The average dimension of the GNRs was 46 nm in length and 15 nm in diameter, which corresponds to aspect ratio of c.a. 3.0. Then, their both ends were further grown at the presence of silver precursor (AgNO3), resulting in formation of arrow-head GNRs. By tuning the amount of the silver precursor, the end-shape of the GNRs was changed to dumbbell like shape. Moreover, the growth rate of gold could be controlled by tuning the amount of hydrochloric acid (HCl). While arrow-headed GNRs having sharp edges were produced without HCl, the GNRs having dog-bone like or round-head shape at both ends were obtained with HCl. PMID:27427712

  4. Evaluation of thyroid-mediated otolith growth of larval and juvenile tilapia.

    PubMed

    Shiao, Jen-Chieh; Wu, Su-Mei; Hwang, Yi-Ping; Wu, Done-Ping; Hwang, Pung-Pung

    2008-06-01

    Thyroid-mediated otolith growth in tilapia was evaluated by the ontogenic triiodothyronine (T3) profile revealed by radioimmunoassay during the first month after hatching. Thyroid hormone receptor genes (TRalpha and TRbeta) were cloned and only the expression of TRalpha mRNA, quantified by real-time PCR, was similar to the T3 profile. Variations in otolith growth showed median correlation with the T3 profile and TRalpha mRNA expression pattern. Hypothyroidism and hyperthyroidism were induced in tilapia juveniles and larvae by administration of different concentrations of thiourea (TU) and T3, respectively, for 13 days. T3 and TU had little effect on otolith growth during the larval stage. However, T3 increased otolith growth and TU retarded, or stopped, otolith growth during the juvenile stage. Furthermore, TU treatment caused permanent changes in otolith shape in the ventral area. Otolith growth recovered slowly from hypothyroidism, requiring 2 days to form an increment during the first week. These results suggest that otolith growth, at least during the juvenile stage, is regulated by the thyroid hormones and the process may be mediated by TRalpha. PMID:18515722

  5. Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity

    SciTech Connect

    Shen Chong; Meng Qin Schmelzer, Eva; Bader, Augustinus

    2009-07-15

    This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 {mu}M which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 {mu}M. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to {beta}-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.

  6. MicroRNA-194 Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1

    PubMed Central

    Jung, Kwang Hwa; McCarthy, Ryan L.; Zhou, Chong; Uprety, Nadima; Barton, Michelle Craig; Beretta, Laura

    2015-01-01

    MicroRNA expression profiling in human liver progenitor cells following hepatocytic differentiation identified miR-122 and miR-194 as the microRNAs most strongly upregulated during hepatocytic differentiation of progenitor cells. MiR-194 was also highly upregulated following hepatocytic differentiation of human embryonic stem cells (hESCs). Overexpression of miR-194 in progenitor cells accelerated their differentiation into hepatocytes, as measured by morphological features such as canaliculi and expression of hepatocytic markers. Overexpression of miR-194 in hESCs induced their spontaneous differentiation, a phenotype accompanied with accelerated loss of the pluripotent factors OCT4 and NANOG and decrease in mesoderm marker HAND1 expression. We then identified YAP1 as a direct target of miR-194. Inhibition of YAP1 strongly induced hepatocytic differentiation of progenitor cells and YAP1 over expression reversed the miR-194-induced hepatocytic differentiation of progenitor cells. In conclusion, we identified miR-194 as a potent inducer of hepatocytic differentiation of progenitor cells and further identified YAP1 as a mediator of miR-194's effects on hepatocytic differentiation and liver progenitor cell fate. PMID:26731713

  7. Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity.

    PubMed

    Shen, Chong; Meng, Qin; Schmelzer, Eva; Bader, Augustinus

    2009-07-15

    This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 muM which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 muM. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to beta-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs. PMID:19463838

  8. Basal efflux of bile acids contributes to drug-induced bile acid-dependent hepatocyte toxicity in rat sandwich-cultured hepatocytes.

    PubMed

    Susukida, Takeshi; Sekine, Shuichi; Ogimura, Eiichiro; Aoki, Shigeki; Oizumi, Kumiko; Horie, Toshiharu; Ito, Kousei

    2015-10-01

    The bile salt export pump (BSEP or Bsep) functions as an apical transporter to eliminate bile acids (BAs) from hepatocytes into the bile. BSEP or Bsep inhibitors engender BA retention, suggested as an underlying mechanism of cholestatic drug-induced liver injury. We previously reported a method to evaluate BSEP-mediated BA-dependent hepatocyte toxicity by using sandwich-cultured hepatocytes (SCHs). However, basal efflux transporters, including multidrug resistance-associated proteins (MRP or Mrp) 3 and 4, also participate in BA efflux. This study examined the contribution of basal efflux transporters to BA-dependent hepatocyte toxicity in rat SCHs. The apical efflux of [(3)H]taurocholic acid (TC) was potently inhibited by 10 μM cyclosporine A (CsA), with later inhibition of basal [(3)H]TC efflux, while MK571 simultaneously inhibited both apical and basal [(3)H]TC efflux. CsA-induced BA-dependent hepatocyte toxicity was 30% at most at 10 μM CsA and ∼60% at 50 μM, while MK571 exacerbated hepatocyte toxicity at concentrations of ≥50 μM. Quinidine inhibited only basal [(3)H]TC efflux and showed BA-dependent hepatocyte toxicity in rat SCHs. Hence, inhibition of basal efflux transporters as well as Bsep may precipitate BA-dependent hepatocyte toxicity in rat SCHs. PMID:26055650

  9. TAK1 is required for the survival of hematopoietic cells and hepatocytes in mice

    PubMed Central

    Tang, Minghui; Wei, Xudong; Guo, Yinshi; Breslin, Peter; Zhang, Shubin; Zhang, Shanshan; Wei, Wei; Xia, Zhenbiao; Diaz, Manuel; Akira, Shizuo; Zhang, Jiwang

    2008-01-01

    Transforming growth factor β–activated kinase 1 (TAK1), a member of the MAPKKK family, is a key mediator of proinflammatory and stress signals. Activation of TAK1 by proinflammatory cytokines and T and B cell receptors induces the nuclear localization of nuclear factor κB (NF-κB) and the activation of c-Jun N-terminal kinase (JNK)/AP1 and P38, which play important roles in mediating inflammation, immune responses, T and B cell activation, and epithelial cell survival. Here, we report that TAK1 is critical for the survival of both hematopoietic cells and hepatocytes. Deletion of TAK1 results in bone marrow (BM) and liver failure in mice due to the massive apoptotic death of hematopoietic cells and hepatocytes. Hematopoietic stem cells and progenitors were among those hematopoietic cells affected by TAK1 deletion–induced cell death. This apoptotic cell death is autonomous, as demonstrated by reciprocal BM transplantation. Deletion of TAK1 resulted in the inactivation of both JNK and NF-κB signaling, as well as the down-regulation of expression of prosurvival genes. PMID:18573910

  10. Hormone-Mediated Pattern Formation in Seedling of Plants: a Competitive Growth Dynamics Model

    NASA Astrophysics Data System (ADS)

    Kawaguchi, Satoshi; Mimura, Masayasu; Ohya, Tomoyuki; Oikawa, Noriko; Okabe, Hirotaka; Kai, Shoichi

    2001-10-01

    An ecologically relevant pattern formation process mediated by hormonal interactions among growing seedlings is modeled based on the experimental observations on the effects of indole acetic acid, which can act as an inhibitor and activator of root growth depending on its concentration. In the absence of any lateral root with constant hormone-sensitivity, the edge effect phenomenon is obtained depending on the secretion rate of hormone from the main root. Introduction of growth-stage-dependent hormone-sensitivity drastically amplifies the initial randomness, resulting in spatially irregular macroscopic patterns. When the lateral root growth is introduced, periodic patterns are obtained whose periodicity depends on the length of lateral roots. The growth-stage-dependent hormone-sensitivity and the lateral root growth are crucial for macroscopic periodic-pattern formation.

  11. Coping Strategies as a Mediator of Posttraumatic Growth among Adult Survivors of the Wenchuan Earthquake

    PubMed Central

    He, Lili; Xu, Jiuping; Wu, Zhibin

    2013-01-01

    Objective By testing the mediating effect of coping strategies on the relationship between social support (SS) and posttraumatic growth (PTG), the aim of this research was to develop a new approach for the study of post-disaster psychological intervention. Methods A mediating effect model analysis was conducted on 2080 adult survivors selected from 19 of the counties hardest-hit by the 2008 Wenchuan earthquake. The Social Support Rating Scale and the Coping Scale were used to predict the PTG. Results A bivariate correlation analysis showed that there was a correlation between posttraumatic growth, social support and coping strategies. The mediation analysis revealed that coping strategies played a mediating role between social support and posttraumatic growth in survivors after the earthquake. Conclusion The results demonstrated that mental health programs for survivors need to focus on the establishment of a good social support network, which was found to be conductive to maintaining and increasing mental health levels. At the same time, adequate social support is able to assist survivors in adopting mature coping strategies, such as problem solving and asking for help. Hence, social support was found to play a vital role in balancing and protecting mental health. PMID:24386345

  12. Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

    SciTech Connect

    Yamaguchi, Kazuki; Feril, Loreto B.; Tachibana, Katsuro; Takahashi, Akira; Matsuo, Miki; Endo, Hitomi; Harada, Yoshimi; Nakayama, Juichiro

    2011-07-22

    Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

  13. Insulin internalization in isolated rat hepatocytes

    SciTech Connect

    Galan, J.; Trankina, M.; Noel, R.; Ward, W. )

    1990-02-26

    This project was designed to determine whether neomycin, an aminoglycoside antibiotic, has a significant effect upon the pathways of ligand endocytosis in isolated rat hepatocytes. The pathways studied include receptor-mediated endocytosis and fluid-phase endocytosis. Neomycin causes a dose-dependent acceleration of {sup 125}I-insulin internalization. Since fluid-phase endocytosis can also be a significant factor in {sup 125}I-insulin internalization, lucifer yellow (LY), a marker for fluid-phase endocytosis, was incorporated into an assay similar to the {sup 125}I-insulin internalization procedure. In the presence of 5 mM neomycin, a significant increase in LY uptake was evident at 0.2 and 0.4 mg/ml of LY. At 0.8 mg/ml, a decrease in LY uptake was observed. The increased rate of {sup 125}I-insulin internalization in the presence of neomycin was intriguing. Since one action of neomycin is to inhibit phosphoinositidase C, it suggests that the phosphotidylinositol cycle may be involved in ligand internalization by hepatocytes. At low insulin concentrations, receptor-mediated uptake predominates. Fluid-phase uptake can become an important uptake route as insulin concentrations are increased. Since neomycin stimulates fluid-phase endocytosis, it must also be taken into account when measuring ligand internalization.

  14. A Comprehensive Analysis of Plasmodium Circumsporozoite Protein Binding to Hepatocytes

    PubMed Central

    Zhao, Jinghua; Bhanot, Purnima; Hu, Junjie; Wang, Qian

    2016-01-01

    Circumsporozoite protein (CSP) is the dominant protein on the surface of Plasmodium sporozoites and plays a critical role in the invasion by sporozoites of hepatocytes. Contacts between CSP and heparin sulfate proteoglycans (HSPGs) lead to the attachment of sporozoites to hepatocytes and trigger signaling events in the parasite that promote invasion of hepatocytes. The precise sequence elements in CSP that bind HSPGs have not been identified. We performed a systematic in vitro analysis to dissect the association between Plasmodium falciparum CSP (PfCSP) and hepatocytes. We demonstrate that interactions between PfCSP and heparin or a cultured hepatoma cell line, HepG2, are mediated primarily by a lysine-rich site in the amino terminus of PfCSP. Importantly, the carboxyl terminus of PfCSP facilitates heparin-binding by the amino-terminus but does not interact directly with heparin. These findings provide insights into how CSP recognizes hepatocytes and useful information for further functional studies of CSP. PMID:27560376

  15. Cryptotanshinone Suppresses Androgen Receptor-mediated Growth in Androgen Dependent and Castration Resistant Prostate Cancer Cells

    PubMed Central

    Xu, Defeng; Lin, Tzu-Hua; Li, Shaoshun; Da, Jun; Wen, Xing-Qiao; Ding, Jiang; Chang, Chawnshang; Yeh, Shuyuan

    2012-01-01

    Androgen receptor (AR) is the major therapeutic target for the treatment of prostate cancer (PCa). Anti-androgens to reduce or prevent androgens binding to AR are widely used to suppress AR-mediated PCa growth; however, the androgen depletion therapy is only effective for a period of time. Here we found a natural product/Chinese herbal medicine cryptotanshinone (CTS), with a structure similar to dihydrotestosterone (DHT), can effectively inhibit the DHT-induced AR transactivation and prostate cancer cell growth. Our results indicated that 0.5 µM CTS effectively suppresses the growth of AR-positive PCa cells, but has little effect on AR negative PC-3 cells and non-malignant prostate epithelial cells. Furthermore, our data indicated that CTS could modulate AR transactivation and suppress the DHT-mediated AR target genes (PSA, TMPRSS2, and TMEPA1) expression in both androgen responsive PCa LNCaP cells and castration resistant CWR22rv1 cells. Importantly, CTS selective inhibits AR without repressing the activities of other nuclear receptors, including ERα, GR, and PR. The mechanistic studies indicate that CTS functions as an AR inhibitor to suppress androgen/AR-mediated cell growth and PSA expression by blocking AR dimerization and the AR–coregulator complex formation. Furthermore, we showed that CTS effectively inhibits CWR22Rv1 cell growth in the xenograft animal model. The previously un-described mechanisms of CTS may explain how CTS inhibits the growth of PCa cells and help us to establish new therapeutic concepts for the treatment of PCa. PMID:22154085

  16. Posttraumatic stress and growth among Tibetan refugees: the mediating role of cognitive-emotional regulation strategies.

    PubMed

    Hussain, Dilwar; Bhushan, Braj

    2011-07-01

    This study examined posttraumatic stress (PTS) and posttraumatic growth (PTG) among 226 Tibetan refugees across two generations. Additional objectives were to (i) examine the sex and generation differences on the scores of trauma, PTS, and PTG, (ii) explore the relationship between traumatic experiences, PTS and PTG, and (iii) investigate the mediating effect of cognitive-emotional regulation strategies between the traumatic experiences and PTS as well as PTG. Females scored higher on trauma, PTS, and PTG. The trauma, PTS, and PTG scores of the two generations were significantly different. Acceptance and putting into perspective partially mediated the relationship between traumatic experience and PTS. Positive refocusing, refocus on planning, putting into perspective, and catastrophisizing partially mediated the relationship between traumatic experiences and PTG. PMID:21455959

  17. The bioactivation of 1,2-dibromoethane in rat hepatocytes: deuterium isotope effect.

    PubMed

    White, R D; Petry, T W; Sipes, I G

    1984-04-01

    The metabolism and genotoxicity of 1,2-dibromoethane (EDB) and its deuterium substituted analog ( d4EDB ) were studied in isolated rat hepatocytes. There was a marked isotope effect on the metabolism of EDB by hepatocytes. This was due to decreased microsomal oxidation of d4EDB . Cytosolic metabolism of EDB, as measured by bromide ion release, was unaffected by deuterium substitution. The genotoxicity of the two analogs was assessed by assaying for the presence of EDB induced single-strand breaks in DNA. As measured by the alkaline elution technique, both compounds caused DNA single-strand breaks when incubated at a concentration of 0.1 mM with hepatocytes. No difference in the degree of DNA damage could be demonstrated between hepatocytes incubated with EDB or d4EDB . These data suggest that the GSH transferase mediated metabolism of EDB is responsible for the genotoxic effects of EDB observed in hepatocytes. PMID:6373030

  18. Evidence for multiple pathways of sup 125 I-insulin internalization in isolated rat hepatocytes

    SciTech Connect

    Moss, A.L.

    1988-01-01

    Insulin internalization has been characterized frequently as occurring by the coated pit pathway of receptor-mediated endocytosis. The present study in rat hepatocytes demonstrates that insulin internalization is, in part, receptor-mediated, but also occurs by nonreceptor-mediated or fluid-phase endocytosis. Endocytosis was probed with four perturbations: depletion of metabolic energy with anoxia, inhibition of endocytosis with phenylarsine oxide, disruption of coated pits with hyperosmolar sucrose, and inhibition of receptor recycling or ligand-receptor dissociation with monensin. Internalization of {sup 125}I-epidermal growth factor and {sup 125}I-asialofetuin was compared to {sup 125}I-insulin internalization. Pretreatment of cells with anoxia or hyperosmolarity inhibited {sup 125}I-insulin internalization by 40%; pretreatment with phenylarsine oxide resulted in inhibition by 54%. Monensin has no effect on uptake or degradation of a high insulin concentration, but inhibited degradation of a low insulin concentration resulting in intracellular accumulation of insulin. In contract, all four perturbations inhibited {sup 125}I-asialofetuin internalization by greater than 90%. Phenylarsine oxide almost completely abolished {sup 125}I-epidermal growth factor uptake; the other perturbations caused partial inhibition. Competition studies demonstrated that insulin internalization was receptor-mediated over a wide concentration range.

  19. Chirality-dependent boron-mediated growth of nitrogen-doped single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Wiltshire, Joseph G.; Li, Lain-Jong; Herz, Laura M.; Nicholas, Robin J.; Glerup, Marianne; Sauvajol, Jean-Louis; Khlobystov, Andrei N.

    2005-11-01

    A change in the relative abundance of single-walled carbon nanotubes, due to the presence of both nitrogen and boron during synthesis, has been identified through Raman and absorption spectroscopy. Raman spectroscopy shows that for two specific branches boron mediates the growth of smaller-diameter zigzag or near-zigzag nanotubes. We combine our experimental results with an improved Kataura model to identify two of the preferentially grown species as (16,0) and (14,1).

