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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

    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.

  2. 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

  3. Hepatocyte growth factor induces Mcl-1 in primary human hepatocytes and inhibits CD95-mediated apoptosis via Akt.

    PubMed

    Schulze-Bergkamen, Henning; Brenner, Dirk; Krueger, Andreas; Suess, Dorothee; Fas, Stefanie C; Frey, Christian R; Dax, Andreas; Zink, Dorothea; Büchler, Peter; Müller, Martina; Krammer, Peter H

    2004-03-01

    CD95 (APO-1/Fas)-mediated apoptosis of hepatocytes plays a central role in the pathophysiology of various human liver diseases. Hepatocyte growth factor (HGF) was shown to exert antiapoptotic functions in rodent hepatocytes. We previously showed that primary human hepatocytes (PHH) are a valuable tool for the investigation of apoptotic processes in liver cells. In this study, we analyzed the influence of HGF on CD95-mediated apoptosis of PHH and its molecular determinants. HGF significantly inhibited CD95-mediated apoptosis of PHH as well as cleavage of caspase-8 and poly (ADP-ribose)polymerase. HGF transcriptionally induced the expression of the anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1). In contrary, HGF did not alter the expression levels of Bcl-2 or Bcl-x(L). HGF activated survival pathways such as the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK and the signal transducer and activator of transcription 3 (STAT3) pathway. Notably, HGF triggered serine(727)--but not tyrosine(705)--phosphorylation of STAT3. Pretreatment of PHH with the PI3K inhibitor LY294002 as well as adenoviral transduction of dominant negative Akt1 prevented HGF-mediated Mcl-1 induction and reversed the antiapoptotic effects of HGF. In conclusion, HGF confers survival of PHH by activation of the PI3K/Akt pathway. PI3K/Akt activation by HGF results in the induction of antiapoptotic proteins such as Mcl-1. Thus, application of HGF may be a therapeutic approach to prevent CD95-mediated hepatocellular damage in human liver diseases. PMID:14999683

  4. 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

    2016-03-17

    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.

  5. 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

  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. 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

  8. 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.

  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. Gelsolin-mediated activation of PI3K/Akt pathway is crucial for hepatocyte growth factor-induced cell scattering in gastric carcinoma

    PubMed Central

    Huang, Baohua; Deng, Shuo; Loo, Ser Yue; Datta, Arpita; Yap, Yan Lin; Yan, Benedict; Ooi, Chia Huey; Dinh, Thuy Duong; Zhuo, Jingli; Tochhawng, Lalchhandami; Gopinadhan, Suma; Jegadeesan, Tamilarasi; Tan, Patrick; Salto-Tellez, Manuel; Yong, Wei Peng; Soong, Richie; Yeoh, Khay Guan; Goh, Yaw Chong; Lobie, Peter E.; Yang, Henry; Kumar, Alan Prem; Maciver, Sutherland K.; So, Jimmy B.Y.; Yap, Celestial T.

    2016-01-01

    In gastric cancer (GC), the main subtypes (diffuse and intestinal types) differ in pathological characteristics, with diffuse GC exhibiting early disseminative and invasive behaviour. A distinctive feature of diffuse GC is loss of intercellular adhesion. Although widely attributed to mutations in the CDH1 gene encoding E-cadherin, a significant percentage of diffuse GC do not harbor CDH1 mutations. We found that the expression of the actin-modulating cytoskeletal protein, gelsolin, is significantly higher in diffuse-type compared to intestinal-type GCs, using immunohistochemical and microarray analysis. Furthermore, in GCs with wild-type CDH1, gelsolin expression correlated inversely with CDH1 gene expression. Downregulating gelsolin using siRNA in GC cells enhanced intercellular adhesion and E-cadherin expression, and reduced invasive capacity. Interestingly, hepatocyte growth factor (HGF) induced increased gelsolin expression, and gelsolin was essential for HGF-medicated cell scattering and E-cadherin transcriptional repression through Snail, Twist and Zeb2. The HGF-dependent effect on E-cadherin was found to be mediated by interactions between gelsolin and PI3K-Akt signaling. This study reveals for the first time a function of gelsolin in the HGF/cMet oncogenic pathway, which leads to E-cadherin repression and cell scattering in gastric cancer. Our study highlights gelsolin as an important pro-disseminative factor contributing to the aggressive phenotype of diffuse GC. PMID:27058427

  11. Gelsolin-mediated activation of PI3K/Akt pathway is crucial for hepatocyte growth factor-induced cell scattering in gastric carcinoma.

    PubMed

    Huang, Baohua; Deng, Shuo; Loo, Ser Yue; Datta, Arpita; Yap, Yan Lin; Yan, Benedict; Ooi, Chia Huey; Dinh, Thuy Duong; Zhuo, Jingli; Tochhawng, Lalchhandami; Gopinadhan, Suma; Jegadeesan, Tamilarasi; Tan, Patrick; Salto-Tellez, Manuel; Yong, Wei Peng; Soong, Richie; Yeoh, Khay Guan; Goh, Yaw Chong; Lobie, Peter E; Yang, Henry; Kumar, Alan Prem; Maciver, Sutherland K; So, Jimmy B Y; Yap, Celestial T

    2016-05-01

    In gastric cancer (GC), the main subtypes (diffuse and intestinal types) differ in pathological characteristics, with diffuse GC exhibiting early disseminative and invasive behaviour. A distinctive feature of diffuse GC is loss of intercellular adhesion. Although widely attributed to mutations in the CDH1 gene encoding E-cadherin, a significant percentage of diffuse GC do not harbor CDH1 mutations. We found that the expression of the actin-modulating cytoskeletal protein, gelsolin, is significantly higher in diffuse-type compared to intestinal-type GCs, using immunohistochemical and microarray analysis. Furthermore, in GCs with wild-type CDH1, gelsolin expression correlated inversely with CDH1 gene expression. Downregulating gelsolin using siRNA in GC cells enhanced intercellular adhesion and E-cadherin expression, and reduced invasive capacity. Interestingly, hepatocyte growth factor (HGF) induced increased gelsolin expression, and gelsolin was essential for HGF-medicated cell scattering and E-cadherin transcriptional repression through Snail, Twist and Zeb2. The HGF-dependent effect on E-cadherin was found to be mediated by interactions between gelsolin and PI3K-Akt signaling. This study reveals for the first time a function of gelsolin in the HGF/cMet oncogenic pathway, which leads to E-cadherin repression and cell scattering in gastric cancer. Our study highlights gelsolin as an important pro-disseminative factor contributing to the aggressive phenotype of diffuse GC.

  12. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    PubMed

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  13. Deletion of Smad2 in Mouse Liver Reveals Novel Functions in Hepatocyte Growth and Differentiation

    PubMed Central

    Ju, Wenjun; Ogawa, Atsushi; Heyer, Joerg; Nierhof, Dirk; Yu, Liping; Kucherlapati, Raju; Shafritz, David A.; Böttinger, Erwin P.

    2006-01-01

    Smad family proteins Smad2 and Smad3 are activated by transforming growth factor β (TGF-β)/activin/nodal receptors and mediate transcriptional regulation. Although differential functional roles of Smad2 and Smad3 are apparent in mammalian development, the relative functional roles of Smad2 and Smad3 in postnatal systems remain unclear. We used Cre/loxP-mediated gene targeting for hepatocyte-specific deletion of Smad2 (S2HeKO) in adult mice and generated hepatocyte-selective Smad2/Smad3 double knockouts by intercrossing AlbCre/Smad2f/f (S2HeKO) and Smad3-deficient Smad3ex8/ex8 (S3KO) mice. All strains were viable and had normal adult liver. However, necrogenic CCL4-induced hepatocyte proliferation was significantly increased in S2HeKO compared to Ctrl and S3KO livers, and transplanted S2HeKO hepatocytes repopulated recipient liver at dramatically increased rates compared to Ctrl hepatocytes in vivo. Using primary hepatocytes, we found that TGF-β-induced G1 arrest, apoptosis, and epithelial-to-mesenchymal transition in Ctrl and S2HeKO but not in S3KO hepatocytes. Interestingly, S2HeKO cells spontaneously acquired mesenchymal features characteristic of epithelial-to-mesenchymal transition (EMT). Collectively, these results demonstrate that Smad2 suppresses hepatocyte growth and dedifferentiation independent of TGF-β signaling. Smad2 is not required for TGF-β-stimulated apoptosis, EMT, and growth inhibition in hepatocytes. PMID:16382155

  14. Activation of PI3K-Akt-GSK3{beta} pathway mediates hepatocyte growth factor inhibition of RANTES expression in renal tubular epithelial cells

    SciTech Connect

    Gong Rujun . E-mail: rgong@Brown.edu; Rifai, Abdalla; Dworkin, Lance D.

    2005-04-29

    Hepatocyte growth factor (HGF) was recently reported to ameliorate renal inflammation in a rat model of chronic renal failure. HGF exerted its action through suppression of RANTES expression in renal tubules. In the present study, we utilized an in vitro model of human kidney proximal tubule epithelial cells (HKC) to elucidate the mechanisms of RANTES suppression by HGF. HGF significantly suppressed basal and TNF-{alpha}-induced mRNA and protein expression of RANTES in a time and dose dependent fashion. HGF elicited PI3K-Akt activation and inhibited GSK3, a downstream transducer of PI3K-Akt, by inhibitory phosphorylation at Ser-9. When the PI3K-Akt pathway was blocked by wortmannin, HGF inhibition of RANTES was abrogated, demonstrating that the PI3K-Akt pathway is necessary for HGF action. In addition, specific inhibition of GSK3 activity by lithium ion suppressed basal and TNF-{alpha}-induced RANTES expression, reminiscent of the action of HGF. To further investigate the role of GSK3 in modulating RANTES expression, we examined the effect of forced expression of wild type GSK3{beta} or an uninhibitable mutant GSK3{beta}, in which the regulatory Ser-9 residue is changed to alanine (S9A-GSK3{beta}) in HKC. Overexpression of wild type GSK3{beta} did not alter the inhibitory action of HGF on RANTES. In contrast, expression of S9A-GSK3{beta} abolished HGF inhibition of basal and TNF-{alpha} stimulated RANTES expression. These findings suggest that PI3K-Akt activation and subsequent inhibitory phosphorylation of GSK3{beta} are required for HGF-induced suppression of RANTES in HKC.

  15. 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.