  20. MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers

    PubMed Central

    Mohseni, Morassa; Cidado, Justin; Croessmann, Sarah; Cravero, Karen; Cimino-Mathews, Ashley; Wong, Hong Yuen; Scharpf, Rob; Zabransky, Daniel J.; Abukhdeir, Abde M.; Garay, Joseph P.; Wang, Grace M.; Beaver, Julia A.; Cochran, Rory L.; Blair, Brian G.; Rosen, D. Marc; Erlanger, Bracha; Argani, Pedram; Hurley, Paula J.; Lauring, Josh; Park, Ben Ho

    2014-01-01

    Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy. PMID:25422431

  1. CD166-mediated epidermal growth factor receptor phosphorylation promotes the growth of oral squamous cell carcinoma.

    PubMed

    Jia, Guodong; Wang, Xu; Yan, Ming; Chen, Wantao; Zhang, Ping

    2016-08-01

    CD166 has been considered a relatively specific marker of stem cells and cancer stem cells, and the altered expression of CD166 has also been reported as a prognostic marker of several other types of cancer. However, the molecular functions of CD166 in these cancer cells are largely unknown. In this study, we found that CD166 significantly enhanced epidermal growth factor receptor (EGFR) phosphorylation and prolonged epidermal growth factor (EGF)/EGFR signalling activation. In addition, EGF stimulation in CD166-overexpressing oral squamous carcinoma cells led to enhanced colony formation, invasion capacity and cytoskeletal re-organization in vitro and elevated tumourigenesis in vivo. Taken together, the results of our study identify CD166 as an intriguing therapeutic target for patients suffering from oral squamous cell carcinoma (OSCC). PMID:27424177

  2. Trauma or growth after a natural disaster? The mediating role of rumination processes

    PubMed Central

    García, Felipe E.; Cova, Félix; Rincón, Paulina; Vázquez, Carmelo

    2015-01-01

    The aim of this study was to test a cognitive model of posttraumatic symptoms (PTS) and posttraumatic growth (PTG) after exposure to a natural disaster. It was hypothesized that although subjective severity of trauma would be related to the severity of PTS, this relation would be mediated by brooding and cognitive strategies related to the presence of repetitive negative content in thoughts. Furthermore, the relation between severity and PTG would be fully mediated by deliberate rumination (DR), cognitive strategies related to conscious efforts focused on handling the event. To evaluate the cognitive model, adults (N=351) who lost their homes as a result of the earthquake and tsunami that occurred in Chile on February 27, 2010, were selected. Structural equation modeling was used to analyze the data. The resulting model had adequate indices of goodness adjustment and showed that brooding completely mediated the relation between subjective severity and PTS, and DR completely mediated the relation between subjective severity, brooding, and PTG. These results highlight the role of both the content and process of rumination in mediating the association between subjective severity of trauma, PTS, and PTG. The implications of these results for a more comprehensive model of symptom severity that occurs after trauma are discussed. PMID:26234365

  3. Differentiation of Bone Marrow: Derived Mesenchymal Stem Cells into Hepatocyte-like Cells.

    PubMed

    Al Ghrbawy, Nesrien M; Afify, Reham Abdel Aleem Mohamed; Dyaa, Nehal; El Sayed, Asmaa A

    2016-09-01

    Cirrhosis is the end-stage liver fibrosis, whereby normal liver architecture is disrupted by fibrotic bands, parenchymal nodules and vascular distortion. Portal hypertension and hepatocyte dysfunction are the end results and give rise to major systemic complications and premature death. Mesenchymal stem cells (MSC) have the capacity of self-renew and to give rise to cells of various lineages, so MSC can be isolated from bone marrow (BM) and induced to differentiate into hepatocyte-like cells. MSC were induced to differentiate into hepatocyte-like cells by hepatotic growth factor (HGF) and fibroblast growth factor-4 (FGF-4). Differentiated cells were examined for the expression of hepatocyte-specific markers and hepatocyte functions. MSC were isolated. Flow cytometry analysis showed that they expressed the MSC-specific markers, reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that MSC expressed the hepatocyte-specific marker cytokeratin 18 (CK-18) following hepatocyte induction. This study demonstrates that BM-derived-MSC can differentiate into functional hepatocyte-like cells following the induction of HGF and FGF-4. MSC can serve as a favorable cell source for tissue engineering in the treatment of liver disease. PMID:27429519

  4. A 3-D constrained mixture model for mechanically mediated vascular growth and remodeling

    PubMed Central

    Wan, William; Hansen, Laura

    2010-01-01

    In contrast to the widely applied approach to model soft tissue remodeling employing the concept of volumetric growth, microstructurally motivated models are capable of capturing many of the underlying mechanisms of growth and remodeling; i.e., the production, removal, and remodeling of individual constituents at different rates and to different extents. A 3-dimensional constrained mixture computational framework has been developed for vascular growth and remodeling, considering new, microstructurally motivated kinematics and constitutive equations and new stress and muscle activation mediated evolution equations. Our computational results for alterations in flow and pressure, using reasonable physiological values for rates of constituent growth and turnover, concur with findings in the literature. For example, for flow-induced remodeling, our simulations predict that, although the wall shear stress is restored completely, the circumferential stress is not restored employing realistic physiological rate parameters. Also, our simulations predict different levels of thickening on inner versus outer wall locations, as shown in numerous reports of pressure-induced remodeling. Whereas the simulations are meant to be illustrative, they serve to highlight the experimental data currently lacking to fully quantify mechanically mediated adaptations in the vasculature. PMID:20039091

  5. Serum level of hepatocyte growth factor is a novel marker of predicting the outcome and resistance to the treatment with trastuzumab in HER2-positive patients with metastatic gastric cancer

    PubMed Central

    Takahashi, Naoki; Furuta, Koh; Taniguchi, Hirokazu; Sasaki, Yusuke; Shoji, Hirokazu; Honma, Yoshitaka; Iwasa, Satoru; Okita, Natsuko; Takashima, Atsuo; Kato, Ken; Hamaguchi, Tetsuya; Shimada, Yasuhiro; Yamada, Yasuhide

    2016-01-01

    HER2-overexpression in tumor tissue is observed in 6 to 23% of advanced gastric cancer (GC) cases, and trastuzumab is an active molecular drug for these patients. There are no data available on whether serum levels of ligands are associated with the response and resistance to trastuzumab in HER2-positive patients with metastatic GC. HER2 screening of 502 patients with advanced gastric cancer was performed in our institution. Among these patients, 84 patients (16.8%) were diagnosed as HER2-positive, and those who were treated with trastuzumab and met the inclusion criteria were enrolled in the present study. Serum levels of ligands that affect the HER2 signal pathway were measured by an enzyme-linked immunosorbent assay. Forty-six HER2-positive patients were enrolled in this study, and 26 patients (56.5%) achieved a partial response to treatment with trastuzumab. Among several ligands, the serum level of hepatocyte growth factor (HGF) was significantly lower in responders compared with that in non-responders (p = 0.014). Multivariate analyses showed that a high level of serum HGF was a poor prognostic factor for overall survival (OS) compared with low levels of HGF (adjusted HR: 3.857, 95% CI: 1.309–11.361, p = 0.014). Among 25 patients without initial disease progression on the treatment with trastuzumab, the mean value of serum HGF at disease progression was significantly higher than that at pre-treatment (p = 0.041). As novel findings, our study indicated that serum level of HGF was associated with tumor shrinkage and time to progression of trastuzumab in HER2-positive patients with metastatic GC. PMID:26716644

  6. Intrasplenic transplantation of allogeneic hepatocytes prolongs survival in anhepatic rats.

    PubMed

    Arkadopoulos, N; Lilja, H; Suh, K S; Demetriou, A A; Rozga, J

    1998-11-01

    To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n = 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n = 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor beta1 (TGF-beta1) levels. Group 3 (n = 16) rats received intrasplenic injection of isolated hepatocytes (2.5 x 10(7) cells/rat) followed by total hepatectomy after 3 days. Group 4 (n = 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 +/- 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-beta1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 +/- 8.5 vs. 15.5 +/- 4.8 hrs, P < .01). Additionally, at 12 hours post-hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF-beta1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-beta1 levels in rats rendered anhepatic. PMID:9794923

  7. Drosophila tribbles antagonizes insulin signaling-mediated growth and metabolism via interactions with Akt kinase.

    PubMed

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

  8. Drosophila Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

    PubMed Central

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L.

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

  9. Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

    PubMed Central

    Dewaele, Michael; Tabaglio, Tommaso; Willekens, Karen; Bezzi, Marco; Teo, Shun Xie; Low, Diana H.P.; Koh, Cheryl M.; Rambow, Florian; Fiers, Mark; Rogiers, Aljosja; Radaelli, Enrico; Al-Haddawi, Muthafar; Tan, Soo Yong; Hermans, Els; Amant, Frederic; Yan, Hualong; Lakshmanan, Manikandan; Koumar, Ratnacaram Chandrahas; Lim, Soon Thye; Derheimer, Frederick A.; Campbell, Robert M.; Bonday, Zahid; Tergaonkar, Vinay; Shackleton, Mark; Blattner, Christine; Marine, Jean-Christophe; Guccione, Ernesto

    2015-01-01

    MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient–derived xenograft (PDX) mouse models, antisense oligonucleotide–mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target. PMID:26595814

  10. The Pollen Receptor Kinase LePRK2 Mediates Growth-Promoting Signals and Positively Regulates Pollen Germination and Tube Growth

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In flowering plants, the process of pollen germination and tube growth is required for successful fertilization. A pollen receptor kinase from tomato, LePRK2, has been implicated in signaling during pollen germination and tube growth as well as in mediating pollen (tube)-pistil communication. Here w...

  11. Growth Control in Colon Epithelial Cells: Gadolinium Enhances Calcium-Mediated Growth Regulation

    PubMed Central

    Attili, Durga; Jenkins, Brian; Aslam, Muhammad Nadeem; Dame, Michael K.

    2013-01-01

    Gadolinium, a member of the lanthanoid family of transition metals, interacts with calcium-binding sites on proteins and other biological molecules. The overall goal of the present investigation was to determine if gadolinium could enhance calcium-induced epithelial cell growth inhibition in the colon. Gadolinium at concentrations as low as 1–5 µM combined with calcium inhibits proliferation of human colonic epithelial cells more effectively than calcium alone. Gadolinium had no detectable effect on calcium-induced differentiation in the same cells based on change in cell morphology, induction of E-cadherin synthesis, and translocation of E-cadherin from the cytosol to the cell surface. When the colon epithelial cells were treated with gadolinium and then exposed to increased calcium concentrations, movement of extracellular calcium into the cell was suppressed. In contrast, gadolinium treatment had no effect on ionomycin-induced release of stored intracellular calcium into the cytoplasm. Whether these in vitro observations can be translated into an approach for reducing abnormal proliferation in the colonic mucosa (including polyp formation) is not known. These results do, however, provide an explanation for our recent findings that a multi-mineral supplement containing all of the naturally occurring lanthanoid metals including gadolinium are more effective than calcium alone in preventing colon polyp formation in mice on a high-fat diet. PMID:23008064

  12. Hepatocyte cell therapy in liver disease.

    PubMed

    Bartlett, David Christopher; Newsome, Philip N

    2015-01-01

    Liver disease is a leading cause of morbidity and mortality. Liver transplantation remains the only proven treatment for end-stage liver failure but is limited by the availability of donor organs. Hepatocyte cell therapy, either with bioartificial liver devices or hepatocyte transplantation, may help address this by delaying or preventing liver transplantation. Early clinical studies have shown promising results, however in most cases, the benefit has been short lived and so further research into these therapies is required. Alternative sources of hepatocytes, including stem cell-derived hepatocytes, are being investigated as the isolation of primary human hepatocytes is limited by the same shortage of donor organs. This review summarises the current clinical experience of hepatocyte cell therapy together with an overview of possible alternative sources of hepatocytes. Current and future areas for research that might lead towards the realisation of the full potential of hepatocyte cell therapy are discussed. PMID:26212798

  13. Differential effects of STAT proteins on growth hormone-mediated IGF-I gene expression.

    PubMed

    Varco-Merth, Ben; Rotwein, Peter

    2014-11-01

    Growth hormone (GH) plays a key role regulating somatic growth and in controlling metabolism and other physiological processes in humans and other animal species. GH acts by binding to the extracellular part of its transmembrane receptor, leading to induction of multiple intracellular signal transduction pathways that culminate in changes in gene and protein expression. A key agent in GH-stimulated growth is the latent transcription factor signal transducer and activator of transcription (STAT) 5B, one of four STAT proteins induced by the GH receptor in cultured cells and in vivo. As shown by genetic and biochemical studies, GH-activated STAT5B promotes transcription of the gene encoding the critical growth peptide, insulin-like growth factor-I (IGF-I), and natural null mutations of STAT5B in humans lead to growth failure accompanied by diminished IGF-I expression. Here we have examined the possibility that other GH-activated STATs can enhance IGF-I gene transcription, and thus potentially contribute to GH-regulated somatic growth. We find that human STAT5A is nearly identical to STAT5B in its biochemical and functional responses to GH but that STAT1 and STAT3 show a weaker profile of in vitro binding to STAT DNA elements from the IGF-I gene than STAT5B, and are less potent inducers of gene transcription through these elements. Taken together, our results offer a molecular explanation for why STAT5B is a key in vivo mediator of GH-activated IGF-I gene transcription and thus of GH-regulated somatic growth. PMID:25205818

  14. Conditional Cooperativity of Toxin - Antitoxin Regulation Can Mediate Bistability between Growth and Dormancy

    PubMed Central

    Cataudella, Ilaria; Sneppen, Kim; Gerdes, Kenn; Mitarai, Namiko

    2013-01-01

    Many toxin-antitoxin operons are regulated by the toxin/antitoxin ratio by mechanisms collectively coined “conditional cooperativity”. Toxin and antitoxin form heteromers with different stoichiometric ratios, and the complex with the intermediate ratio works best as a transcription repressor. This allows transcription at low toxin level, strong repression at intermediate toxin level, and then again transcription at high toxin level. Such regulation has two interesting features; firstly, it provides a non-monotonous response to the concentration of one of the proteins, and secondly, it opens for ultra-sensitivity mediated by the sequestration of the functioning heteromers. We explore possible functions of conditional regulation in simple feedback motifs, and show that it can provide bistability for a wide range of parameters. We then demonstrate that the conditional cooperativity in toxin-antitoxin systems combined with the growth-inhibition activity of free toxin can mediate bistability between a growing state and a dormant state. PMID:24009488

  15. Conditional cooperativity of toxin - antitoxin regulation can mediate bistability between growth and dormancy.

    PubMed

    Cataudella, Ilaria; Sneppen, Kim; Gerdes, Kenn; Mitarai, Namiko

    2013-01-01

    Many toxin-antitoxin operons are regulated by the toxin/antitoxin ratio by mechanisms collectively coined "conditional cooperativity". Toxin and antitoxin form heteromers with different stoichiometric ratios, and the complex with the intermediate ratio works best as a transcription repressor. This allows transcription at low toxin level, strong repression at intermediate toxin level, and then again transcription at high toxin level. Such regulation has two interesting features; firstly, it provides a non-monotonous response to the concentration of one of the proteins, and secondly, it opens for ultra-sensitivity mediated by the sequestration of the functioning heteromers. We explore possible functions of conditional regulation in simple feedback motifs, and show that it can provide bistability for a wide range of parameters. We then demonstrate that the conditional cooperativity in toxin-antitoxin systems combined with the growth-inhibition activity of free toxin can mediate bistability between a growing state and a dormant state. PMID:24009488

  16. Transforming growth factor-beta1 mediates cellular response to DNA damage in situ

    NASA Technical Reports Server (NTRS)

    Ewan, Kenneth B.; Henshall-Powell, Rhonda L.; Ravani, Shraddha A.; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J.; Barcellos-Hoff, Mary Helen

    2002-01-01

    Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

  17. Pharmacological inhibition of fibroblast growth factor (FGF) receptor signaling ameliorates FGF23-mediated hypophosphatemic rickets.

    PubMed

    Wöhrle, Simon; Henninger, Christine; Bonny, Olivier; Thuery, Anne; Beluch, Noemie; Hynes, Nancy E; Guagnano, Vito; Sellers, William R; Hofmann, Francesco; Kneissel, Michaela; Graus Porta, Diana

    2013-04-01

    Fibroblast growth factor 23 (FGF23) is a circulating factor secreted by osteocytes that is essential for phosphate homeostasis. In kidney proximal tubular cells FGF23 inhibits phosphate reabsorption and leads to decreased synthesis and enhanced catabolism of 1,25-dihydroxyvitamin D3 (1,25[OH]2 D3 ). Excess levels of FGF23 cause renal phosphate wasting and suppression of circulating 1,25(OH)2 D3 levels and are associated with several hereditary hypophosphatemic disorders with skeletal abnormalities, including X-linked hypophosphatemic rickets (XLH) and autosomal recessive hypophosphatemic rickets (ARHR). Currently, therapeutic approaches to these diseases are limited to treatment with activated vitamin D analogues and phosphate supplementation, often merely resulting in partial correction of the skeletal aberrations. In this study, we evaluate the use of FGFR inhibitors for the treatment of FGF23-mediated hypophosphatemic disorders using NVP-BGJ398, a novel selective, pan-specific FGFR inhibitor currently in Phase I clinical trials for cancer therapy. In two different hypophosphatemic mouse models, Hyp and Dmp1-null mice, resembling the human diseases XLH and ARHR, we find that pharmacological inhibition of FGFRs efficiently abrogates aberrant FGF23 signaling and normalizes the hypophosphatemic and hypocalcemic conditions of these mice. Correspondingly, long-term FGFR inhibition in Hyp mice leads to enhanced bone growth, increased mineralization, and reorganization of the disturbed growth plate structure. We therefore propose NVP-BGJ398 treatment as a novel approach for the therapy of FGF23-mediated hypophosphatemic diseases. PMID:23129509

  18. Fetal production of growth factors and inflammatory mediators predicts pulmonary hypertension in congenital diaphragmatic hernia

    PubMed Central

    Fleck, Shannon; Bautista, Geoanna; Keating, Sheila M.; Lee, Tzong-Hae; Keller, Roberta L.; Moon-Grady, Anita J.; Gonzales, Kelly; Norris, Philip J.; Busch, Michael P.; Kim, CJ; Romero, Roberto; Lee, Hanmin; Miniati, Doug; MacKenzie, Tippi C.