  16. A crucial function of PDGF in TGF-beta-mediated cancer progression of hepatocytes.

    PubMed

    Gotzmann, J; Fischer, A N M; Zojer, M; Mikula, M; Proell, V; Huber, H; Jechlinger, M; Waerner, T; Weith, A; Beug, H; Mikulits, W

    2006-05-25

    Polarized hepatocytes expressing hyperactive Ha-Ras adopt an invasive and metastatic phenotype in cooperation with transforming growth factor (TGF)-beta. This dramatic increase in malignancy is displayed by an epithelial to mesenchymal transition (EMT), which mimics the TGF-beta-mediated progression of human hepatocellular carcinomas. In culture, hepatocellular EMT occurs highly synchronously, facilitating the analysis of molecular events underlying the various stages of this process. Here, we show that in response to TGF-beta, phosphorylated Smads rapidly translocated into the nucleus and activated transcription of target genes such as E-cadherin repressors of the Snail superfamily, causing loss of cell adhesion. Within the TGF-beta superfamily of cytokines, TGF-beta1, -beta2 and -beta3 were specific for the induction of hepatocellular EMT. Expression profiling of EMT kinetics revealed 78 up- and 235 downregulated genes, which preferentially modulate metabolic activities, extracellular matrix composition, transcriptional activities and cell survival. Independent of the genetic background, platelet-derived growth factor (PDGF)-A ligand and both PDGF receptor subunits were highly elevated, together with autocrine secretion of bioactive PDGF. Interference with PDGF signalling by employing hepatocytes expressing the dominant-negative PDGF-alpha receptor revealed decreased TGF-beta-induced migration in vitro and efficient suppression of tumour growth in vivo. In conclusion, these results provide evidence for a crucial role of PDGF in TGF-beta-mediated tumour progression of hepatocytes and suggest PDGF as a target for therapeutic intervention in liver cancer.

  17. 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

  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. Transcription-dependent epidermal growth factor receptor activation by hepatocyte growth factor.

    PubMed

    Reznik, Thomas E; Sang, Yingying; Ma, Yongxian; Abounader, Roger; Rosen, Eliot M; Xia, Shuli; Laterra, John

    2008-01-01

    The mechanisms and biological implications of coordinated receptor tyrosine kinase coactivation remain poorly appreciated. Epidermal growth factor receptor (EGFR) and c-Met are frequently coexpressed in cancers, including those associated with hepatocyte growth factor (HGF) overexpression, such as malignant astrocytoma. In a previous analysis of the HGF-induced transcriptome, we found that two EGFR agonists, transforming growth factor-alpha and heparin-binding epidermal growth factor-like growth factor (HB-EGF), are prominently up-regulated by HGF in human glioma cells. We now report that stimulating human glioblastoma cells with recombinant HGF induces biologically relevant EGFR activation. EGFR phosphorylation at Tyr(845) and Tyr(1068) increased 6 to 24 h after cell stimulation with HGF and temporally coincided with the induction of transforming growth factor-alpha (~5-fold) and HB-EGF (~23-fold) expression. Tyr(845) and Tyr(1068) phosphorylation, in response to HGF, was inhibited by cycloheximide and actinomycin D, consistent with a requirement for DNA transcription and RNA translation. Specifically, blocking HB-EGF binding to EGFR with the antagonist CRM197 inhibited HGF-induced EGFR phosphorylation by 60% to 80% and inhibited HGF-induced S-G(2)-M transition. CRM197 also inhibited HGF-induced anchorage-dependent cell proliferation but had no effect on HGF-mediated cytoprotection. These findings establish that EGFR can be activated with functional consequences by HGF as a result of EGFR ligand expression. This transcription-dependent cross-talk between the HGF receptor c-Met and EGFR expands our understanding of receptor tyrosine kinase signaling networks and may have considerable consequences for oncogenic mechanisms and cancer therapeutics.

  2. 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.

  3. Asialoglycoprotein Receptor-Mediated Gene Delivery to Hepatocytes Using Galactosylated Polymers.

    PubMed

    Thapa, Bindu; Kumar, Piyush; Zeng, Hongbo; Narain, Ravin

    2015-09-14

    Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition-fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free asialofetuin or by adding free asialofetuin together with polyplexes into hepatocytes. The results confirmed that polyplexes with these polymers were taken up specifically by hepatocytes via ASGPR-mediated endocytosis. The results from transfection efficiency and uptake of these polymers in cells without ASGPR, such as SK Hep1 and HeLa cells, further support this mechanism. Since in vitro cytotoxicity assays prove these glycopolymers to be nontoxic, they may be useful for delivery of clinically important genes specifically to the liver.