    2014-01-01

    Background Congenital diaphragmatic hernia (CDH) represents a spectrum of lung hypoplasia and consequent pulmonary hypertension is an important cause of postnatal morbidity and mortality. We studied biomarkers at the maternal-fetal interface to understand factors associated with the persistence of pulmonary hypertension. Methods Maternal and cord blood samples from fetuses with CDH and unaffected controls were analyzed using a human 39plex immunoassay kit. Cellular trafficking between the mother and the fetu was quantified using quantitative real-time PCR for non-shared alleles. Biomarker profiles were then correlated with CDH severity based on the degree of pulmonary hypertension. Results Cord blood levels of epidermal growth factor, platelet-derived growth factor, and several inflammatory mediators increased significantly as the severity of CDH increased, while maternal levels growth factors and mediators decreased significantly with CDH severity. Maternal cells were increased in fetuses with severe CDH compared to controls, with elevated levels of the chemokine CXCL-10 in patients with the highest trafficking. Conclusion Patients with CDH demonstrate pro-inflammatory and chemotactic signals in fetal blood at the time of birth. Since some of these molecules have been implicated in the development of pulmonary hypertension, prenatal strategies targeting specific molecular pathways may be useful adjuncts to current fetal therapies. PMID:23770923

  19. 14-3-3 sigma and 14-3-3 zeta plays an opposite role in cell growth inhibition mediated by transforming growth factor-beta 1.

    PubMed

    Hong, Hye-Young; Jeon, Woo-Kwang; Bae, Eun-Jin; Kim, Shin-Tae; Lee, Ho-Jae; Kim, Seong-Jin; Kim, Byung-Chul

    2010-03-01

    The expression of 14-3-3 proteins is dysregulated in various types of cancer. This study was undertaken to investigate the effects of 14-3-3 zeta and 14-3-3 sigma on cell growth inhibition mediated by transforming growth factor-beta 1 (TGF-beta1). Mouse mammary epithelial cells (Eph4) that are transformed with oncogenic c-H-Ras (EpRas) and no longer sensitive to TGF-beta1-mediated growth inhibition displayed increased expression of 14-3-3 zeta and decreased expression of 14-3-3 sigma compared with parental Eph4 cells. Using small interfering RNA-mediated knockdown and overexpression of 14-3-3 sigma or 14-3-3 zeta, we showed that 14-3-3 sigma is required for TGF-beta1-mediated growth inhibition whereas 14-3-3 zeta negatively modulates this growth inhibitory response. Notably, overexpression of 14-3-3 zeta increased the level of Smad3 protein that is phosphorylated at linker regions and cannot mediate the TGF-beta1 growth inhibitory response. Consistent with this finding, mutation of the 14-3-3 zeta phosphorylation sites in Smad3 markedly reduced the 14-3-3 zeta-mediated inhibition of TGF-beta1-induced p15 promoter-reporter activity and cell cycle arrest, suggesting that these residues are critical targets of 14-3-3 zeta in the suppression of TGF-beta1-mediated growth. Taken together, our findings indicate that dysregulation of 14-3-3 sigma or 14-3-3 zeta contributes to TGF-beta1 resistance in cancer cells. PMID:20082218

  20. Synchrotron X-ray and optical studies of the DNA-mediated growth of plasmonic nanostructures

    NASA Astrophysics Data System (ADS)

    Chen, Gang; Wang, Geng; Zhang, Xiaonan; Geng, Heping; Xu, Lifeng; Li, Wenqin; Liu, Xin

    2015-03-01

    Reproducible and controllable growth of nanostructures with well-defined physical and chemical properties is a longstanding problem in nanoscience. A key step to address this issue is to understand their underlying growth mechanism, which is often entangled in the complexity of growth environments and obscured by rapid reaction speeds. Synchrotron x-rays, because of their specific wavelengths (nanometers) and advantages of large flux, high penetration and adjustable photon energy, have a particularly important position in structural and electronic characterizations of nanomaterials. Herein, we demonstrate that the evolution of size, surface morphology, and the optical properties of plasmonic nanostructures could be quantitatively intercepted by dynamic and stoichiometric control of the DNA-mediated growth. By combining synchrotron-based small-angle X-ray scattering with transmission electron microscopy, we reliably obtained quantitative structural parameters for these fine nanostructures that correlate well with their optical properties as identified by UV/Vis absorption and dark-field scattering spectroscopy. We report growth mechanisms for SERS active plasmonic nanostructures, and the remarkable interplay between their morphology and plasmonic properties. Work supported by NNSF of China (11375256) and Sci. and Tech. Commission of Shanghai Municipality (14JC1493300).

  1. Mediation of wound-related Rous sarcoma virus tumorigenesis by TFG (transforming growth factor)-. beta

    SciTech Connect

    Sieweke, M.H.; Bissell, M.J. ); Thompson, N.L.; Sporn, M.B. )

    1990-06-29

    In Rous sarcoma virus (RSV)-infected chickens, wounding leads to tumor formation with nearly 100% frequency in tissues that would otherwise remain tumor-free. Identifying molecular mediators of this phenomenon should yield important clues to the mechanisms involved in RSV tumorigenesis. Immunohistochemical staining showed that TGF-{beta} is present locally shortly after wounding, but not in unwounded controls. In addition, subcutaneous administration of recombinant transforming growth factor {beta}1 (TGF-{beta}1) could substitute completely for wounding in tumor induction. A treatment protocol of four doses of 800 nanograms of TGF-{beta} resulted in v-src-expressing tumors with 100% frequency; four doses of only 10 nanograms still led to tumor formation in 80% of the animals. This effect was specific, as other growth factors with suggested roles in would healing did not elicit the same response. Epidermal growth factor (EGF) or TGF-{alpha} had no effect, and platelet-derived growth factor (PDGF) or insulin-like growth factor-1 (IGF-1) yielded only occasional tumors after longer latency. TGF-{beta} release during the would-healing response may thus be a critical event that creates a conducive environment for RSV tumorigenesis and may act as a cofactor for transformation in this system. 31 refs., 3 figs., 2 tabs.

  2. Vitamin D enhances mitogenesis mediated by keratinocyte growth factor receptor in keratinocytes.

    PubMed

    Gamady, Anat; Koren, Ruth; Ron, Dina; Liberman, Uri A; Ravid, Amiram

    2003-06-01

    The hormonally active vitamin D metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and keratinocyte growth factor (KGF) belong to the network of autocrine and paracrine mediators in the skin. Both were shown to modulate keratinocyte proliferation, to reverse epidermal atrophy, to increase wound healing, and to reduce chemotherapy-induced alopecia. The overlap between their activities may suggest that vitamin D exerts some of its actions by modulation of KGF activities in the skin. This notion was examined by using HaCaT keratinocytes cultured in serum-free medium in the absence of exogenous growth factors and in the presence of the EGF receptor tyrosine kinase inhibitor AG 1478 that blocks their autonomous proliferation. These cells could be stimulated to proliferate by different fibroblast growth factors (FGFs). The relative mitogenic efficacy of basic FGF, acidic FGF, or KGF was in correlation with their affinities for the KGF receptor (KGFR). Forty-eight hour co-treatment with 1,25(OH)(2)D(3) enhanced KGFR-mediated cell proliferation in a dose dependent manner. Both ERK1/2 and c-Jun N-terminal kinase (JNK) were activated by the FGFs. Treatment with 1,25(OH)(2)D(3) increased the activation of ERK but reduced the activation of JNK. Treatment with 1,25(OH)(2)D(3) increased the levels of KGFR in the presence but not in the absence of KGF, probably due to inhibition of ligand-induced receptor degradation. Inhibition of protein kinase C with bisindolylmaleimide did not interfere with the effect of 1,25(OH)(2)D(3) on KGFR-mediated ERK activation. Our results support the notion that the paracrine KGF-KGFR system in the skin can act in concert with the autocrine vitamin D system in keratinocytes to promote keratinocyte proliferation and survival under situations of stress and injury. PMID:12761878

  3. Vitamin K enhancement of Sorafenib-mediated HCC cell growth inhibition in vitro and in vivo

    PubMed Central

    Wei, Gang; Wang, Meifanf; Hyslop, Terry; Wang, Ziqiu; Carr, Brian I.

    2010-01-01

    The multi-kinase inhibitor Sorafenib, is the first oral agent to show activity against human hepatocellular carcinoma (HCC). Apoptosis has been shown to be induced in HCC by several agents, including Sorafenib, as well as by the naturally occurring K vitamins (VKs). Since few non toxic agents have activity against HCC growth, we evaluated the activity of Sorafenib and K vitamins, both independently and together on the growth in vitro and in vivo of HCC cells. We found that when VK was combined with Sorafenib, the concentration of Sorafenib required for growth inhibition was substantially reduced. Conversely, VK enhanced Sorafenib effects in several HCC cell lines on growth inhibition. Growth could be inhibited at doses of VK plus Sorafenib that were ineffective with either agent alone,using vitamins K1, K2 and K5. Combination VK1 plus Sorafenib induced apoptosis on FACS, TUNEL staining and caspase activation. Phospho-ERK levels were decreased, as was Mcl-1, an ERK target. Sorafenib alone inhibited growth of transplantable HCC in vivo. At sub-effective Sorafenib doses in vivo, addition of VK1 caused major tumor regression, with decreased phospho-ERK and Mcl-1 staining. Thus, combination VK1 plus Sorafenib strongly induced growth inhibition and apoptosis in rodent and human HCC and inhibited the RAF/MEK/ERK pathway. VK1 alone activated PKA, a mediator of inhibitory Raf phosphorylation. Thus, each agent can antagonize Raf; Sorafenib as a direct inhibitor and VK1 through inhibitory Raf phosphorylation. Since both agents are available for human use, the combination has potential for improving Sorafenib effects in HCC. PMID:21351273

  4. Hammerhead Ribozyme-Mediated Knockdown of mRNA for Fibrotic Growth Factors: Transforming Growth Factor-Beta 1 and Connective Tissue Growth Factor

    PubMed Central

    Robinson, Paulette M.; Blalock, Timothy D.; Yuan, Rong; Lewin, Alfred S.; Schultz, Gregory S.

    2013-01-01

    Excessive scarring (fibrosis) is a major cause of pathologies in multiple tissues, including lung, liver, kidney, heart, cornea, and skin. The transforming growth factor- β (TGF- β) system has been shown to play a key role in regulating the formation of scar tissue throughout the body. Furthermore, connective tissue growth factor (CTGF) has been shown to mediate most of the fibrotic actions of TGF- β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. Currently, no approved drugs selectively and specifically regulate scar formation. Thus, there is a need for a drug that selectively targets the TGF- β cascade at the molecular level and has minimal off-target side effects. This chapter focuses on the design of hammerhead ribozymes, measurement of kinetic activity, and assessment of knockdown mRNAs of TGF- β and CTGF in cell cultures. PMID:22131029

  5. Seed-mediated growth of palladium nanocrystals: the effect of pseudo-halide thiocyanate ions.

    PubMed

    Zhang, Ling; Niu, Wenxin; Xu, Guobao

    2011-02-01

    In synthesis in a solution phase, adsorbates such as halides can interact selectively with different metal crystal facets and affect the final morphology of nanocrystals. Pseudo-halide thiocyanate ions (SCN-) can also adsorb on the metal surface, but they have never been used for the synthesis of shape-controlled colloidal metal nanocrystals. In this study, we first investigated the effect of SCN- on the morphology of palladium nanocrystals through a seed-mediated growth method. The presence of 1 µM SCN- in the growth solutions could lead to the formation of palladium polyhedra: truncated rhombic dodecahedra enclosed by twelve {110}, eight {111} and six {100} facets. The products were nanocubes enclosed with six {100} facets if cetyltrimethylammonium bromide (CTAB) was the only capping agent. Meanwhile, the mechanism of the effect of SCN- on the morphology of Pd nanocrystals is discussed. PMID:21170425

  6. Antimony mediated growth of high-density InAs quantum dots for photovoltaic cells

    SciTech Connect

    Tutu, F. K.; Wu, J.; Lam, P.; Tang, M.; Liu, H.; Miyashita, N.; Okada, Y.; Wilson, J.; Allison, R.

    2013-07-22

    We report enhanced solar cell performance using high-density InAs quantum dots. The high-density quantum dot was grown by antimony mediated molecular beam epitaxy. In-plane quantum dot density over 1 × 10{sup 11} cm{sup −2} was achieved by applying a few monolayers of antimony on the GaAs surface prior to quantum dot growth. The formation of defective large clusters was reduced by optimization of the growth temperature and InAs coverage. Comparing with a standard quantum dot solar cell without the incorporation of antimony, the high-density quantum dot solar cell demonstrates a distinct improvement in short-circuit current from 7.4 mA/cm{sup 2} to 8.3 mA/cm{sup 2}.

  7. Dnmt2/Trdmt1 as Mediator of RNA Polymerase II Transcriptional Activity in Cardiac Growth

    PubMed Central

    Polo, Beatrice; Baudouy, Delphine; Kiani, Jafar; Michiels, Jean-François; Cuzin, François; Rassoulzadegan, Minoo

    2016-01-01

    Dnmt2/Trdmt1 is a methyltransferase, which has been shown to methylate tRNAs. Deficient mutants were reported to exhibit various, seemingly unrelated, defects in development and RNA-mediated epigenetic heredity. Here we report a role in a distinct developmental regulation effected by a noncoding RNA. We show that Dnmt2-deficiency in mice results in cardiac hypertrophy. Echocardiographic measurements revealed that cardiac function is preserved notwithstanding the increased dimensions of the organ due to cardiomyocyte enlargement. Mechanistically, activation of the P-TEFb complex, a critical step for cardiac growth, results from increased dissociation of the negatively regulating Rn7sk non-coding RNA component in Dnmt2-deficient cells. Our data suggest that Dnmt2 plays an unexpected role for regulation of cardiac growth by modulating activity of the P-TEFb complex. PMID:27270731

  8. Property of hepatitis B virus replication in Tupaia belangeri hepatocytes.

    PubMed

    Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

    2016-01-01

    The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3-6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10(5) copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10(4)-10(6) copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10(3) copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. PMID:26654952

  9. Constitutive Smad linker phosphorylation in melanoma: a mechanism of resistance to transforming growth factor-β-mediated growth inhibition.

    PubMed

    Cohen-Solal, Karine A; Merrigan, Kim T; Chan, Joseph L-K; Goydos, James S; Chen, Wenjin; Foran, David J; Liu, Fang; Lasfar, Ahmed; Reiss, Michael

    2011-06-01

    Melanoma cells are resistant to transforming growth factor-β (TGFβ)-induced cell-cycle arrest. In this study, we investigated a mechanism of resistance involving a regulatory domain, called linker region, in Smad2 and Smad3, main downstream effectors of TGFβ. Melanoma cells in culture and tumor samples exhibited constitutive Smad2 and Smad3 linker phosphorylation. Treatment of melanoma cells with the MEK1/2 inhibitor, U0126, or the two pan-CDK and GSK3 inhibitors, Flavopiridol and R547, resulted in decreased linker phosphorylation of Smad2 and Smad3. Overexpression of the linker phosphorylation-resistant Smad3 EPSM mutant in melanoma cells resulted in an increase in expression of p15(INK4B) and p21(WAF1) , as compared with cells transfected with wild-type (WT) Smad3. In addition, the cell numbers of EPSM Smad3-expressing melanoma cells were significantly reduced compared with WT Smad3-expressing cells. These results suggest that the linker phosphorylation of Smad3 contributes to the resistance of melanoma cells to TGFβ-mediated growth inhibition. PMID:21477078

  10. Hepatocytes: a key cell type for innate immunity.

    PubMed

    Zhou, Zhou; Xu, Ming-Jiang; Gao, Bin

    2016-05-01

    Hepatocytes, the major parenchymal cells in the liver, play pivotal roles in metabolism, detoxification, and protein synthesis. Hepatocytes also activate innate immunity against invading microorganisms by secreting innate immunity proteins. These proteins include bactericidal proteins that directly kill bacteria, opsonins that assist in the phagocytosis of foreign bacteria, iron-sequestering proteins that block iron uptake by bacteria, several soluble factors that regulate lipopolysaccharide signaling, and the coagulation factor fibrinogen that activates innate immunity. In this review, we summarize the wide variety of innate immunity proteins produced by hepatocytes and discuss liver-enriched transcription factors (e.g. hepatocyte nuclear factors and CCAAT/enhancer-binding proteins), pro-inflammatory mediators (e.g. interleukin (IL)-6, IL-22, IL-1β and tumor necrosis factor-α), and downstream signaling pathways (e.g. signal transducer and activator of transcription factor 3 and nuclear factor-κB) that regulate the expression of these innate immunity proteins. We also briefly discuss the dysregulation of these innate immunity proteins in chronic liver disease, which may contribute to an increased susceptibility to bacterial infection in patients with cirrhosis. PMID:26685902

  11. Hepatocytes: a key cell type for innate immunity

    PubMed Central

    Zhou, Zhou; Xu, Ming-Jiang; Gao, Bin

    2016-01-01

    Hepatocytes, the major parenchymal cells in the liver, play pivotal roles in metabolism, detoxification, and protein synthesis. Hepatocytes also activate innate immunity against invading microorganisms by secreting innate immunity proteins. These proteins include bactericidal proteins that directly kill bacteria, opsonins that assist in the phagocytosis of foreign bacteria, iron-sequestering proteins that block iron uptake by bacteria, several soluble factors that regulate lipopolysaccharide signaling, and the coagulation factor fibrinogen that activates innate immunity. In this review, we summarize the wide variety of innate immunity proteins produced by hepatocytes and discuss liver-enriched transcription factors (e.g. hepatocyte nuclear factors and CCAAT/enhancer-binding proteins), pro-inflammatory mediators (e.g. interleukin (IL)-6, IL-22, IL-1β and tumor necrosis factor-α), and downstream signaling pathways (e.g. signal transducer and activator of transcription factor 3 and nuclear factor-κB) that regulate the expression of these innate immunity proteins. We also briefly discuss the dysregulation of these innate immunity proteins in chronic liver disease, which may contribute to an increased susceptibility to bacterial infection in patients with cirrhosis. PMID:26685902

  12. The Involvement of Gibberellins in 1,8-Cineole-Mediated Inhibition of Sprout Growth in Russet Burbank Tubers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The involvement of gibberellins in 1,8-cineole-mediated inhibition of tuber sprout growth was investigated in non-dormant field- and greenhouse-grown tubers of Russet Burbank. Continuous exposure of tubers to cineole in the vapor-phase resulted in a dose-dependent inhibition of sprout growth. Comp...

  13. Seed-mediated growth of palladium nanocrystals: The effect of pseudo-halide thiocyanate ions

    NASA Astrophysics Data System (ADS)

    Zhang, Ling; Niu, Wenxin; Xu, Guobao

    2011-02-01

    In synthesis in a solution phase, adsorbates such as halides can interact selectively with different metal crystal facets and affect the final morphology of nanocrystals. Pseudo-halide thiocyanate ions (SCN-) can also adsorb on the metal surface, but they have never been used for the synthesis of shape-controlled colloidal metal nanocrystals. In this study, we first investigated the effect of SCN- on the morphology of palladium nanocrystals through a seed-mediated growth method. The presence of 1 µM SCN- in the growth solutions could lead to the formation of palladium polyhedra: truncated rhombic dodecahedra enclosed by twelve {110}, eight {111} and six {100} facets. The products were nanocubes enclosed with six {100} facets if cetyltrimethylammonium bromide (CTAB) was the only capping agent. Meanwhile, the mechanism of the effect of SCN- on the morphology of Pd nanocrystals is discussed.In synthesis in a solution phase, adsorbates such as halides can interact selectively with different metal crystal facets and affect the final morphology of nanocrystals. Pseudo-halide thiocyanate ions (SCN-) can also adsorb on the metal surface, but they have never been used for the synthesis of shape-controlled colloidal metal nanocrystals. In this study, we first investigated the effect of SCN- on the morphology of palladium nanocrystals through a seed-mediated growth method. The presence of 1 µM SCN- in the growth solutions could lead to the formation of palladium polyhedra: truncated rhombic dodecahedra enclosed by twelve {110}, eight {111} and six {100} facets. The products were nanocubes enclosed with six {100} facets if cetyltrimethylammonium bromide (CTAB) was the only capping agent. Meanwhile, the mechanism of the effect of SCN- on the morphology of Pd nanocrystals is discussed. Electronic supplementary information (ESI) available: Additional SEM, TEM and XRD data. See DOI: 10.1039/c0nr00622j

  14. Modeling of Hepatocytes Proliferation Isolated from Proximal and Distal Zones from Human Hepatocellular Carcinoma Lesion

    PubMed Central

    Montalbano, Mauro; Curcurù, Giuseppe; Shirafkan, Ali; Vento, Renza; Rastellini, Cristiana; Cicalese, Luca

    2016-01-01

    Isolation of hepatocytes from cirrhotic human livers and subsequent primary culture are important new tools for laboratory research and cell-based therapeutics in the study of hepatocellular carcinoma (HCC). Using such techniques, we have previously identified different subpopulations of human hepatocytes and among them one is showing a progressive transformation of hepatocytes in HCC-like cells. We have hypothesized that increasing the distance from the neoplastic lesion might affect hepatocyte function and transformation capacity. However, limited information is available in comparing the growth and proliferation of human hepatocytes obtained from different areas of the same cirrhotic liver in relation to their distance from the HCC lesion. In this study, hepatocytes from 10 patients with cirrhosis and HCC undergoing surgical resections from specimens obtained at a proximal (CP) and distal (CD) distance from the HCC lesion were isolated and placed in primary culture. CP hepatocytes (CP-Hep) were isolated between 1 to 3 cm (leaving at least 1cm margin to avoid cancer cells and/or satellite lesions), while CD hepatocytes (CD-Hep) were isolated from more than 5 cm or from the contralateral-lobe. A statistical model was built to analyze the proliferation rates of these cells and we evaluated expression of HCC markers (Glypican-3 (GPC3), αSmooth Muscle Actin (α-SMA) and PCNA). We observed a significant difference in proliferation and in-vitro growth showing that CP-Hep had a proliferation pattern and rate significantly different than CD-Hep. Based on these data, this model can provide information to predict growth of human hepatocytes in primary culture in relation to their pre-cancerous state with significant differences in the HCC markers expression. This model provides an important innovative tool for in-vitro analysis of HCC. PMID:27074018

  15. The effects of anxiety and depression on stress-related growth among Chinese army recruits: Resilience and coping as mediators.

    PubMed

    Yu, Yongju; Peng, Li; Liu, Botao; Liu, Yunbo; Li, Min; Chen, Long; Xie, Junrun; Li, Jing; Li, Jiawen

    2016-09-01

    Stress-related growth can occur after various traumas or stressful events. In order to investigate how anxiety and depression relate to stress-related growth, this study was conducted with 443 Chinese army recruits who had just finished a 3-month recruit training program. Path analyses revealed that resilience and positive/negative coping partially mediated the effect of anxiety on perceived stress-related growth, while negative coping fully mediated the relationship between depression and perceived stress-related growth. Moreover, positive coping partially carried the influence of resilience on perceived stress-related growth. Anxiety and depression may be potential targets for intervention to enhance the development of stress-related growth among Chinese army recruits. PMID:25631664

  16. Does social status within a dominance hierarchy mediate individual growth, residency and relocation?

    PubMed

    Akbaripasand, Abbas; Ramezani, J; Krkosek, Martin; Lokman, P Mark; Closs, Gerard P

    2014-11-01

    The availability of food, and hence energy, is known to influence the abundance, habitat choice and growth of individuals. In contrast, there is a paucity of knowledge on how the interaction of energy supply and social status determines patterns of residency and movement. This study tests whether the presence of conspecifics and an individual's social status in relation to food supply influence the fitness and movement of a drift-feeding fish (Galaxias fasciatus). Using an information-theoretic approach (AIC), our analysis indicated that the most parsimonious model of fish movement among pools was one that included food supply, social rank and fish relative growth rate. Our results indicated that subordinate fish relocated more frequently compared to dominant fish, most likely as a consequence of intra-specific competition that limited the access of these smaller fish to resources and constrained their growth. Our results suggest that energy constraints may force individuals to explore new habitats in an effort to find more energetically profitable patches. We conclude that intra-specific competition mediated through the social hierarchy amongst closely interacting individuals plays a key role in determining individual growth, residency and relocation. PMID:25159213

  17. Brevundimonas diminuta mediated alleviation of arsenic toxicity and plant growth promotion in Oryza sativa L.

    PubMed

    Singh, Namrata; Marwa, Naina; Mishra, Shashank K; Mishra, Jyoti; Verma, Praveen C; Rathaur, Sushma; Singh, Nandita

    2016-03-01

    Arsenic (As), a toxic metalloid adversely affects plant growth in polluted areas. In the present study, we investigated the possibility of improving phytostablization of arsenic through application of new isolated strain Brevundimonas diminuta (NBRI012) in rice plant [Oryza sativa (L.) Var. Sarju 52] at two different concentrations [10ppm (low toxic) and 50ppm (high toxic)] of As. The plant growth promoting traits of bacterial strains revealed the inherent ability of siderophores, phosphate solubilisation, indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production which may be associated with increased biomass, chlorophyll and MDA content of rice and thereby promoting plant growth. The study also revealed the As accumulation property of NBRI012 strain which could play an important role in As removal from contaminated soil. Furthermore, NBRI012 inoculation significantly restored the hampered root epidermal and cortical cell growth of rice plant and root hair elimination. Altogether our study highlights the multifarious role of B. diminuta in mediating stress tolerance and modulating translocation of As in edible part of rice plant. PMID:26650422

  18. Human Sarcoma growth is sensitive to small-molecule mediated AXIN stabilization.

    PubMed

    De Robertis, Alessandra; Mennillo, Federica; Rossi, Marco; Valensin, Silvia; Tunici, Patrizia; Mori, Elisa; Caradonna, Nicola; Varrone, Maurizio; Salerno, Massimiliano

    2014-01-01

    Sarcomas are mesenchymal tumors showing high molecular heterogeneity, reflected at the histological level by the existence of more than fifty different subtypes. Genetic and epigenetic evidences link aberrant activation of the Wnt signaling to growth and progression of human sarcomas. This phenomenon, mainly accomplished by autocrine loop activity, is sustained by gene amplification, over-expression of Wnt ligands and co-receptors or epigenetic silencing of endogenous Wnt antagonists. We previously showed that pharmacological inhibition of Wnt signaling mediated by Axin stabilization produced in vitro and in vivo antitumor activity in glioblastoma tumors. Here, we report that targeting different sarcoma cell lines with the Wnt inhibitor/Axin stabilizer SEN461 produces a less transformed phenotype, as supported by modulation of anchorage-independent growth in vitro. At the molecular level, SEN461 treatment enhanced the stability of the scaffold protein Axin1, a key negative regulator of the Wnt signaling with tumor suppressor function, resulting in downstream effects coherent with inhibition of canonical Wnt signaling. Genetic phenocopy of small molecule Axin stabilization, through Axin1 over-expression, coherently resulted in strong impairment of soft-agar growth. Importantly, sarcoma growth inhibition through pharmacological Axin stabilization was also observed in a xenograft model in vivo in female CD-1 nude mice. Our findings suggest the usefulness of Wnt inhibitors with Axin stabilization activity as a potentialyl clinical relevant strategy for certain types of sarcomas. PMID:24842792

  19. Human Sarcoma Growth Is Sensitive to Small-Molecule Mediated AXIN Stabilization

    PubMed Central

    Rossi, Marco; Valensin, Silvia; Tunici, Patrizia; Mori, Elisa; Caradonna, Nicola; Varrone, Maurizio; Salerno, Massimiliano

    2014-01-01

    Sarcomas are mesenchymal tumors showing high molecular heterogeneity, reflected at the histological level by the existence of more than fifty different subtypes. Genetic and epigenetic evidences link aberrant activation of the Wnt signaling to growth and progression of human sarcomas. This phenomenon, mainly accomplished by autocrine loop activity, is sustained by gene amplification, over-expression of Wnt ligands and co-receptors or epigenetic silencing of endogenous Wnt antagonists. We previously showed that pharmacological inhibition of Wnt signaling mediated by Axin stabilization produced in vitro and in vivo antitumor activity in glioblastoma tumors. Here, we report that targeting different sarcoma cell lines with the Wnt inhibitor/Axin stabilizer SEN461 produces a less transformed phenotype, as supported by modulation of anchorage-independent growth in vitro. At the molecular level, SEN461 treatment enhanced the stability of the scaffold protein Axin1, a key negative regulator of the Wnt signaling with tumor suppressor function, resulting in downstream effects coherent with inhibition of canonical Wnt signaling. Genetic phenocopy of small molecule Axin stabilization, through Axin1 over-expression, coherently resulted in strong impairment of soft-agar growth. Importantly, sarcoma growth inhibition through pharmacological Axin stabilization was also observed in a xenograft model in vivo in female CD-1 nude mice. Our findings suggest the usefulness of Wnt inhibitors with Axin stabilization activity as a potentialyl clinical relevant strategy for certain types of sarcomas. PMID:24842792

  20. Platelet-derived growth factor-BB-mediated glycosaminoglycan synthesis is transduced through Akt.

    PubMed

    Cartel, Nicholas J; Wang, Jinxia; Post, Martin

    2002-04-01

    Previously we have demonstrated that the phosphoinositide 3-kinase (PI-3K) signal-transduction pathway mediates platelet-derived growth factor (PDGF)-BB-induced glycosaminoglycan (GAG) synthesis in fetal lung fibroblasts. In the present study we further investigated the signal-transduction pathway(s) that results in PDGF-BB-induced GAG synthesis. Over-expression of a soluble PDGF beta-receptor as well as a mutated form of the beta-receptor, unable to bind PI-3K, diminished GAG synthesis in fetal lung fibroblasts subsequent to PDGF-BB stimulation. The PI-3K inhibitor wortmannin blocked PDGF-BB-induced Akt activity as well as significantly diminishing PDGF-BB-mediated GAG synthesis. Expression of dominant-negative PI-3K also abrogated Akt activity and GAG synthesis. Furthermore, expression of dominant-negative Akt abrogated endogenous Akt activity, Rab3D phosphorylation and GAG synthesis, whereas expression of constitutively activated Akt stimulated Rab3D phosphorylation and GAG synthesis in the absence of PDGF-BB. Over-expression of wild-type PTEN (phosphatase and tensin homologue deleted in chromosome 10) inhibited Akt activity and concomitantly attenuated GAG synthesis in fibroblasts stimulated with PDGF-BB. These data suggest that Akt is an integral protein involved in PDGF-BB-mediated GAG regulation in fetal lung fibroblasts. PMID:11903042

  1. Inhibition of tumor cell growth by Sigma1 ligand mediated translational repression

    SciTech Connect

    Kim, Felix J.; Schrock, Joel M.; Spino, Christina M.; Marino, Jacqueline C.; Pasternak, Gavril W.

    2012-09-21

    Highlights: Black-Right-Pointing-Pointer Sigma1 ligand treatment mediates decrease in tumor cell mass. Black-Right-Pointing-Pointer Identification of a Sigma1 ligand with reversible translational repressor actions. Black-Right-Pointing-Pointer Demonstration of a role for Sigma1 in cellular protein synthesis. -- Abstract: Treatment with sigma1 receptor (Sigma1) ligands can inhibit cell proliferation in vitro and tumor growth in vivo. However, the cellular pathways engaged in response to Sigma1 ligand treatment that contribute to these outcomes remain largely undefined. Here, we show that treatment with putative antagonists of Sigma1 decreases cell mass. This effect corresponds with repressed cap-dependent translation initiation in multiple breast and prostate cancer cell lines. Sigma1 antagonist treatment suppresses phosphorylation of translational regulator proteins p70S6K, S6, and 4E-BP1. RNAi-mediated knockdown of Sigma1 also results in translational repression, consistent with the effects of antagonist treatment. Sigma1 antagonist mediated translational repression and decreased cell size are both reversible. Together, these data reveal a role for Sigma1 in tumor cell protein synthesis, and demonstrate that small molecule Sigma1 ligands can be used as modulators of protein translation.

  2. The Wedelolactone Derivative Inhibits Estrogen Receptor-Mediated Breast, Endometrial, and Ovarian Cancer Cells Growth

    PubMed Central

    Xu, Defeng; Lin, Tzu-Hua; Cheng, Max A.; Chen, Lu-Min; Chang, Chawnshang; Yeh, Shuyuan

    2014-01-01

    Estrogen and estrogen receptor (ER)-mediated signaling pathways play important roles in the etiology and progression of human breast, endometrial, and ovarian cancers. Attenuating ER activities by natural products and their derivatives is a relatively practical strategy to control and reduce breast, endometrial, and ovarian cancer risk. Here, we found 3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one (BTB), a new derivative of wedelolactone, could effectively inhibit the 17-estradiol (E2)-induced ER transactivation and suppress the growth of breast cancer as well as endometrial and ovarian cancer cells. Our results indicate that 2.5 μM BTB effectively suppresses ER-positive, but not ER-negative, breast, endometrial, and ovarian cancer cells. Furthermore, our data indicate that BTB can modulate ER transactivation and suppress the expression of E2-mediated ER target genes (Cyclin D1, E2F1, and TERT) in the ER-positive MCF-7, Ishikawa, and SKOV-3 cells. Importantly, this BTB mediated inhibition of ER activity is selective since BTB does not suppress the activities of other nuclear receptors, including glucocorticoid receptor and progesterone receptor, suggesting that BTB functions as a selective ER signaling inhibitor with the potential to treat breast, endometrial, and ovarian cancers. PMID:25221777

  3. Tyk2 mediates effects of urokinase on human vascular smooth muscle cell growth

    SciTech Connect

    Patecki, Margret; Schaewen, Markus von; Tkachuk, Sergey; Jerke, Uwe; Dietz, Rainer; Dumler, Inna; Kusch, Angelika . E-mail: angelika.kusch@charite.de

    2007-08-03

    The urokinase (uPA)/uPA receptor (uPAR) system plays a role in the response of the vessel wall to injury, presumably by modulating vascular smooth muscle cell (VSMC) functional behaviour. The Jak/Stat signaling pathway has been implicated to mediate the uPA/uPAR-directed cell migration and proliferation in VSMC. We have therefore investigated the underlying molecular mechanisms, which remained not completely understood. In particular, we aimed at identification of the kinase involved in the signaling cascade leading to Stat1 phosphorylation by uPA and its impact on VSMC growth. We performed expression in VSMC of kinase-deficient mutant forms of the Janus kinases Jak1 and Tyk2 and used different cell culture models imitating the response to vascular injury. We provide evidence that Tyk2, but not Jak1, mediates uPA-induced Stat1 phosphorylation and VSMC growth inhibition and suggest a novel function for Tyk2 as an important modulator of the uPA-directed VSMC functional behaviour at the place of injury.