  4. Instant Blood-Mediated Inflammatory Reaction in Hepatocyte Transplantation: Current Status and Future Perspectives.

    PubMed

    Lee, Charlotte A; Dhawan, Anil; Smith, Richard A; Mitry, Ragai R; Fitzpatrick, Emer

    2016-01-01

    Hepatocyte transplantation (HT) is emerging as a promising alternative to orthotopic liver transplantation (OLT) in patients with certain liver-based metabolic disease and acute liver failure. Hepatocytes are generally infused into the portal venous system, from which they migrate into the liver cell plates of the native organ. One of the major hurdles to the sustained success of this therapy is early cell loss, with up to 70% of hepatocytes lost immediately following infusion. This is largely thought to be due to the instant blood-mediated inflammatory reaction (IBMIR), resulting in the activation of complement and coagulation pathways. Transplanted hepatocytes produce and release tissue factor (TF), which activates the coagulation pathway, leading to the formation of thrombin and fibrin clots. Thrombin can further activate a number of complement proteins, leading to the activation of the membrane attack complex (MAC) and subsequent hepatocyte cell death. Inflammatory cells including granulocytes, monocytes, Kupffer cells, and natural killer (NK) cells have been shown to cluster around transplanted hepatocytes, leading to their rapid clearance shortly after transplantation. Current research aims to improve cell engraftment and prevent early cell loss. This has been proven successful in vitro using pharmacological interventions such as melagatran, low-molecular-weight dextran sulphate, and N-acetylcysteine (NAC). Effective inhibition of IBMIR would significantly improve hepatocyte engraftment, proliferation, and function, providing successful treatment for patients with liver-based metabolic diseases. PMID:26996786

  5. Antibody-directed complement-mediated cytotoxicity to hepatocytes from patients with chronic hepatitis B.

    PubMed Central

    Michalak, T I; Lau, J Y; McFarlane, B M; Alexander, G J; Eddleston, A L; Williams, R

    1995-01-01

    The susceptibility of hepatocytes from patients with chronic hepatitis B to complement-dependent cytotoxicity mediated by heterologous antibodies to hepatitis B virus core (anti-HBc) and surface (anti-HBs) antigens and to hepatic asialoglycoprotein receptor was examined using a microcytotoxicity assay. The anti-HBc-induced cytotoxicity was found to be markedly enhanced against hepatocytes isolated from patients with chronic active hepatitis (72.6 +/- 9.5% (mean +/- s.e.m.); n = 6) over that against hepatocytes from individuals with chronic persistent hepatitis or inactive liver cirrhosis (40.6 +/- 18.6%; n = 4) (P = 0.019). Overall, values of the anti-HBc-directed cytotoxicity were higher in patients positive for HBcAg in hepatocytes and seropositive for hepatitis B virus e antigen (HBeAg). Hepatocytotoxicity was also exerted by anti-HBs and anti-asialoglycoprotein receptor antibodies in the presence of complement, but it was not seemingly related to disease activity. These results indicate that hepatitis B virus core and surface antigens and asialoglycoprotein receptor at the hepatocyte surface can be recognized by antibodies, and raise the possibility that complement-dependent cytolysis may contribute to the injury of hepatitis B virus-infected hepatocytes. The data also suggest that liver cells of patients with severe chronic hepatitis might be more susceptible to anti-HBc antibody-directed complement-mediated cytotoxicity than those with inactive liver histology. PMID:7743660

  6. Comparison of mutagenicities in a Salmonella reversion assay mediated by uninduced hepatocytes and hepatocytes from rats pretreated for 1 or 5 days with Aroclor 1254.

    PubMed

    Hass, B S; Heflich, R H; Shaddock, J G; Casciano, D A

    1985-01-01

    Hepatocytes prepared from rats pretreated for 5 days with 500 mg/kg Aroclor 1254 were found to be unsuitable for use in a modified Salmonella mutagenicity assay. These hepatocytes exhibited low viability, did not readily attach to plastic culture dishes, and produced mutagenicity responses with benzo[a]pyrene (B[a]P) and 2-aminofluorene (2AF) that were greatly enhanced by the addition of an NADPH-regenerating system (NADPH-RS). Shortening the Aroclor pretreatment time to 1 day resulted in hepatocytes that exhibited high viability and readily attached to plastic culture dishes. These hepatocytes produced higher numbers of revertants when used to assay the mutagenicities of B[a]P and 2AF than were produced using hepatocytes from animals that were pretreated for 5 days. These reversion frequencies were also higher than those produced using uninduced hepatocytes and were much less affected by the addition of NADPH-RS than were the reversions mediated by the 5-day preinduced hepatocytes. Liver homogenate postmitochondrial fractions (S9s), which were prepared from rats pretreated with Aroclor for 1 or 5 days, were nearly equal in their ability to mediate the mutagenicity of B[a]P and 2AF in the Salmonella/microsome reversion assay. Qualitative differences between the S9- and hepatocyte-mediated mutagenicity of 2AF were found, however. These results indicate that employing hepatocytes from rats pretreated with Aroclor for 1 day, rather than 5 days, results in an enzymatically induced, more-intact cell population that is capable of detecting the mutagenicity of B[a]P and 2AF in a modified Salmonella reversion assay.