  4. INO80 governs superenhancer-mediated oncogenic transcription and tumor growth in melanoma.

    PubMed

    Zhou, Bingying; Wang, Li; Zhang, Shu; Bennett, Brian D; He, Fan; Zhang, Yan; Xiong, Chengliang; Han, Leng; Diao, Lixia; Li, Pishun; Fargo, David C; Cox, Adrienne D; Hu, Guang

    2016-06-15

    Superenhancers (SEs) are large genomic regions with a high density of enhancer marks. In cancer, SEs are found near oncogenes and dictate cancer gene expression. However, how oncogenic SEs are regulated remains poorly understood. Here, we show that INO80, a chromatin remodeling complex, is required for SE-mediated oncogenic transcription and tumor growth in melanoma. The expression of Ino80, the SWI/SNF ATPase, is elevated in melanoma cells and patient melanomas compared with normal melanocytes and benign nevi. Furthermore, Ino80 silencing selectively inhibits melanoma cell proliferation, anchorage-independent growth, tumorigenesis, and tumor maintenance in mouse xenografts. Mechanistically, Ino80 occupies >90% of SEs, and its occupancy is dependent on transcription factors such as MITF and Sox9. Ino80 binding reduces nucleosome occupancy and facilitates Mediator recruitment, thus promoting oncogenic transcription. Consistently, genes co-occupied by Ino80 and Med1 are selectively expressed in melanomas compared with melanocytes. Together, our results reveal an essential role of INO80-dependent chromatin remodeling in SE function and suggest a novel strategy for disrupting SEs in cancer treatment. PMID:27340176

  5. Activation of signalling pathways during hepatocyte isolation: relevance to toxicology in vitro.

    PubMed

    Paine, Alan J; Andreakos, Evangelos

    2004-04-01

    The "Holy Grail" of in vitro toxicology is to develop assay systems that mimic the in vivo situation and hence reduce the need for toxicity tests employing experimental animals. However a major problem to be overcome with cell culture models is the rapid loss of differentiated phenotype that markedly limits extrapolation of results to the whole animal (i.e. human) situation. This limitation is most obvious in the application of hepatocyte cultures to predict pathways of metabolism mediated toxicity and results from the rapid loss of cytochrome P450 content. Here we demonstrate that changes in hepatocyte gene expression (e.g. MAP kinase and NF-kappaB activation) occur very early into the well established hepatocyte isolation procedure employing collagenase suggesting that hepatocytes are undergoing a pro-inflammatory ('acute phase') response before they are cultured. Data is presented indicating that the stimulus is, in part, due to oxidative stress but the demonstration of endotoxins in collagenase preparations is likely to exacerbate the situation. Thus appreciation of these early events during hepatocyte isolation represents the surest foundation for the successful application of cultured hepatocytes to toxicology rather than relying on traditional manipulations of hepatocyte culture medium/substratum once differentiated phenotype has already been lost. PMID:14757109

  6. ER stress induces NLRP3 inflammasome activation and hepatocyte death

    PubMed Central

    Lebeaupin, C; Proics, E; de Bieville, C H D; Rousseau, D; Bonnafous, S; Patouraux, S; Adam, G; Lavallard, V J; Rovere, C; Le Thuc, O; Saint-Paul, M C; Anty, R; Schneck, A S; Iannelli, A; Gugenheim, J; Tran, A; Gual, P; Bailly-Maitre, B

    2015-01-01

    The incidence of chronic liver disease is constantly increasing, owing to the obesity epidemic. However, the causes and mechanisms of inflammation-mediated liver damage remain poorly understood. Endoplasmic reticulum (ER) stress is an initiator of cell death and inflammatory mechanisms. Although obesity induces ER stress, the interplay between hepatic ER stress, NLRP3 inflammasome activation and hepatocyte death signaling has not yet been explored during the etiology of chronic liver diseases. Steatosis is a common disorder affecting obese patients; moreover, 25% of these patients develop steatohepatitis with an inherent risk for progression to hepatocarcinoma. Increased plasma LPS levels have been detected in the serum of patients with steatohepatitis. We hypothesized that, as a consequence of increased plasma LPS, ER stress could be induced and lead to NLRP3 inflammasome activation and hepatocyte death associated with steatohepatitis progression. In livers from obese mice, administration of LPS or tunicamycin results in IRE1α and PERK activation, leading to the overexpression of CHOP. This, in turn, activates the NLRP3 inflammasome, subsequently initiating hepatocyte pyroptosis (caspase-1, -11, interleukin-1β secretion) and apoptosis (caspase-3, BH3-only proteins). In contrast, the LPS challenge is blocked by the ER stress inhibitor TUDCA, resulting in: CHOP downregulation, reduced caspase-1, caspase-11, caspase-3 activities, lowered interleukin-1β secretion and rescue from cell death. The central role of CHOP in mediating the activation of proinflammatory caspases and cell death was characterized by performing knockdown experiments in primary mouse hepatocytes. Finally, the analysis of human steatohepatitis liver biopsies showed a correlation between the upregulation of inflammasome and ER stress markers, as well as liver injury. We demonstrate here that ER stress leads to hepatic NLRP3 inflammasome pyroptotic death, thus contributing as a novel mechanism of

  7. Macrophage activation by factors released from acetaminophen-injured hepatocytes: Potential role of HMGB1

    SciTech Connect

    Dragomir, Ana-Cristina; Laskin, Jeffrey D.; Laskin, Debra L.

    2011-06-15

    Toxic doses of acetaminophen (AA) cause hepatocellular necrosis. Evidence suggests that activated macrophages contribute to the pathogenic process; however, the factors that activate these cells are unknown. In these studies, we assessed the role of mediators released from AA-injured hepatocytes in macrophage activation. Treatment of macrophages with conditioned medium (CM) collected 24 hr after treatment of mouse hepatocytes with 5 mM AA (CM-AA) resulted in increased production of reactive oxygen species (ROS). Macrophage expression of heme oxygenase-1 (HO-1) and catalase mRNA was also upregulated by CM-AA, as well as cyclooxygenase (COX)-2 and 12/15-lipoxygenase (LOX). CM-AA also upregulated expression of the proinflammatory chemokines, MIP-1{alpha} and MIP-2. The effects of CM-AA on expression of COX-2, MIP-1{alpha} and MIP-2 were inhibited by blockade of p44/42 MAP kinase, suggesting a biochemical mechanism mediating macrophage activation. Hepatocytes injured by AA were found to release HMGB1, a potent macrophage activator. This was inhibited by pretreatment of hepatocytes with ethyl pyruvate (EP), which blocks HMGB1 release. EP also blocked CM-AA induced ROS production and antioxidant expression, and reduced expression of COX-2, but not MIP-1{alpha} or MIP-2. These findings suggest that HMGB1 released by AA-injured hepatocytes contributes to macrophage activation. This is supported by our observation that expression of the HMGB1 receptor RAGE is upregulated in macrophages in response to CM-AA. These data indicate that AA-injured hepatocytes contribute to the inflammatory environment in the liver through the release of mediators such as HMGB1. Blocking HMGB1/RAGE may be a useful approach to limiting classical macrophage activation and AA-induced hepatotoxicity. - Research Highlights: > These studies analyze macrophage activation by mediators released from acetaminophen-damaged hepatocytes. > Factors released from acetaminophen-injured hepatocytes induce

  8. Hormone-mediated growth dynamics of the barley pericarp as revealed by magnetic resonance imaging and transcript profiling

    PubMed Central

    Pielot, Rainer; Kohl, Stefan; Manz, Bertram; Rutten, Twan; Weier, Diana; Tarkowská, Danuše; Rolčík, Jakub; Strnad, Miroslav; Volke, Frank; Weber, Hans

    2015-01-01

    The shape of the maternal pericarp affects cereal grain mass and yield. Pericarp growth was analysed by magnetic resonance imaging (MRI), revealing topological maps of mobile water in developing pericarp of barley (Hordeum vulgare) and displaying tissue regions actively elongating in specific temporal–spatial patterns. Correlation analysis of MRI signals and growth rates reveals that growth in length is mediated by dorsal and also lateral rather than ventral regions. Growth in thickness is related to ventral regions. Switching from dorsal to ventral growth is associated with differential expression of axial regulators of the HD-ZipIII and Kanadi/Ettin types, and NPH3 photoreceptors, suggesting light-mediated auxin re-distribution. Auxin increases with the highest levels in the basal pericarp at 6 days after fertilization (DAF), together with transcriptionally up-regulated auxin transport and signalling. Gibberellin biosynthesis is transcriptionally up-regulated only later, and levels of bioactive gibberellins increase from 7 to 13 DAF, with higher levels in ventral than dorsal regions. Differential gene expression related to cell expansion indicates genes related to apoplast acidification, wall relaxation, sugar cleavage, water transport, and cell wall biosynthesis. Candidate genes potentially involved in pericarp extension are distinguished by their temporal expression, representing potential isoforms responsible for dorsal-mediated early growth in length or ventral-mediated late growth in thickness. PMID:26276866

  9. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    SciTech Connect

    Ilowski, Maren; Kleespies, Axel; Toni, Enrico N. de; Donabauer, Barbara; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang E.

    2011-01-07

    Research highlights: {yields} ALR decreases cytochrome c release from mitochondria. {yields} ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. {yields} ALR exerts a liver-specific anti-apoptotic effect. {yields} A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-{beta}, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  10. Eugenol-inhibited root growth in Avena fatua involves ROS-mediated oxidative damage.

    PubMed

    Ahuja, Nitina; Singh, Harminder Pal; Batish, Daizy Rani; Kohli, Ravinder Kumar

    2015-02-01

    Plant essential oils and their constituent monoterpenes are widely known plant growth retardants but their mechanism of action is not well understood. We explored the mechanism of phytotoxicity of eugenol, a monoterpenoid alcohol, proposed as a natural herbicide. Eugenol (100-1000 µM) retarded the germination of Avena fatua and strongly inhibited its root growth compared to the coleoptile growth. We further investigated the underlying physiological and biochemical alterations leading to the root growth inhibition. Eugenol induced the generation of reactive oxygen species (ROS) leading to oxidative stress and membrane damage in the root tissue. ROS generation measured in terms of hydrogen peroxide, superoxide anion and hydroxyl radical content increased significantly in the range of 24 to 144, 21 to 91, 46 to 173% over the control at 100 to 1000 µM eugenol, respectively. The disruption in membrane integrity was indicated by 25 to 125% increase in malondialdehyde (lipid peroxidation byproduct), and decreased conjugated diene content (~10 to 41%). The electrolyte leakage suggesting membrane damage increased both under light as well as dark conditions measured over a period from 0 to 30 h. In defense to the oxidative damage due to eugenol, a significant upregulation in the ROS-scavenging antioxidant enzyme machinery was observed. The activities of superoxide dismutases, catalases, ascorbate peroxidases, guaiacol peroxidases and glutathione reductases were elevated by ~1.5 to 2.8, 2 to 4.3, 1.9 to 5.0, 1.4 to 3.9, 2.5 to 5.5 times, respectively, in response to 100 to 1000 µM eugenol. The study concludes that eugenol inhibits early root growth through ROS-mediated oxidative damage, despite an activation of the antioxidant enzyme machinery. PMID:25752432

  11. Activated carbon decreases invasive plant growth by mediating plant–microbe interactions

    PubMed Central

    Nolan, Nicole E.; Kulmatiski, Andrew; Beard, Karen H.; Norton, Jeanette M.

    2015-01-01

    There is growing appreciation for the idea that plant–soil interactions (e.g. allelopathy and plant–microbe feedbacks) may explain the success of some non-native plants. Where this is the case, native plant restoration may require management tools that change plant–soil interactions. Activated carbon (AC) is one such potential tool. Previous research has shown the potential for high concentrations of AC to restore native plant growth to areas dominated by non-natives on a small scale (1 m × 1 m plots). Here we (i) test the efficacy of different AC concentrations at a larger scale (15 m × 15 m plots), (ii) measure microbial responses to AC treatment and (iii) use a greenhouse experiment to identify the primary mechanism, allelopathy versus microbial changes, through which AC impacts native and non-native plant growth. Three years after large-scale applications, AC treatments decreased non-native plant cover and increased the ratio of native to non-native species cover, particularly at concentrations >400 g m−2. Activated carbon similarly decreased non-native plant growth in the greenhouse. This effect, however, was only observed in live soils, suggesting that AC effects were microbially mediated and not caused by direct allelopathy. Bacterial community analysis of field soils indicated that AC increased the relative abundance of an unidentified bacterium and an Actinomycetales and decreased the relative abundance of a Flavobacterium, suggesting that these organisms may play a role in AC effects on plant growth. Results support the idea that manipulations of plant–microbe interactions may provide novel and effective ways of directing plant growth and community development (e.g. native plant restoration). PMID:25387751

  12. Activated carbon decreases invasive plant growth by mediating plant-microbe interactions.

    PubMed

    Nolan, Nicole E; Kulmatiski, Andrew; Beard, Karen H; Norton, Jeanette M

    2014-01-01

    There is growing appreciation for the idea that plant-soil interactions (e.g. allelopathy and plant-microbe feedbacks) may explain the success of some non-native plants. Where this is the case, native plant restoration may require management tools that change plant-soil interactions. Activated carbon (AC) is one such potential tool. Previous research has shown the potential for high concentrations of AC to restore native plant growth to areas dominated by non-natives on a small scale (1 m × 1 m plots). Here we (i) test the efficacy of different AC concentrations at a larger scale (15 m × 15 m plots), (ii) measure microbial responses to AC treatment and (iii) use a greenhouse experiment to identify the primary mechanism, allelopathy versus microbial changes, through which AC impacts native and non-native plant growth. Three years after large-scale applications, AC treatments decreased non-native plant cover and increased the ratio of native to non-native species cover, particularly at concentrations >400 g m(-2). Activated carbon similarly decreased non-native plant growth in the greenhouse. This effect, however, was only observed in live soils, suggesting that AC effects were microbially mediated and not caused by direct allelopathy. Bacterial community analysis of field soils indicated that AC increased the relative abundance of an unidentified bacterium and an Actinomycetales and decreased the relative abundance of a Flavobacterium, suggesting that these organisms may play a role in AC effects on plant growth. Results support the idea that manipulations of plant-microbe interactions may provide novel and effective ways of directing plant growth and community development (e.g. native plant restoration). PMID:25387751

  13. Disentangling direct and growth-mediated influences on early survival: a mechanistic approach.

    PubMed

    Plard, Floriane; Yoccoz, Nigel G; Bonenfant, Christophe; Klein, François; Warnant, Claude; Gaillard, Jean-Michel

    2015-09-01

    1. Early survival is a key life-history trait that often accounts for a large part of the variation in individual fitness and shapes population dynamics. The factors influencing early survival are multiple in large herbivores, including malnutrition, predation, cohort variation or maternal effects. However, the mechanistic pathways connecting these drivers to variation in early survival are much less studied. Indeed, whether these factors influence early survival directly or indirectly through early growth remains to be disentangled. 2. In this study, we used a path analysis to separate the direct and indirect (i.e. mediated by early growth) pathways through which sex, birth date, cohort and family effects influence early survival. We used a large data set of marked roe deer newborns collected from 1985 to 2010 in the intensively monitored population of Trois Fontaines (France). 3. We found that most drivers have indirect influences on early survival through early growth. Indeed, cohort effects influenced early survival through the indirect effect of precipitation around birth on early growth. Precipitation also had direct effects on early survival. Family effects indirectly influenced early survival. Twins from the same litter grew at about the same rate, so they had the same fate. Moreover, some factors, such as birth date, had both direct and indirect effects on roe deer early survival, with fawns born early in the season benefiting from high early survival both because they have more time to grow before the harsh season and because they grow faster during their first days of life than late-born fawns. 4. These findings suggest that most drivers of early survival previously identified in large mammalian herbivores may affect early survival primarily through their influence on early growth. Disentangling the direct and indirect pathways by which different factors influence early survival is of crucial importance to understand the mechanisms shaping this key

  14. Molecular-mediated crystal growth of PbTiO 3 nanostructure on silicon substrate

    NASA Astrophysics Data System (ADS)

    Chao, Chunying; Ren, Zhaohui; Liu, Zhenya; Xiao, Zhen; Xu, Gang; Li, Xiang; Wei, Xiao; Shen, Ge; Han, Gaorong

    2011-09-01

    A simple approach based on an organically modified sol-gel process has been developed to fabricate PbTiO3 (PT) nanocrystals on Si (1 0 0) substrate, where the amorphous powder modified by acetylacetone (acac) was used as precursor. After dropping the amorphous powder precursor prepared by freeze-drying process, PT nanocrystals on Si (1 0 0) substrate were obtained after heat treatment at 720 °C for 30 min in air. PT nanocrystals have been detected by XRD to be tetragonal perovskite structure. With the increase of acac/Pb molar ratio, the relative (1 0 0)/(0 0 1) diffraction peak intensity gradually increases, which probably suggested an oriented growth of PT nanocrystal along [1 0 0] on Si (1 0 0) substrates. In addition, Atomic force microscopy (AFM) results indicated that the height and the average lateral size of PT nanocrystal increased and then decreased as the acac/Pb molar ratio increased. Piezoelectric force microscopy (PFM) results demonstrated that all the samples show obvious piezoelectric activity. These results implied that the acetylacetone molecular mediated the growth of PT nanocrystals on Si (1 0 0) substrates possibly by the acac/Pb molar ratio. This simple method has been suggested to be attractive for tailoring an oriented growth of the nanostructures of perovskite oxide systems on Si substrates.

  15. Repression of multiple CYP2D genes in mouse primary hepatocytes with a single siRNA construct.

    PubMed

    Elraghy, Omaima; Baldwin, William S

    2015-01-01

    The Cyp2d subfamily is the second most abun-dant subfamily of hepatic drug-metabolizing CYPs. In mice, there are nine Cyp2d members that are believed to have redundant catalytic activity. We are testing and optimizing the ability of one short interfering RNA (siRNA) construct to knockdown the expression of multiple mouse Cyp2ds in primary hepatocytes. Expression of Cyp2d10, Cyp2d11, Cyp2d22, and Cyp2d26 was observed in the primary male mouse hepatocytes. Cyp2d9, which is male-specific and growth hormone-dependent, was not expressed in male primary hepatocytes, potentially because of its dependence on pulsatile growth hormone release from the anterior pituitary. Several different siRNAs at different concentrations and with different reagents were used to knockdown Cyp2d expression. siRNA constructs designed to repress only one construct often mildly repressed several Cyp2d isoforms. A construct designed to knockdown every Cyp2d isoform provided the best results, especially when incubated with transfection reagents designed specifically for primary cell culture. Interestingly, a construct designed to knockdown all Cyp2d isoforms, except Cyp2d10, caused a 2.5× increase in Cyp2d10 expression, presumably because of a compensatory response. However, while RNA expression is repressed 24 h after siRNA treatment, associated changes in Cyp2d-mediated metabolism are tenuous. Overall, this study provides data on the expression of murine Cyp2ds in primary cell lines, valuable information on designing siRNAs for silencing multiple murine CYPs, and potential pros and cons of using siRNA as a tool for repressing Cyp2d and estimating Cyp2d's role in murine xenobiotic metabolism. PMID:25124873

  16. PGE{sub 2}-induced colon cancer growth is mediated by mTORC1

    SciTech Connect

    Dufour, Marc Faes, Seraina Dormond-Meuwly, Anne Demartines, Nicolas Dormond, Olivier

    2014-09-05

    Highlights: • PGE{sub 2} activates mTORC1 in colon cancer cells. • Inhibition of mTORC1 blocks PGE{sub 2} induced colon cancer cell growth. • mTORC1 is a signaling intermediary in PGE{sub 2} induced colon cancer cell responses. - Abstract: The inflammatory prostaglandin E{sub 2} (PGE{sub 2}) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE{sub 2} directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE{sub 2}-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE{sub 2} increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE{sub 2} EP{sub 4} receptor was responsible for transducing the signal to mTORC1. Moreover, PGE{sub 2} increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE{sub 2}-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE{sub 2} increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE{sub 2} mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.