  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. Kupffer cells induce Notch-mediated hepatocyte conversion in a common mouse model of intrahepatic cholangiocarcinoma

    PubMed Central

    Terada, Maiko; Horisawa, Kenichi; Miura, Shizuka; Takashima, Yasuo; Ohkawa, Yasuyuki; Sekiya, Sayaka; Matsuda-Ito, Kanae; Suzuki, Atsushi

    2016-01-01

    Intrahepatic cholangiocarcinoma (ICC) is a malignant epithelial neoplasm composed of cells resembling cholangiocytes that line the intrahepatic bile ducts in portal areas of the hepatic lobule. Although ICC has been defined as a tumor arising from cholangiocyte transformation, recent evidence from genetic lineage-tracing experiments has indicated that hepatocytes can be a cellular origin of ICC by directly changing their fate to that of biliary lineage cells. Notch signaling has been identified as an essential factor for hepatocyte conversion into biliary lineage cells at the onset of ICC. However, the mechanisms underlying Notch signal activation in hepatocytes remain unclear. Here, using a mouse model of ICC, we found that hepatic macrophages called Kupffer cells transiently congregate around the central veins in the liver and express the Notch ligand Jagged-1 coincident with Notch activation in pericentral hepatocytes. Depletion of Kupffer cells prevents the Notch-mediated cell-fate conversion of hepatocytes to biliary lineage cells, inducing hepatocyte apoptosis and increasing mortality in mice. These findings will be useful for uncovering the pathogenic mechanism of ICC and developing prevenient and therapeutic strategies for this refractory disease. PMID:27698452

  9. The Slit/Robo System Suppresses Hepatocyte Growth Factor-dependent Invasion and Morphogenesis

    PubMed Central

    Comoglio, Paolo M.

    2009-01-01

    The Slit protein acts through the Roundabout receptor as a paracrine chemorepellent in axon guidance and as an inhibitor in leukocyte chemotaxis, but its role in epithelial cell motility and morphogenesis remains largely unexplored. We report that nontransformed epithelial cells and cancerous cells empower the Slit-2/Robo1 signaling system to limit outward migration in response to motogenic attractants and to remain positionally confined within their primitive location. Short hairpin RNA-mediated depletion of SLIT-2 or ectopic expression of a soluble decoy Robo enhance hepatocyte growth factor (HGF)-induced migration, matrix invasion, and tubulogenesis, concomitantly with the up-regulation of Cdc-42 and the down-modulation of Rac-1 activities. Accordingly, autocrine overexpression or exogenous administration of Slit-2 prevent HGF-triggered motile responses, reduce Cdc-42 activation, and stimulate Rac-1. This antimigratory activity of Slit-2 derives from the inhibition of actin-based protrusive forces and from an increased adhesive strength of cadherin-mediated intercellular contacts. These results disclose a novel function for Slit and Robo in the inhibition of growth factor-mediated epithelial cell motility and morphogenesis, invoking a critical role for both molecules as natural antagonists of neoplastic invasive growth. PMID:19005219

  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. 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

  12. Presence of urokinase in serum-free primary rat hepatocyte cultures and its role in activating hepatocyte growth factor.

    PubMed

    Mars, W M; Kim, T H; Stolz, D B; Liu, M L; Michalopoulos, G K

    1996-06-15

    Serum-free rat hepatocyte cultures can be stimulated to divide by the inactive, single-chain form of hepatocyte growth factor (scHGF), suggesting that hepatocytes contain a protein that can cleave scHGF to its biologically active, two-chain (tcHGF) form. We added radiolabeled scHGF to serum-free cultures and confirmed that tcHGF was being generated. Because scHGF can be cleaved to tcHGF by plasminogen activators (PAs), we next tested the cultures for active PA. Although little PA activity was initially present, the majority was of the urokinase type (u-PA) as determined by neutralization studies using either a polyclonal antibody against u-PA or, since u-PA functions in the context of its receptor (u-PAR), a monoclonal antibody against u-PAR. Considerable PA activity developed within 24 h, which was also neutralizable with antibody. To test whether the active, receptor-bound u-PA from the cell cultures was cleaving scHGF, iodinated scHGF was added to intact cells in the presence of the antibody against u-PAR. Comparison to control cultures determined that the antibody prevented scHGF cleavage. Analysis of cultures treated with HGF, epidermal growth factor, and transforming growth factor alpha (TGF-alpha) alpha showed these growth factors increased the hepatocyte PA activity in parallel with the mRNA for u-PA. TGF-beta had the opposite effect, and when TGF-beta was added to the culture system, conversion of scHGF to tcHGF was prevented in concert with the production of the type 1 PA inhibitor. When liver remnants from hepatectomized animals were assayed for active TGF-beta, elevated protein was found just prior to the appearance of PA inhibitor 1 message and protein. Collectively, our data show that in culture, active u-PA is present and cleaves scHGF to tcHGF in the context of its receptor. It also suggests that modulation of u-PA activity by various growth factors is relevant for regulating cleavage of scHGF to tcHGF both in vitro and in vivo.

  13. Hepatocyte-mediated cytotoxicity and host defense mechanisms in the alcohol-injured liver.

    PubMed

    McVicker, Benita L; Thiele, Geoffrey M; Tuma, Dean J; Casey, Carol A

    2014-09-01

    The consumption of alcohol is associated with many health issues including alcoholic liver disease (ALD). The natural history of ALD involves the development of steatosis, inflammation (steatohepatitis), fibrosis and cirrhosis. During the stage of steatohepatitis, the combination of inflammation and cellular damage can progress to a severe condition termed alcoholic hepatitis (AH). Unfortunately, the pathogenesis of AH remains uncharacterized. Some modulations have been identified in host defense and liver immunity mechanisms during AH that highlight the role of intrahepatic lymphocyte accumulation and associated inflammatory cytokine responses. Also, it is hypothesized that alcohol-induced injury to liver cells may significantly contribute to the aberrant lymphocytic distribution that is seen in AH. In particular, the regulation of lymphocytes by hepatocytes may be disrupted in the alcoholic liver resulting in altered immunologic homeostasis and perpetuation of disease. In recent studies, it was demonstrated that the direct killing of activated T lymphocytes by hepatocytes is facilitated by the asialoglycoprotein receptor (ASGPR). The ASGPR is a well-characterized glycoprotein receptor that is exclusively expressed by hepatocytes. This hepatic receptor is known for its role in the clearance of desialylated glycoproteins or cells, yet neither its physiological function nor its role in disease states has been determined. Interestingly, alcohol markedly impairs ASGPR function; however, the effect alcohol has on ASGPR-mediated cytotoxicity of lymphocytes remains to be elucidated. This review discusses the contribution of hepatocytes in immunological regulation and, importantly, how pathological effects of ethanol disrupt hepatocellular-mediated defense mechanisms.

  14. 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

  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. Hepatocyte growth factor/cMET pathway activation enhances cancer hallmarks in adrenocortical carcinoma

    PubMed Central

    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

    2015-01-01

    Adrenocortical carcinoma (ACC) 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 ACC has not been examined. In this study, we investigated the association between HGF/cMET expression and cancer hallmarks of ACC. Transcriptomic and immunohistochemical analyses indicated that increased HGF/cMET expression in human ACC samples was positively associated with cancer-related biological processes including proliferation and angiogenesis, and negatively correlated with apoptosis. Accordingly, treatment of ACC cells with exogenous HCG resulted in increased cell proliferation in vitro and in vivo while short hairpin RNA-mediated knockdown or pharmacological 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 ACC 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

  17. Transcriptional induction of the agp/ebp (c/ebp beta) gene by hepatocyte growth factor.

    PubMed

    Shen, B J; Chang, C J; Lee, H S; Tsai, W H; Miau, L H; Lee, S C

    1997-06-01

    Hepatocyte growth factor (HGF) is a pleiotropic factor with mitogenic, morphogenic, motogenic, cytotoxic, or growth inhibitory activity. Although the signaling of HGF is mediated through the cell membrane receptor c-Met, the molecular mechanism of downstream signal transduction remains obscure. In this report, we present evidence that shows HGF can stimulate the expression of AGP/EBP (C/EBP beta) and NF-kappaB, which are both key transcription factors responsible for the regulation of many genes under stress conditions or during the acute-phase response. Biochemical and functional analysis indicates that the HGF-responsive element is located in the region -376 to -352 (URE1) of the 5'-upstream regulatory sequence of agp/ebp. Activation of NF-kappaB by HGF was observed to precede the induction of agp/ebp. Further studies indicate that NF-kappaB can cooperate with AGP/EBP or other members of the C/EBP family to activate the agp/ebp gene in both URE1 and URE2-dependent manner. These results suggest that the induction of the agp/ebp gene by HGF is mediated at least in part by its activation of NF-kappaB. The activated NF-kappaB then interacts with AGP/EBP, resulting in the induction of agp/ebp.

  18. Effects of methylmercury on primary cultured rat hepatocytes: Cell injury and inhibition of growth factor stimulated DNA synthesis

    SciTech Connect

    Tanno, Keiichi; Fukazawa, Toshiyuki; Tajima, Shizuko; Fujiki, Motoo )

    1992-08-01

    Many more studies deal with the toxicity of methylmercury on nervous tissue than on its toxicity to the liver. Methylmercury accumulates in the liver in higher concentrations than brain and the liver has the primary function of detoxifying methylmercury. According to recent studies, hepatocyte mitochondrial membranes are destroyed by methylmercury and DNA synthesis is inhibited by methylmercury during hepatocyte regeneration. Methylmercury alters the membrane ion permeability of isolate skate hepatocytes, and inhibits the metal-sensitive alcohol dehydrogenase and glutathione reductase of primary cultured rat hepatocytes. However, little is known about the effect of methylmercury on hepatocyte proliferation in primary cultured rat hepatocytes. We therefore used the primary cultured rat hepatocytes to investigate the effects of methylmercury on cell injury and growth factor stimulate DNA synthesis. The primary effect of methylmercury is to inhibit hepatocyte proliferation rather than to cause direct cell injury. 16 refs., 4 figs.

  19. Epidermal growth factor receptor numbers in male and female mouse primary hepatocyte cultures.

    PubMed

    Benveniste, R; Danoff, T M; Ilekis, J; Craig, H R

    1988-10-01

    Epidermal growth factor receptors (EGF-R) were measured in adult male and female mouse primary hepatocyte cultures. On culture day 1, female hepatocytes had significantly fewer EGF-R than male hepatocytes (1.3 x 10(4) versus 6.2 x 10(5) per cell). Over the next three days, morphological changes consistent with progressive heptocyte dedifferentiation were observed. During this period, EGF-R numbers progressively increased in female cultures and decreased in male cultures, and by day 4 the sexual difference in EGF-R numbers was obliterated. These results indicate that a relationship exists between the degree of differentiation in hepatocyte cultures and the expression of EGF-R on the cell surface.