  17. Increased reprogramming of human fetal hepatocytes compared with adult hepatocytes in feeder-free conditions.

    PubMed

    Hansel, Marc C; Gramignoli, Roberto; Blake, William; Davila, Julio; Skvorak, Kristen; Dorko, Kenneth; Tahan, Veysel; Lee, Brian R; Tafaleng, Edgar; Guzman-Lepe, Jorge; Soto-Gutierrez, Alejandro; Fox, Ira J; Strom, Stephen C

    2014-01-01

    Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes, and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome, type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors, while fetal hepatocytes could be reprogrammed with three (OCT4, SOX2, NANOG) or four factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation. PMID:23394081

  18. Transforming growth factor-β: an important mediator in Helicobacter pylori-associated pathogenesis

    PubMed Central

    Li, Nianshuang; Xie, Chuan; Lu, Nong-Hua

    2015-01-01

    Helicobacter pylori (H.pylori) is a Gram-negative, microaerophilic, helical bacillus that specifically colonizes the gastric mucosa. The interaction of virulence factors, host genetic factors, and environmental factors contributes to the pathogenesis of H. pylori-associated conditions, such as atrophic gastritis and intestinal metaplasia. Infection with H. pylori has recently been recognized as the strongest risk factor for gastric cancer. As a pleiotropic cytokine, transforming growth factor (TGF)-β regulates various biological processes, including cell cycle, proliferation, apoptosis, and metastasis. Recent studies have shed new light on the involvement of TGF-β signaling in the pathogenesis of H. pylori infection. This review focuses on the potential etiological roles of TGF-β in H. pylori-mediated gastric pathogenesis. PMID:26583078

  19. Cathepsin L knockdown enhances curcumin-mediated inhibition of growth, migration, and invasion of glioma cells.

    PubMed

    Fei, Yao; Xiong, Yajie; Zhao, Yifan; Wang, Wenjuan; Han, Meilin; Wang, Long; Tan, Caihong; Liang, Zhongqin

    2016-09-01

    Curcumin can be used to prevent and treat cancer. However, its exact underlying molecular mechanisms remain poorly understood. Cathepsin L, a lysosomal cysteine protease, is overexpressed in several cancer types. This study aimed to determine the role of cathepsin L in curcumin-mediated inhibition of growth, migration, and invasion of glioma cells. Results revealed that the activity of cathepsin L was enhanced in curcumin-treated glioma cells. Cathepsin L knockdown induced by RNA interference significantly promoted curcumin-induced cytotoxicity, apoptosis, and cell cycle arrest. The knockdown also inhibited the migration and invasion of glioma cells. Our results suggested that the inhibition of cathepsin L can enhance the sensitivity of glioma cells to curcumin. Therefore, cathepsin L may be a new target to enhance the efficacy of curcumin against cancers. PMID:27373979

  20. The Role of Endogenous Epidermal Growth Factor Receptor Ligands in Mediating Corneal Epithelial Homeostasis

    PubMed Central

    Peterson, Joanne L.; Phelps, Eric D.; Doll, Mark A.; Schaal, Shlomit; Ceresa, Brian P.

    2014-01-01

    Purpose. To provide a comprehensive study of the biological role and therapeutic potential of six endogenous epidermal growth factor receptor (EGFR) ligands in corneal epithelial homeostasis. Methods. Kinetic analysis and dose response curves were performed by using in vitro and in vivo wound-healing assays. Biochemical assays were used to determine receptor expression and activity. Human tears were collected and quantitatively analyzed by multianalyte profiling for endogenous EGFR ligands. Results. Epidermal growth factor receptor ligands improved wound closure and activated EGFR, but betacellulin (BTC) was the most efficacious promoter of wound healing in vitro. In contrast, only epidermal growth factor (EGF) promoted wound healing in vivo. Human tears from 25 healthy individuals showed EGFR ligands at these average concentrations: EGF at 2053 ± 312.4 pg/mL, BTC at 207 ± 39.4 pg/mL, heparin-binding EGF at 44 ± 5.8 pg/mL, amphiregulin at 509 ± 28.8 pg/mL, transforming growth factor-α at 84 ± 19 pg/mL, and epiregulin at 52 ± 15 pg/mL. Conclusions. Under unwounded conditions, only EGF was present at concentrations near the ligand's Kd for the receptor, indicating it is the primary mediator of corneal epithelial homeostasis. Other ligands were present but at concentrations 11- to 7500-fold less their Kd, preventing significant ligand binding. Further, the high levels of EGF and its predicted binding preclude receptor occupancy by exogenous ligand and can explain the discrepancy between the in vitro and in vivo data. Therefore, therapeutic use of EGFR ligands may be unpredictable and impractical. PMID:24722692

  1. Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion.

    PubMed

    Zarember, Kol A; Sugui, Janyce A; Chang, Yun C; Kwon-Chung, Kyung J; Gallin, John I

    2007-05-15

    Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus. PMID:17475866

  2. Stromal inhibition of prostatic epithelial cell proliferation not mediated by transforming growth factor beta.

    PubMed Central

    Kooistra, A.; van den Eijnden-van Raaij, A. J.; Klaij, I. A.; Romijn, J. C.; Schröder, F. H.

    1995-01-01

    The paracrine influence of prostatic stroma on the proliferation of prostatic epithelial cells was investigated. Stromal cells from the human prostate have previously been shown to inhibit anchorage-dependent as well as anchorage-independent growth of the prostatic tumour epithelial cell lines PC-3 and LNCaP. Antiproliferative activity, mediated by a diffusible factor in the stromal cell conditioned medium, was found to be produced specifically by prostatic stromal cells. In the present study the characteristics of this factor were examined. It is demonstrated that prostate stroma-derived inhibiting factor is an acid- and heat-labile, dithiothreitol-sensitive protein. Although some similarities with type beta transforming growth factor (TGF-beta)-like inhibitors are apparent, evidence is presented that the factor is not identical to TGF-beta or to the TGF-beta-like factors activin and inhibin. Absence of TGF-beta activity was shown by the lack of inhibitory response of the TGF-beta-sensitive mink lung cell line CCL-64 to prostate stromal cell conditioned medium and to concentrated, partially purified preparations of the inhibitor. Furthermore, neutralising antibodies against TGF-beta 1 or TGF-beta 2 did not cause a decline in the level of PC-3 growth inhibition caused by partially purified inhibitor. Using Northern blot analyses, we excluded the involvement of inhibin or activin. It is concluded that the prostate stroma-derived factor may be a novel growth inhibitor different from any of the currently described inhibiting factors. Images Figure 5 PMID:7543773

  3. Computational comparison of mediated current generation capacity of Chlamydomonas reinhardtii in photosynthetic and respiratory growth modes.

    PubMed

    Mao, Longfei; Verwoerd, Wynand S

    2014-11-01

    Chlamydomonas reinhardtii possesses many potential advantages to be exploited as a biocatalyst in microbial fuel cells (MFCs) for electricity generation. In the present study, we performed computational studies based on flux balance analysis (FBA) to probe the maximum potential of C. reinhardtii for current output and identify the metabolic mechanisms supporting a high current generation in three different cultivation conditions, i.e., heterotrophic, photoautotrophic and mixotrophic growth. The results showed that flux balance limitations allow the highest current output for C. reinhardtii in the mixotrophic growth mode (2.368 A/gDW), followed by heterotrophic growth (1.141 A/gDW) and photoautotrophic growth the lowest (0.7035 A/gDW). The significantly higher mediated electron transfer (MET) rate in the mixotrophic mode is in complete contrast to previous findings for a photosynthetic cyanobacterium, and was attributed to the fact that for C. reinhardtii the photophosphorylation improved the efficiency of converting the acetate into biomass and NADH production. Overall, the cytosolic NADH-dependent current production was mainly associated with five reactions in both mixotrophic and photoautotrophic nutritional modes, whereas four reactions participated in the heterotrophic mode. The mixotrophic and photoautotrophic metabolisms were alike and shared the same set of reactions for maximizing current production, whereas in the heterotrophic mode, the current production was additionally contributed by the metabolic activities in the two organelles: glyoxysome and chloroplast. In conclusion, C. reinhardtii has a potential to be exploited in MFCs of MET mode to produce a high current output. PMID:24875305

  4. Polyethylene glycol-mediated colorectal cancer chemoprevention: roles of epidermal growth factor receptor and Snail.

    PubMed

    Wali, Ramesh K; Kunte, Dhananjay P; Koetsier, Jennifer L; Bissonnette, Marc; Roy, Hemant K

    2008-09-01

    Polyethylene glycol (PEG) is a clinically widely used agent with profound chemopreventive properties in experimental colon carcinogenesis. We reported previously that Snail/beta-catenin signaling may mediate the suppression of epithelial proliferation by PEG, although the upstream events remain unclear. We report herein the role of epidermal growth factor receptor (EGFR), a known mediator of Snail and overexpressed in approximately 80% of human colorectal cancers, on PEG-mediated antiproliferative and hence antineoplastic effects in azoxymethane (AOM) rats and HT-29 colon cancer cells. AOM rats were randomized to either standard diet or one with 10% PEG-3350 and euthanized 8 weeks later. The colonic samples were subjected to immunohistochemical or Western blot analyses. PEG decreased mucosal EGFR by 60% (P < 0.001). Similar PEG effects were obtained in HT-29 cells. PEG suppressed EGFR protein via lysosmal degradation with no change in mRNA levels. To show that EGFR antagonism per se was responsible for the antiproliferative effect, we inhibited EGFR by either pretreating cells with gefitinib or stably transfecting with EGFR-short hairpin RNA and measured the effect of PEG on proliferation. In either case, PEG effect was blunted, suggesting a vital role of EGFR. Flow cytometric analysis revealed that EGFR-short hairpin RNA cells, besides having reduced membrane EGFR, also expressed low Snail levels (40%), corroborating a strong association. Furthermore, in EGFR silenced cells, PEG effect on EGFR or Snail was muted, similar to that on proliferation. In conclusion, we show that EGFR is the proximate membrane signaling molecule through which PEG initiates antiproliferative activity with Snail/beta-catenin pathway playing the central intermediary function. PMID:18790788

  5. External-beam radiotherapy as preparative regimen for hepatocyte transplantation after partial hepatectomy

    SciTech Connect

    Christiansen, Hans . E-mail: hchrist@gwdg.de; Koenig, Sarah; Krause, Petra; Hermann, Robert Michael; Rave-Frank, Margret; Proehl, Thomas; Becker, Heinz; Hess, Clemens Friedrich; Schmidberger, Heinz

    2006-06-01

    Purpose: The transplantation of donor hepatocytes is considered a promising option to correct chronic liver failure through repopulation of the diseased organ. This study describes a novel selective external-beam irradiation technique as a preparative regimen for hepatocyte transplantation. Methods and Materials: Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with external-beam single-dose irradiation (25 Gy) delivered to two thirds of the liver. Four days later, a one-third partial hepatectomy (PH) was performed to resect the untreated liver section, and 15 million wild-type (DPPIV{sup +}) hepatocytes were transplanted via the spleen into the recipient livers. The degree of donor-cell integration and growth was studied 8 h, 3 days, and 5 and 12 weeks after transplantation. Results: Transplanted hepatocytes integrated rapidly into the irradiated liver and proliferated as clusters, finally repopulating the host liver to approximately 20% hepatocyte mass. After 12 weeks, donor cells and their numerous descendents were fully integrated and expressed functional markers to the same extent as host hepatocytes. Conclusions: We demonstrate that external-beam liver irradiation is sufficient to achieve partial repopulation of the host liver after hepatocyte transplantation, under the additional stimulus of one-third PH. The method described has potentially good prospects for its application in a clinically viable form of treatment.

  6. CHIP-mediated degradation of transglutaminase 2 negatively regulates tumor growth and angiogenesis in renal cancer.

    PubMed

    Min, B; Park, H; Lee, S; Li, Y; Choi, J-M; Lee, J Y; Kim, J; Choi, Y D; Kwon, Y-G; Lee, H-W; Bae, S-C; Yun, C-O; Chung, K C

    2016-07-14

    The multifunctional enzyme transglutaminase 2 (TG2) primarily catalyzes cross-linking reactions of proteins via (γ-glutamyl) lysine bonds. Several recent findings indicate that altered regulation of intracellular TG2 levels affects renal cancer. Elevated TG2 expression is observed in renal cancer. However, the molecular mechanism underlying TG2 degradation is not completely understood. Carboxyl-terminus of Hsp70-interacting protein (CHIP) functions as an ubiquitin E3 ligase. Previous studies reveal that CHIP deficiency mice displayed a reduced life span with accelerated aging in kidney tissues. Here we show that CHIP promotes polyubiquitination of TG2 and its subsequent proteasomal degradation. In addition, TG2 upregulation contributes to enhanced kidney tumorigenesis. Furthermore, CHIP-mediated TG2 downregulation is critical for the suppression of kidney tumor growth and angiogenesis. Notably, our findings are further supported by decreased CHIP expression in human renal cancer tissues and renal cancer cells. The present work reveals that CHIP-mediated TG2 ubiquitination and proteasomal degradation represent a novel regulatory mechanism that controls intracellular TG2 levels. Alterations in this pathway result in TG2 hyperexpression and consequently contribute to renal cancer. PMID:26568304

  7. Targeting Six1 by lentivirus-mediated RNA interference inhibits colorectal cancer cell growth and invasion

    PubMed Central

    Li, Zhaoming; Tian, Tian; Hu, Xiaopeng; Zhang, Xudong; Li, Lifeng; Nan, Feifei; Chang, Yu; Wang, Xinhua; Sun, Zhenchang; Lv, Feng; Zhang, Mingzhi

    2014-01-01

    The Six1 homeodomain protein is a developmental transcription factor that has been implicated in tumor onset and progression. Recently, it’s reported that overexpression of Six1 is sufficient to induce epithelial-to-mesenchymal transition (EMT) and metastasis of colorectal cancer. Moreover, its expression is significantly associated with poorer overall survival probability in advanced-stage colorectal cancer. To address whether Six1 could serve as a therapeutic target for human colorectal cancer, we used a lentivirus-mediated short hairpin RNA (shRNA) gene knockdown method to suppress the expression of Six1 in colorectal cancer cells. We showed that lentivirusmediated shRNA targeted to Six1 gene efficiently reduced its expression in colorectal cancer cells at both mRNA and protein levels. In vitro functional assays revealed that knockdown of Six1 significantly suppressed cell proliferation, and inhibited cell migration and invasion of colorectal cancer cells. Furthermore, tumor xenograft model demonstrated that downregulation of Six1 dramatically inhibited colorectal cancer growth in vivo. In conclusion, these findings suggest that lentivirus-mediated Six1 inhibition may represent a novel therapeutic approach for treatment of colorectal cancer. PMID:24551283

  8. Tumor necrosis factor primes hepatocytes for DNA replication in the rat.

    PubMed

    Webber, E M; Bruix, J; Pierce, R H; Fausto, N

    1998-11-01

    Signaling through tumor necrosis factor receptor type 1 (TNFR-1) using a pathway that involves nuclear factor kappaB (NF-kappaB), interleukin-6 (IL-6), and STAT3 is required for the initiation of liver regeneration. We have proposed that TNF primes hepatocytes to respond to the mitogenic effect of growth factors, but so far, there has been no experimental demonstration that TNF enhances growth factor responses of hepatocytes. To test this hypothesis, we infused hepatocyte growth factor (HGF) and transforming growth factor (TGF-) (40 microgram/24 h) directly into the portal vein of rats for 24 hours using osmotic pumps and determined whether TNF injection (5 microgram per rat) would significantly increase hepatocyte DNA labeling in these animals. All rats received 5-bromo-2'-deoxyuridine (BrdU) by intraperitoneal delivery during a 48-hour period (i.e., BrdU infusion continued for 24 hours after the end of growth factor administration). BrdU labeling in the liver was measured by both immunohistochemistry and flow cytometry, and the results obtained by these methods showed excellent concordance. The results demonstrate that TNF transiently activates NF-kappaB and STAT3 and increases the proliferative response of hepatocytes to HGF or TGF- by fourfold. Priming effects on hepatocyte DNA replication were also obtained with injection of lipopolysaccharide (LPS) and gadolinium chloride (GdCl3), agents that release TNF in the liver. Similarly to TNF, GdCl3 injection caused the activation of NF-kappaB and STAT3, reaching a maximum 8 to 12 hours after the injection. The results show that TNF acts as a primer to sensitize hepatocytes to the proliferative effects of growth factors and offers a mechanism to explain the initiation and progression phases of liver regeneration after partial hepatectomy (PH). PMID:9794905

  9. Desacetyl nimbinene inhibits breast cancer growth and metastasis through reactive oxygen species mediated mechanisms.

    PubMed

    Arumugam, Arunkumar; Subramani, Ramadevi; Nandy, Sushmita; Powell, Sara; Velazquez, Marissa; Orozco, Alexis; Galvez, Adriana; Lakshmanaswamy, Rajkumar

    2016-05-01

    Accumulation of reactive oxygen species (ROS) has been implicated in induction of apoptosis and regulation of key signaling molecules in cancer cells. Phytochemicals are potent source of anticancer drugs as wells as potential inducers of ROS. Neem (Azadirachta indica) is a medicinal plant used for the treatment of various diseases. The main objective of this study is to investigate the anticancer effect of desacetyl nimbinene (DAN; an active ingredient of neem) against breast cancer. Normal and breast cancer cell lines were used for the study. The effect of DAN on cell proliferation, apoptosis, ROS generation, migration, and invasion was analyzed. Antioxidant enzymes superoxide dismutase (SOD)1 and SOD2 were overexpressed to test the effect of DAN-induced ROS generation on breast cancer growth. Key survival and apoptotic protein markers were analyzed to validate the anticancer effect of DAN. Our data demonstrated that DAN inhibited the growth of breast cancer cells by inducing ROS generation. Further investigations revealed that DAN treatment lead to the loss of mitochondrial membrane potential resulting in mitochondria-dependent apoptotic cell death. Increased phosphorylation of c-Jun-N-terminal kinase (JNK) and reduced phosphorylation of p38 were also observed in response to DAN treatment. Inhibition of ROS production by overexpressing antioxidant enzymes SOD1 and SOD2 reduced the DAN-induced cytotoxicity. Additionally, DAN significantly inhibited migration and invasion of MDA-MB-231 breast cancer cells. Overall, our data suggest that DAN exerts its anticancer effect on breast cancer by induction of mitochondria-mediated apoptosis mediated by ROS accumulation. PMID:26637227

  10. The use of primary rat hepatocytes to achieve metabolic activation of promutagens in the Chinese hamster ovary/hypoxantine-guanine phosphoribosyl transferase mutational assay

    SciTech Connect

    Bermudez, E.; Couch, D.B.; Tillery, D.