  20. Hepatocyte growth factor induces proliferation and differentiation of multipotent and erythroid hemopoietic progenitors

    PubMed Central

    1994-01-01

    Hepatocyte growth factor (HGF) is a mesenchymal derived growth factor known to induce proliferation and "scattering" of epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c- MET protooncogene. Here we show that highly purified recombinant HGF stimulates hemopoietic progenitors to form colonies in vitro. In the presence of erythropoietin, picomolar concentrations of HGF induced the formation of erythroid burst-forming unit colonies from CD34-positive cells purified from human bone marrow, peripheral blood, or umbilical cord blood. The growth stimulatory activity was restricted to the erythroid lineage. HGF also stimulated the formation of multipotent CFU- GEMM colonies. This effect is synergized by stem cell factor, the ligand of the tyrosine kinase receptor encoded by the c-KIT protooncogene, which is active on early hemopoietic progenitors. By flow cytometry analysis, the receptor for HGF was found to be expressed on the cell surface in a fraction of CD34+ progenitors. Moreover, in situ hybridization experiments showed that HGF receptor mRNA is highly expressed in embryonic erythroid cells (megaloblasts). HGF mRNA was also found to be produced in the embryonal liver. These data show that HGF plays a direct role in the control of proliferation and differentiation of erythroid progenitors, and they suggest that it may be one of the long-sought mediators of paracrine interactions between stromal and hemopoietic cells within the hemopoietic microenvironment. PMID:7528222

  1. Dual delivery of vascular endothelial growth factor and hepatocyte growth factor coacervate displays strong angiogenic effects.

    PubMed

    Awada, Hassan K; Johnson, Noah R; Wang, Yadong

    2014-05-01

    Controlled delivery of multiple growth factors (GFs) holds great potential for the clinical treatment of ischemic diseases and might be more therapeutically effective to reestablish vasculature than the provision of a single GF. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are two potent angiogenic factors. However, due to rapid degradation and dilution in the body, their clinical potential will rely on an effective mode of delivery. A coacervate, composed of heparin and a biodegradable polycation, which protects GFs from proteolysis and potentiates their bioactivities, is developed. Here, the coacervate incorporates VEGF and HGF and sustains their release for at least three weeks. Their strong angiogenic effects on endothelial cell proliferation and tube formation in vitro are confirmed. Furthermore, it is demonstrated that coacervate-based delivery of these factors has stronger effects than free application of both factors and to coacervate delivery of each GF separately.

  2. Hepatocyte growth factor, a determinant of airspace homeostasis in the murine lung.

    PubMed

    Calvi, Carla; Podowski, Megan; Lopez-Mercado, Armando; Metzger, Shana; Misono, Kaori; Malinina, Alla; Dikeman, Dustin; Poonyagariyon, Hataya; Ynalvez, Leslie; Derakhshandeh, Roshanak; Le, Anne; Merchant, Mark; Schwall, Ralph; Neptune, Enid R

    2013-01-01

    The alveolar compartment, the fundamental gas exchange unit in the lung, is critical for tissue oxygenation and viability. We explored hepatocyte growth factor (HGF), a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, and resistance to apoptosis, as a candidate mediator of alveolar formation and regeneration. Mice deficient in the expression of the HGF receptor Met in lung epithelial cells demonstrated impaired airspace formation marked by a reduction in alveolar epithelial cell abundance and survival, truncation of the pulmonary vascular bed, and enhanced oxidative stress. Administration of recombinant HGF to tight-skin mice, an established genetic emphysema model, attenuated airspace enlargement and reduced oxidative stress. Repair in the TSK/+ mouse was punctuated by enhanced akt and stat3 activation. HGF treatment of an alveolar epithelial cell line not only induced proliferation and scattering of the cells but also conferred protection against staurosporine-induced apoptosis, properties critical for alveolar septation. HGF promoted cell survival was attenuated by akt inhibition. Primary alveolar epithelial cells treated with HGF showed improved survival and enhanced antioxidant production. In conclusion, using both loss-of-function and gain-of-function maneuvers, we show that HGF signaling is necessary for alveolar homeostasis in the developing lung and that augmentation of HGF signaling can improve airspace morphology in murine emphysema. Our studies converge on prosurvival signaling and antioxidant protection as critical pathways in HGF-mediated airspace maintenance or repair. These findings support the exploration of HGF signaling enhancement for diseases of the airspace.

  3. Amniotic fluid-borne hepatocyte growth factor protects rat pups against experimental necrotizing enterocolitis.

    PubMed

    Jain, Sunil K; Baggerman, Eric W; Mohankumar, Krishnan; Namachivayam, Kopperuncholan; Jagadeeswaran, Ramasamy; Reyes, Victor E; Maheshwari, Akhil

    2014-03-01

    Fetal swallowing of amniotic fluid, which contains numerous cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid can provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (HGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC6 intestinal epithelial cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Amniotic fluid supplementation in oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by HGF and blocked by anti-HGF antibody or a PI3K inhibitor. HGF transactivated several RTKs in IEC6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, recombinant HGF also reduced the frequency and severity of NEC-like injury in rat pups. Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF.