    1982-01-01

    A method is described in which primary rat hepatocytes have been cocultured with chinese hamster ovary (CHO) cells to provide metabolic activation of promutgens in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) mutational assay. Single cell hepatocyte suspensions were prepared from male Fisher-344 rats using the in situ collagenase perfusion technique. Hepatocytes were allowed to attach for 1.5 hours in tissue culture dishes containing an approximately equal number of CHO cells in log growth. The cocultures were exposed to promutagens for up to 20 hours in serum-free medium. The survival and 6-thioguanine-resistant fraction of treated CHO cells were then determined as in the standard CHO/HGPRT assay. Aflatoxin B/sub 1/ (AFB/sub 1/) 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B(a)P) were found to produce increases in the mutant fractions of treated CHO cells as a function of concentration. The time required for optimum expression of the mutant phenotype following exposure to DMBA and AFB/sub 1/ was approximately 8 days. Primary cell-mediated mutagenesis may be useful in elucidating methobolic pathways important in the production and detoxification of genotoxic products in vivo.

  11. Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes

    SciTech Connect

    Xue, Tao; Luo, Peihua; Zhu, Hong; Zhao, Yuqin; Wu, Honghai; Gai, Renhua; Wu, Youping; Yang, Bo; Yang, Xiaochun; He, Qiaojun

    2012-06-15

    Dasatinib, a multitargeted inhibitor of BCR–ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague–Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 and cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity. -- Highlights: ►Dasatinib shows potential hepatotoxicity both in vitro and in vivo. ►Apoptosis plays a vital role in Dasatinib

  12. The immobilization of hepatocytes on 24 nm-sized gold colloid for enhanced hepatocytes proliferation.

    PubMed

    Gu, Hai-Ying; Chen, Zhong; Sa, Rong-Xiao; Yuan, Su-Su; Chen, Hong-Yuan; Ding, Yi-Tao; Yu, Ai-Min

    2004-08-01

    Bioartificial liver and hepatocyte transplantation is anticipated to supply a temporary metabolic support for candidates of liver transplantation or for patients with fulminant liver failure. An essential restriction of this form is the inability to acquire an enough amount of hepatocytes. Enhancement of the proliferation and differentiated function of hepatocytes is becoming a pursued target. Here, porcine hepatocytes were successfully immobilized on nano-sized gold colloid particles to construct a "hepatocyte/gold colloid" interface at which hepatocytes can be quickly proliferated. The properties of this resulting interface were characterized and confirmed by scanning electron microscopy and atomic force microscopy. The proliferative mechanism of hepatocytes was also discussed. The proliferated hepatocytes could be applied to the clinic based on their excellent functions for the synthesis of protein, glucose and urea as well as lower lactate dehydrogenase release. PMID:15020118

  13. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    SciTech Connect

    Nagata, Yosuke Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

  14. Growth suppression of Leydig TM3 cells mediated by aryl hydrocarbon receptor

    SciTech Connect

    Iseki, Minoru; Ikuta, Togo; Kobayashi, Tetsuya; Kawajiri, Kaname . E-mail: kawajiri@cancer-c.pref.saitama.jp

    2005-06-17

    Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental toxicity in reproductive organs. To elucidate the function of AhR, we generated stable transformants of TM3 cells overexpressing wild-type aryl hydrocarbon receptor (AhR) or its mutants which carried mutations in nuclear localization signal or nuclear export signal. In the presence of 3-methylcholanthrene (MC), proliferation of the cells transfected with wild-type AhR was completely suppressed, whereas cells expressing AhR mutants proliferated in a manner equivalent to control TM3 cells, suggesting AhR-dependent growth inhibition. The suppression was associated with up-regulation of cyclin-dependent kinase inhibitor p21{sup Cip1}, which was abolished by pretreatment with actinomycin D. A p38 MAPK specific inhibitor, SB203580, blocked the increase of p21{sup Cip1} mRNA in response to MC. Treatment with indigo, another AhR ligand, failed to increase of p21{sup Cip1} mRNA, although up-regulation of mRNA for CYP1A1 was observed. These data suggest AhR in Leydig cells mediates growth inhibition by inducing p21{sup Cip1}.

  15. TGF-β1 mediates the hypertrophic cardiomyocyte growth induced by angiotensin II

    PubMed Central

    Schultz, Jo El J.; Witt, Sandra A.; Glascock, Betty J.; Nieman, Michelle L.; Reiser, Peter J.; Nix, Stacey L.; Kimball, Thomas R.; Doetschman, Thomas

    2002-01-01

    Angiotensin II (Ang II), a potent hypertrophic stimulus, causes significant increases in TGFb1 gene expression. However, it is not known whether there is a causal relationship between increased levels of TGF-β1 and cardiac hypertrophy. Echocardiographic analysis revealed that TGF-β1–deficient mice subjected to chronic subpressor doses of Ang II had no significant change in left ventricular (LV) mass and percent fractional shortening during Ang IItreatment. In contrast, Ang II–treated wild-type mice showed a >20% increase in LV mass and impaired cardiac function. Cardiomyocyte cross-sectional area was also markedly increased in Ang II–treated wild-type mice but unchanged in Ang II–treated TGF-β1–deficient mice. No significant levels of fibrosis, mitotic growth, or cytokine infiltration were detected in Ang II–treated mice. Atrial natriuretic factor expression was ∼6-fold elevated in Ang II–treated wild-type, but not TGF-β1–deficient mice. However, the α- to β-myosin heavy chain switch did not occur in Ang II–treated mice, indicating that isoform switching is not obligatorily coupled with hypertrophy or TGF-β1. The Ang IIeffect on hypertrophy was shown not to result from stimulation of the endogenous renin-angiotensis system. These results indicate that TGF-β1 is an important mediator of the hypertrophic growth response of the heart to Ang II. PMID:11901187

  16. Transgenic expression in the liver of truncated Met blocks apoptosis and permits immortalization of hepatocytes.

    PubMed Central

    Amicone, L; Spagnoli, F M; Späth, G; Giordano, S; Tommasini, C; Bernardini, S; De Luca, V; Della Rocca, C; Weiss, M C; Comoglio, P M; Tripodi, M

    1997-01-01

    Hepatocyte growth factor induces proliferation, motility and differentiation of epithelial cells through the tyrosine kinase receptor encoded by the MET protooncogene. The cytoplasmic portion of Met (referred to as cyto-Met) is activated but only weakly transforming. In order to determine the effect of activated Met on hepatocytes, we have targeted truncated Met expression to the liver by incorporating the cDNA into a vector carrying the entire human alpha-1-antitrypsin transcriptional unit. Transgenic expression in the liver of truncated human Met, containing the regulatory and the catalytic cytoplasmic domains, renders hepatocytes constitutively resistant to apoptosis and reproducibly permits immortalization. The emerging stable cell lines are not transformed and maintain a highly differentiated phenotype judged by the retention of epithelial cell polarity and the expression of hepatocyte-enriched transcription factors as well as hepatic products. PMID:9034332

  17. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.

    PubMed Central

    Overturf, K.; al-Dhalimy, M.; Ou, C. N.; Finegold, M.; Grompe, M.

    1997-01-01

    Previous work has shown that adult mouse hepatocytes can divide at least 18 times in vivo. To test whether this represents the upper limit of their regenerative capacity, we performed serial transplantation of hepatocytes in the fumarylacetoacetate hydrolase deficiency murine model of liver repopulation. Hepatocytes from adult donors were serially transplanted in limiting numbers six times and resulted in complete repopulation during each cycle. This corresponds to a minimal number of 69 cell doublings or a 7.3 x 10(20)-fold expansion. No evidence for abnormal liver function or altered hepatic architecture was found in repopulated animals. We conclude that a fraction of adult mouse hepatocytes have growth potential similar to that of hematopoietic stem cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9358753

  18. Parasite-induced ER stress response in hepatocytes facilitates Plasmodium liver stage infection.

    PubMed

    Inácio, Patricia; Zuzarte-Luís, Vanessa; Ruivo, Margarida T G; Falkard, Brie; Nagaraj, Nagarjuna; Rooijers, Koos; Mann, Matthias; Mair, Gunnar; Fidock, David A; Mota, Maria M

    2015-08-01

    Upon infection of a mammalian host, Plasmodium parasites first replicate inside hepatocytes, generating thousands of new parasites. Although Plasmodium intra-hepatic development represents a substantial metabolic challenge to the host hepatocyte, how infected cells respond to and integrate this stress remains poorly understood. Here, we present proteomic and transcriptomic analyses, revealing that the endoplasmic reticulum (ER)-resident unfolded protein response (UPR) is activated in host hepatocytes upon Plasmodium berghei infection. The expression of XBP1s--the active form of the UPR mediator XBP1--and the liver-specific UPR mediator CREBH is induced by P. berghei infection in vivo. Furthermore, this UPR induction increases parasite liver burden. Altogether, our data suggest that ER stress is a central feature of P. berghei intra-hepatic development, contributing to the success of infection. PMID:26113366

  19. Parasite-induced ER stress response in hepatocytes facilitates Plasmodium liver stage infection

    PubMed Central

    Inácio, Patricia; Zuzarte-Luís, Vanessa; Ruivo, Margarida TG; Falkard, Brie; Nagaraj, Nagarjuna; Rooijers, Koos; Mann, Matthias; Mair, Gunnar; Fidock, David A; Mota, Maria M

    2015-01-01

    Upon infection of a mammalian host, Plasmodium parasites first replicate inside hepatocytes, generating thousands of new parasites. Although Plasmodium intra-hepatic development represents a substantial metabolic challenge to the host hepatocyte, how infected cells respond to and integrate this stress remains poorly understood. Here, we present proteomic and transcriptomic analyses, revealing that the endoplasmic reticulum (ER)-resident unfolded protein response (UPR) is activated in host hepatocytes upon Plasmodium berghei infection. The expression of XBP1s—the active form of the UPR mediator XBP1—and the liver-specific UPR mediator CREBH is induced by P. berghei infection in vivo. Furthermore, this UPR induction increases parasite liver burden. Altogether, our data suggest that ER stress is a central feature of P. berghei intra-hepatic development, contributing to the success of infection. PMID:26113366

  20. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS.

    PubMed

    Momen-Heravi, Fatemeh; Bala, Shashi; Kodys, Karen; Szabo, Gyongyi

    2015-01-01

    Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS. PMID:25973575

  1. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS

    PubMed Central

    Momen-Heravi, Fatemeh; Bala, Shashi; Kodys, Karen; Szabo, Gyongyi

    2015-01-01

    Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS. PMID:25973575

  2. A Structural Model of Parental Alcoholism, Family Functioning, and Psychological Health: The Mediating Effects of Hardiness and Personal Growth Orientation.

    ERIC Educational Resources Information Center

    Robitschek, Christine; Kashubeck, Susan

    1999-01-01

    This study sought to: (a) determine whether personal-growth orientation and hardiness mediated the relations of parental alcoholism and family functioning to psychological well-being and distress; (b) determine whether this model was invariant across men and women; and (c) examine the role of parental alcoholism in a model that included family…

  3. LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.

    PubMed

    Homolya, László; Fu, Dong; Sengupta, Prabuddha; Jarnik, Michal; Gillet, Jean-Pierre; Vitale-Cross, Lynn; Gutkind, J Silvio; Lippincott-Schwartz, Jennifer; Arias, Irwin M

    2014-01-01

    Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation. PMID:24643070

  4. LKB1/AMPK and PKA Control ABCB11 Trafficking and Polarization in Hepatocytes

    PubMed Central

    Homolya, László; Fu, Dong; Sengupta, Prabuddha; Jarnik, Michal; Gillet, Jean-Pierre; Vitale-Cross, Lynn; Gutkind, J. Silvio; Lippincott-Schwartz, Jennifer; Arias, Irwin M.

    2014-01-01

    Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation. PMID:24643070

  5. Hepatocyte-specific deletion of hepatocyte nuclear factor-4α in adult mice results in increased hepatocyte proliferation

    PubMed Central

    Walesky, Chad; Gunewardena, Sumedha; Terwilliger, Ernest F.; Edwards, Genea; Borude, Prachi

    2013-01-01

    Hepatocyte nuclear factor-4α (HNF4α) is known as the master regulator of hepatocyte differentiation. Recent studies indicate that HNF4α may inhibit hepatocyte proliferation via mechanisms that have yet to be identified. Using a HNF4α knockdown mouse model based on delivery of inducible Cre recombinase via an adeno-associated virus 8 viral vector, we investigated the role of HNF4α in the regulation of hepatocyte proliferation. Hepatocyte-specific deletion of HNF4α resulted in increased hepatocyte proliferation. Global gene expression analysis showed that a majority of the downregulated genes were previously known HNF4α target genes involved in hepatic differentiation. Interestingly, ≥500 upregulated genes were associated with cell proliferation and cancer. Furthermore, we identified potential negative target genes of HNF4α, many of which are involved in the stimulation of proliferation. Using chromatin immunoprecipitation analysis, we confirmed binding of HNF4α at three of these genes. Furthermore, overexpression of HNF4α in mouse hepatocellular carcinoma cells resulted in a decrease in promitogenic gene expression and cell cycle arrest. Taken together, these data indicate that, apart from its role in hepatocyte differentiation, HNF4α actively inhibits hepatocyte proliferation by repression of specific promitogenic genes. PMID:23104559

  6. Redox-mediated activation of latent transforming growth factor-beta 1

    NASA Technical Reports Server (NTRS)

    Barcellos-Hoff, M. H.; Dix, T. A.; Chatterjee, A. (Principal Investigator)

    1996-01-01

    Transforming growth factor beta 1 (TGF beta) is a multifunctional cytokine that orchestrates response to injury via ubiquitous cell surface receptors. The biological activity of TGF beta is restrained by its secretion as a latent complex (LTGF beta) such that activation determines the extent of TGF beta activity during physiological and pathological events. TGF beta action has been implicated in a variety of reactive oxygen-mediated tissue processes, particularly inflammation, and in pathologies such as reperfusion injury, rheumatoid arthritis, and atherosclerosis. It was recently shown to be rapidly activated after in vivo radiation exposure, which also generates reactive oxygen species (ROS). In the present studies, the potential for redox-mediated LTGF beta activation was investigated using a cell-free system in which ROS were generated in solution by ionizing radiation or metal ion-catalyzed ascorbate reaction. Irradiation (100 Gray) of recombinant human LTGF beta in solution induced 26% activation compared with that elicited by standard thermal activation. Metal-catalyzed ascorbate oxidation elicited extremely efficient recombinant LTGF beta activation that matched or exceeded thermal activation. The efficiency of ascorbate activation depended on ascorbate concentrations and the presence of transition metal ions. We postulate that oxidation of specific amino acids in the latency-conferring peptide leads to a conformation change in the latent complex that allows release of TGF beta. Oxidative activation offers a novel route for the involvement of TGF beta in tissue processes in which ROS are implicated and endows LTGF beta with the ability to act as a sensor of oxidative stress and, by releasing TGF beta, to function as a signal for orchestrating the response of multiple cell types. LTGF beta redox sensitivity is presumably directed toward recovery of homeostasis; however, oxidation may also be a mechanism of LTGF beta activation that can be deleterious during

  7. Redox-mediated activation of latent transforming growth factor-beta 1.

    PubMed

    Barcellos-Hoff, M H; Dix, T A

    1996-09-01

    Transforming growth factor beta 1 (TGF beta) is a multifunctional cytokine that orchestrates response to injury via ubiquitous cell surface receptors. The biological activity of TGF beta is restrained by its secretion as a latent complex (LTGF beta) such that activation determines the extent of TGF beta activity during physiological and pathological events. TGF beta action has been implicated in a variety of reactive oxygen-mediated tissue processes, particularly inflammation, and in pathologies such as reperfusion injury, rheumatoid arthritis, and atherosclerosis. It was recently shown to be rapidly activated after in vivo radiation exposure, which also generates reactive oxygen species (ROS). In the present studies, the potential for redox-mediated LTGF beta activation was investigated using a cell-free system in which ROS were generated in solution by ionizing radiation or metal ion-catalyzed ascorbate reaction. Irradiation (100 Gray) of recombinant human LTGF beta in solution induced 26% activation compared with that elicited by standard thermal activation. Metal-catalyzed ascorbate oxidation elicited extremely efficient recombinant LTGF beta activation that matched or exceeded thermal activation. The efficiency of ascorbate activation depended on ascorbate concentrations and the presence of transition metal ions. We postulate that oxidation of specific amino acids in the latency-conferring peptide leads to a conformation change in the latent complex that allows release of TGF beta. Oxidative activation offers a novel route for the involvement of TGF beta in tissue processes in which ROS are implicated and endows LTGF beta with the ability to act as a sensor of oxidative stress and, by releasing TGF beta, to function as a signal for orchestrating the response of multiple cell types. LTGF beta redox sensitivity is presumably directed toward recovery of homeostasis; however, oxidation may also be a mechanism of LTGF beta activation that can be deleterious during

  8. Ferritin-stimulated lipid peroxidation, lysosomal leak, and macroautophagy promote lysosomal "metastability" in primary hepatocytes determining in vitro cell survival.

    PubMed

    Krenn, Margit A; Schürz, Melanie; Teufl, Bernhard; Uchida, Koji; Eckl, Peter M; Bresgen, Nikolaus

    2015-03-01

    Several pathologies are associated with elevated levels of serum ferritin, for which growth inhibitory properties have been reported; however, the underlying mechanisms are still poorly defined. Previously we have described cytotoxic properties of isoferritins released from primary hepatocytes in vitro, which induce apoptosis in an iron and oxidative stress-dependent mode. Here we show that this ferritin species stimulates endosome clustering and giant endosome formation in primary hepatocytes accompanied by enhanced lysosomal membrane permeability (LMP). In parallel, protein modification by lipid peroxidation-derived 4-hydroxynonenal (HNE) is strongly promoted by ferritin, the HNE-modified proteins (HNE-P) showing remarkable aggregation. Emphasizing the prooxidant context, GSH is rapidly depleted and the GSH/GSSG ratio is substantially declining in ferritin-treated cells. Furthermore, ferritin triggers a transient upregulation of macroautophagy which is abolished by iron chelation and apparently supports HNE-P clearance. Macroautophagy inhibition by 3-methyladenine strongly amplifies ferritin cytotoxicity in a time- and concentration-dependent mode, suggesting an important role of macroautophagy on cellular responses to ferritin endocytosis. Moreover, pointing at an involvement of lysosomal proteolysis, ferritin cytotoxicity and lysosome fragility are aggravated by the protease inhibitor leupeptin. In contrast, EGF which suppresses ferritin-induced cell death attenuates ferritin-mediated LMP. In conclusion, we propose that HNE-P accumulation, lysosome dysfunction, and macroautophagy stimulated by ferritin endocytosis provoke lysosomal "metastability" in primary hepatocytes which permits cell survival as long as in- and extrinsic determinants (e.g., antioxidant availability, damage repair, EGF signaling) keep the degree of lysosomal destabilization below cell death-inducing thresholds. PMID:25532933

  9. Copper-ion-assisted growth of gold nanorods in seed-mediated growth: significant narrowing of size distribution via tailoring reactivity of seeds.

    PubMed

    Wen, Tao; Hu, Zhijian; Liu, Wenqi; Zhang, Hui; Hou, Shuai; Hu, Xiaona; Wu, Xiaochun

    2012-12-18

    In the well-developed seed-mediated growth of gold nanorods (GNRs), adding the proper amount of Cu(2+) ions in the growth solution leads to significant narrowing in the size distribution of the resultant GNRs, especially for those with shorter aspect ratios (corresponding longitudinal surface plasmon resonance (LSPR) peaks shorter than 750 nm). Cu(2+) ions were found to be able to catalyze the oxidative etching of gold seeds by oxygen, thus mediating subsequent growth kinetics of the GNRs. At proper Cu(2+) concentrations, the size distribution of the original seeds is greatly narrowed via oxidative etching. The etched seeds are highly reactive and grow quickly into desired GNRs with significantly improved size distribution. A similar mechanism can be employed to tune the end cap of the GNRs. Except for copper ions, no observable catalytic effect is observed from other cations presumably due to their lower affinity to oxygen. Considering the widespread use of seed-mediated growth in the morphology-controlled synthesis of noble metal nanostructures, the tailoring in seed reactivity we presented herein could be extended to other systems. PMID:23173599

  10. Effects of acetaldehyde on hepatocyte glycerol uptake and cell size: implication of Aquaporin 9

    PubMed Central

    Potter, James J.; Koteish, Ayman; Hamilton, James; Liu, Xiaopu; Liu, Kun; Agre, Peter; Mezey, Esteban

    2010-01-01

    Background The effects of ethanol and acetaldehyde on uptake of glycerol and on cell size of hepatocytes and a role Aquaporin 9 (AQP9), a glycerol transport channel, were evaluated. Methods The studies were done in primary rat and mouse hepatocytes. The uptake of [14C] glycerol was determined with hepatocytes in suspension. For determination of cell size, rat hepatocytes on coated dishes were incubated with a lipophilic fluorochrome that is incorporated into the cell membrane and examined by confocal microscopy. A three dimensional z scan of the cell was performed, and the middle slice of the z scan was used for area measurements. Results Acute exposure to acetaldehyde, but not to ethanol, causes a rapid increase in the uptake of glycerol and an increase in hepatocyte size, which was inhibited by HgCl2, an inhibitor of aquaporins. This was not observed in hepatocytes from AQP9 knockout mice, nor observed by direct application of acetaldehyde to AQP9 expressed in Xenopus Laevis oocytes. Prolonged 24 hours exposure to either acetaldehyde or ethanol did not result in an increase in glycerol uptake by rat hepatocytes. Acetaldehyde decreased AQP9 mRNA and AQP9 protein, while ethanol decreased AQP9 mRNA but not AQP9 protein. Ethanol, but not acetaldehyde, increased the activities of glycerol kinase and phosphoenolpyruvate carboxykinase. Conclusions The acute effects of acetaldehyde, while mediated by AQP9, are probably influenced by binding of acetaldehyde to hepatocyte membranes and changes in cell permeability. The effects of ethanol in enhancing glucose kinase, and phosphoenolpyruvate carboxykinase leading to increased formation of glycerol-3-phosphate most likely contribute to alcoholic fatty liver. PMID:21294757

  11. Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

    SciTech Connect

    Woolbright, Benjamin L.; Dorko, Kenneth; Antoine, Daniel J.; Clarke, Joanna I.; Gholami, Parviz; Li, Feng; Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson; Fan, Fang; Jenkins, Rosalind E.; Park, B. Kevin; Hagenbuch, Bruno; Olyaee, Mojtaba; Jaeschke, Hartmut

    2015-03-15

    Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury

  12. Subtoxic Alterations in Hepatocyte-Derived Exosomes: An Early Step in Drug-Induced Liver Injury?

    PubMed

    Holman, Natalie S; Mosedale, Merrie; Wolf, Kristina K; LeCluyse, Edward L; Watkins, Paul B

    2016-06-01

    Drug-induced liver injury (DILI) is a significant clinical and economic problem in the United States, yet the mechanisms that underlie DILI remain poorly understood. Recent evidence suggests that signaling molecules released by stressed hepatocytes can trigger immune responses that may be common across DILI mechanisms. Extracellular vesicles released by hepatocytes, principally hepatocyte-derived exosomes (HDEs), may constitute one such signal. To examine HDE alterations as a function of drug-induced stress, this work utilized prototypical hepatotoxicant acetaminophen (APAP) in male Sprague-Dawley (SD) rats, SD rat hepatocytes, and primary human hepatocytes. HDE were isolated using ExoQuick precipitation reagent and analyzed by quantification of the liver-specific RNAs albumin and microRNA-122 (miR-122). In vivo, significant elevations in circulating exosomal albumin mRNA were observed at subtoxic APAP exposures. Significant increases in exosomal albumin mRNA were also observed in primary rat hepatocytes at subtoxic APAP concentrations. In primary human hepatocytes, APAP elicited increases in both exosomal albumin mRNA and exosomal miR-122 without overt cytotoxicity. However, the number of HDE produced in vitro in response to APAP did not increase with exosomal RNA quantity. We conclude that significant drug-induced alterations in the liver-specific RNA content of HDE occur at subtoxic APAP exposures in vivo and in vitro, and that these changes appear to reflect selective packaging rather than changes in exosome number. The current findings demonstrate that translationally relevant HDE alterations occur in the absence of overt hepatocellular toxicity, and support the hypothesis that HDE released by stressed hepatocytes may mediate early immune responses in DILI. PMID:26962055

  13. Ca2+/calmodulin-dependent transcriptional pathways: potential mediators of skeletal muscle growth and development.

    PubMed

    Al-Shanti, Nasser; Stewart, Claire E

    2009-11-01

    The loss of muscle mass with age and disuse has a significant impact on the physiological and social well-being of the aged; this is an increasingly important problem as the population becomes skewed towards older age. Exercise has psychological benefits but it also impacts on muscle protein synthesis and degradation, increasing muscle tissue volume in both young and older individuals. Skeletal muscle hypertrophy involves an increase in muscle mass and cross-sectional area and associated increased myofibrillar protein content. Attempts to understand the molecular mechanisms that underlie muscle growth, development and maintenance, have focused on characterising the molecular pathways that initiate, maintain and regenerate skeletal muscle. Such understanding may aid in improving targeted interventional therapies for age-related muscle loss and muscle wasting associated with diseases. Two major routes through which skeletal muscle development and growth are regulated are insulin-like growth factor I (IGF-I) and Ca(2+)/calmodulin-dependent transcriptional pathways. Many reviews have focused on understanding the signalling pathways of IGF-I and its receptor, which govern skeletal muscle hypertrophy. However, alternative molecular signalling pathways such as the Ca(2+)/calmodulin-dependent transcriptional pathways should also be considered as potential mediators of muscle growth. These latter pathways have received relatively little attention and the purpose herein is to highlight the progress being made in the understanding of these pathways and associated molecules: calmodulin, calmodulin kinases (CaMKs), calcineurin and nuclear factor of activated T-cell (NFAT), which are involved in skeletal muscle regulation. We describe: (1) how conformational changes in the Ca(2+) sensor calmodulin result in the exposure of binding pockets for the target proteins (CaMKs and calcineurin). (2) How Calmodulin consequently activates either the Ca(2+)/calmodulin-dependent kinases

  14. Vascularized subcutaneous human liver tissue from engineered hepatocyte/fibroblast sheets in mice.

    PubMed

    Sakai, Yusuke; Yamanouchi, Kosho; Ohashi, Kazuo; Koike, Makiko; Utoh, Rie; Hasegawa, Hideko; Muraoka, Izumi; Suematsu, Takashi; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Kuroki, Tamotsu; Eguchi, Susumu

    2015-10-01

    Subcutaneous liver tissue engineering is an attractive and minimally invasive approach used to curative treat hepatic failure and inherited liver diseases. However, graft failure occurs frequently due to insufficient infiltration of blood vessels (neoangiogenesis), while the maintenance of hepatocyte phenotype and function requires in vivo development of the complex cellular organization of the hepatic lobule. Here we describe a subcutaneous human liver construction allowing for rapidly vascularized grafts by transplanting engineered cellular sheets consisting of human primary hepatocytes adhered onto a fibroblast layer. The engineered hepatocyte/fibroblast sheets (EHFSs) showed superior expression levels of vascularization-associated growth factors (vascular endothelial growth factor, transforming growth factor beta 1, and hepatocyte growth factor) in vitro. EHFSs developed into vascularized subcutaneous human liver tissues contained glycogen stores, synthesized coagulation factor IX, and showed significantly higher synthesis rates of liver-specific proteins (albumin and alpha 1 anti-trypsin) in vivo than tissues from hepatocyte-only sheets. The present study describes a new approach for vascularized human liver organogenesis under mouse skin. This approach could prove valuable for establishing novel cell therapies for liver diseases. PMID:26142777

  15. A Scabies Mite Serpin Interferes with Complement-Mediated Neutrophil Functions and Promotes Staphylococcal Growth

    PubMed Central

    Swe, Pearl M.; Fischer, Katja

    2014-01-01

    Background Scabies is a contagious skin disease caused by the parasitic mite Sarcoptes scabiei. The disease is highly prevalent worldwide and known to predispose to secondary bacterial infections, in particular by Streptococcus pyogenes and Staphylococcus aureus. Reports of scabies patients co-infected with methicillin resistant S. aureus (MRSA) pose a major concern for serious down-stream complications. We previously reported that a range of complement inhibitors secreted by the mites promoted the growth of S. pyogenes. Here, we show that a recently characterized mite serine protease inhibitor (SMSB4) inhibits the complement-mediated blood killing of S. aureus. Methodology/Principal Findings Blood killing of S. aureus was measured in whole blood bactericidal assays, counting viable bacteria recovered after treatment in fresh blood containing active complement and phagocytes, treated with recombinant SMSB4. SMSB4 inhibited the blood killing of various strains of S. aureus including methicillin-resistant and methicillin-sensitive isolates. Staphylococcal growth was promoted in a dose-dependent manner. We investigated the effect of SMSB4 on the complement-mediated neutrophil functions, namely phagocytosis, opsonization and anaphylatoxin release, by flow cytometry and in enzyme linked immuno sorbent assays (ELISA). SMSB4 reduced phagocytosis of S. aureus by neutrophils. It inhibited the deposition of C3b, C4b and properdin on the bacteria surface, but did not affect the depositions of C1q and MBL. SMSB4 also inhibited C5 cleavage as indicated by a reduced C5b-9 deposition. Conclusions/Significance We postulate that SMSB4 interferes with the activation of all three complement pathways by reducing the amount of C3 convertase formed. We conclude that SMSB4 interferes with the complement-dependent killing function of neutrophils, thereby reducing opsonization, phagocytosis and further recruitment of neutrophils to the site of infection. As a consequence secreted scabies

  16. Dysregulated flow-mediated vasodilatation in the human placenta in fetal growth restriction

    PubMed Central

    Jones, Sarah; Bischof, Helen; Lang, Ingrid; Desoye, Gernot; Greenwood, Sue L; Johnstone, Edward D; Wareing, Mark; Sibley, Colin P; Brownbill, Paul

    2015-01-01

    Increased vascular resistance and reduced fetoplacental blood flow are putative aetiologies in the pathogenesis of fetal growth restriction (FGR); however, the regulating sites and mechanisms remain unclear. We hypothesised that placental vessels dictate fetoplacental resistance and in FGR exhibit endothelial dysfunction and reduced flow-mediated vasodilatation (FMVD). Resistance was measured in normal pregnancies (n = 10) and FGR (n = 10) both in vivo by umbilical artery Doppler velocimetry and ex vivo by dual placental perfusion. Ex vivo FMVD is the reduction in fetal-side inflow hydrostatic pressure (FIHP) following increased flow rate. Results demonstrated a significant correlation between vascular resistance measured in vivo and ex vivo in normal pregnancy, but not in FGR. In perfused FGR placentas, vascular resistance was significantly elevated compared to normal placentas (58 ± 7.7 mmHg and 36.8 ± 4.5 mmHg, respectively; 8 ml min−1; means ± SEM; P < 0.0001) and FMVD was severely reduced (3.9 ± 1.3% and 9.1 ± 1.2%, respectively). In normal pregnancies only, the highest level of ex vivo FMVD was associated with the lowest in vivo resistance. Inhibition of NO synthesis during perfusion (100 μm l-NNA) moderately elevated FIHP in the normal group, but substantially in the FGR group. Human placenta artery endothelial cells from FGR groups exhibited increased shear stress-induced NO generation, iNOS expression and eNOS expression compared with normal groups. In conclusion, fetoplacental resistance is determined by placental vessels, and is increased in FGR. The latter also exhibit reduced FMVD, but with a partial compensatory increased NO generation capacity. The data support our hypothesis, which highlights the importance of FMVD regulation in normal and dysfunctional placentation. Key points A correlation was found between in vivo umbilical artery Doppler velocimetry and resistance to fetal-side flow in the human ex vivo dually

  17. Prolonged Endoplasmic Reticulum-Stressed Hepatocytes Drive an Alternative Macrophage Polarization.

    PubMed

    Xiu, Fangming; Catapano, Michael; Diao, Li; Stanojcic, Mile; Jeschke, Marc G

    2015-07-01

    Relatively little is known about the effects of hepatocytes on hepatic macrophages, particularly under the situation of endoplasmic reticulum (ER) stress. We examined the effects of hepatocytes conditioned media (CM) from HepG2 treated with ER stress inducers, tunicamycin or thapsigargin, on the secretion of cytokines, expression of ER stress markers, and polarization of phorbol myristate acetate-activated THP-1 cells (pTHP-1). We found that CM decreased the production of the proinflammatory cytokines including tumor necrosis factor α, interleukin 6 (IL-6), and IL-1β as well as other cytokines and chemokines from pTHP-1 cells. These effects are mediated by the inhibition of TLR4 expression and nuclear factor κB signaling pathway. In addition, hepatocytes CM increased the expression of binding immunoglobulin protein and the transcription factor C/EBP homologous protein (CHOP) in pTHP-1 cells. Preconditioning with ER stress inhibitor, small molecular chaperone 4-phenylbutyrate before addition of ER stressors, attenuated the ER stress in macrophages, the property of hepatocytes CM to alter tumor necrosis factor α production and nuclear factor κB expression by macrophages. Remarkably, treatment of macrophage with these CM leads to an alternative activation of macrophages mediated by peroxisome proliferator-activated receptor γ signaling pathway, which might be resulted from the secretion of IL-10 and IL-4 as well as releasing apoptotic bodies from hepatocytes under ER stress. Our results highlight a mechanism of ER stress transmission from hepatocytes to macrophage that drives an alternative activation of macrophages, which depends on the exposure of hepatocytes to severe and prolonged ER stress. PMID:25944791

  18. USP47 and C Terminus of Hsp70-Interacting Protein (CHIP) Antagonistically Regulate Katanin-p60-Mediated Axonal Growth

    PubMed Central

    Yang, Seung Wook; Oh, Kyu Hee; Park, Esther; Chang, Hyun Min; Park, Jung Mi; Seong, Min Woo; Ka, Seung Hyeun; Song, Woo Keun; Park, Dong Eun; Baas, Peter W.

    2013-01-01

    Katanin is a heterodimeric enzyme that severs and disassembles microtubules. While the p60 subunit has the enzyme activity, the p80 subunit regulates the p60 activity. The microtubule-severing activity of katanin plays an essential role in axonal growth. However, the mechanisms by which neuronal cells regulate the expression of katanin-p60 remains unknown. Here we showed that USP47 and C terminus of Hsp70-interacting protein (CHIP) antagonistically regulate the stability of katanin-p60 and thereby axonal growth. USP47 was identified as a katanin-p60-specific deubiquitinating enzyme for its stabilization. We also identified CHIP as a ubiquitin E3 ligase that promotes proteasome-mediated degradation of katanin-p60. Moreover, USP47 promoted axonal growth of cultured rat hippocampal neurons, whereas CHIP inhibited it. Significantly, treatment with basic fibroblast growth factor (bFGF), an inducer of axonal growth, increased the levels of USP47 and katanin-p60, but not CHIP. Consistently, bFGF treatment resulted in a marked decrease in the level of ubiquitinated katanin-p60 and thereby in the promotion of axonal growth. On the other hand, the level of USP47, but not CHIP, decreased concurrently with that of katanin-p60 as axons reached their target cells. These results indicate that USP47 plays a crucial role in the control of axonal growth during neuronal development by antagonizing CHIP-mediated katanin-p60 degradation. PMID:23904609

  19. Platelet-Derived Growth Factor CC-Mediated Neuroprotection against HIV Tat Involves TRPC-Mediated Inactivation of GSK 3beta

    PubMed Central

    Peng, Fuwang; Yao, Honghong; Akturk, Halis Kaan; Buch, Shilpa

    2012-01-01

    Platelet-derived growth factor-CC (PDGF-CC) is the third member of the PDGF family, and has been implicated both in embryogenesis and development of the CNS. The biological function of this isoform however, remains largely unexplored in the context of HIV-associated dementia (HAD). In the present study, we demonstrate that exposure of human neuroblastoma cells SH-SY5Y to HIV transactivator protein Tat resulted in decreased intrinsic expression of PDGF-CC as evidenced by RT-PCR and western blot assays. Reciprocally, pretreatment of SH-SY5Y cells with PDGF-CC abrogated Tat-mediated neurotoxicity by mitigating apoptosis and neurite & MAP-2 loss. Using pharmacological and loss of function approaches we identified the role of phosphatidylinositol 3-kinase (PI3K)/Akt signaling