  4. Amniotic fluid-borne hepatocyte growth factor protects rat pups against experimental necrotizing enterocolitis

    PubMed Central

    Baggerman, Eric W.; MohanKumar, Krishnan; Namachivayam, Kopperuncholan; Jagadeeswaran, Ramasamy; Reyes, Victor E.; Maheshwari, Akhil

    2014-01-01

    Fetal swallowing of amniotic fluid, which contains numerous cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid can provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (HGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC6 intestinal epithelial cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Amniotic fluid supplementation in oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by HGF and blocked by anti-HGF antibody or a PI3K inhibitor. HGF transactivated several RTKs in IEC6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, recombinant HGF also reduced the frequency and severity of NEC-like injury in rat pups. Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF. PMID:24407592

  5. Hepatocyte necrosis induced by oxidative stress and IL-1α release mediate carcinogen-induced compensatory proliferation and liver tumorigenesis

    PubMed Central

    Sakurai, Toshiharu; He, Guobin; Matsuzawa, Atsushi; Yu, Guann-Yi; Maeda, Shin; Hardiman, Gary; Karin, Michael

    2009-01-01

    SUMMARY Hepatocyte IκB kinase β (IKKβ) inhibits hepatocarcinogenesis by suppressing accumulation of reactive oxygen species (ROS) and liver damage, whereas JNK1 activation promotes ROS accumulation, liver damage and carcinogenesis. We examined whether hepatocyte p38α, found to inhibit liver carcinogenesis, acts similarly to IKKβ in control of ROS metabolism and cell death. Hepatocyte-specific p38α ablation enhanced ROS accumulation and liver damage, which were prevented upon administration of an antioxidant. In addition to elevated ROS accumulation, hepatocyte death, augmented by loss of either IKKβ or p38α, was associated with release of IL-1α. Inhibition of IL-1α action or ablation of its receptor inhibited carcinogen-induced compensatory proliferation and liver tumorigenesis. IL-1α release by necrotic hepatocytes is therefore an important mediator of liver tumorigenesis. PMID:18691550

  6. 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.

  7. Hepatocyte Growth Factor/Scatter Factor and MET Are Involved in Arterial Repair and Atherogenesis

    PubMed Central

    McKinnon, Heather; Gherardi, Ermanno; Reidy, Michael; Bowyer, David

    2006-01-01

    Several studies have shown that in the arterial wall hepatocyte growth factor/scatter factor (HGF/SF) is expressed by smooth muscle cells (SMCs) but acts on endothelial cells, not SMCs. Other studies, however, have indicated that SMCs can respond to HGF/SF. We have reinvestigated expression and activity of HGF/SF and its receptor MET in arterial SMC and endothelial cell cultures and in whole arteries after superficial or deep injury or atherogenesis. High-density cultures of SMCs produced HGF/SF but did not express MET, whereas SMCs, at the leading edge of injured cultures, expressed both ligand and receptor and showed a dramatic motility and growth response to HGF/SF. In line with these results, HGF/SF and MET expression was undetectable in the media of uninjured carotid arteries but was induced after deep arterial injury in areas of SMC migration in the neointima. Strong MET expression was also observed in the SMCs of the atherosclerotic lesions of homozygous apoE−/− mice, whereas HGF/SF was expressed by macrophage-derived foam cells. These results demonstrate that MET is induced in migrating and proliferating SMCs and that HGF/SF and MET are key mediators of the SMC response in atherogenesis. PMID:16400035

  8. 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.

  9. High glucose potentiates L-FABP mediated fibrate induction of PPARα in mouse hepatocytes.

    PubMed

    Petrescu, Anca D; McIntosh, Avery L; Storey, Stephen M; Huang, Huan; Martin, Gregory G; Landrock, Danilo; Kier, Ann B; Schroeder, Friedhelm

    2013-08-01

    Although liver fatty acid binding protein (L-FABP) binds fibrates and PPARα in vitro and enhances fibrate induction of PPARα in transformed cells, the functional significance of these findings is unclear, especially in normal hepatocytes. Studies with cultured primary mouse hepatocytes show that: 1) At physiological (6mM) glucose, fibrates (bezafibrate, fenofibrate) only weakly activated PPARα transcription of genes in LCFA β-oxidation; 2) High (11-20mM) glucose, but not maltose (osmotic control), significantly potentiated fibrate-induction of mRNA of these and other PPARα target genes to increase LCFA β-oxidation. These effects were associated with fibrate-mediated redistribution of L-FABP into nuclei-an effect prolonged by high glucose-but not with increased de novo fatty acid synthesis from glucose; 3) Potentiation of bezafibrate action by high glucose required an intact L-FABP/PPARα signaling pathway as shown with L-FABP null, PPARα null, PPARα inhibitor-treated WT, or PPARα-specific fenofibrate-treated WT hepatocytes. High glucose alone in the absence of fibrate was ineffective. Thus, high glucose potentiation of PPARα occurred through FABP/PPARα rather than indirectly through other PPARs or glucose induced signaling pathways. These data indicated L-FABP's importance in fibrate-induction of hepatic PPARα LCFA β-oxidative genes, especially in the context of high glucose levels.

  10. Hepatocyte growth factor induction of macrophage chemoattractant protein-1 and osteophyte-inducing factors in osteoarthritis.

    PubMed

    Dankbar, Berno; Neugebauer, Katja; Wunrau, Christina; Tibesku, Carsten O; Skwara, Adrian; Pap, Thomas; Fuchs-Winkelmann, Susanne

    2007-05-01

    In osteoarthritis (OA), hepatocyte growth factor (HGF) is supposed to play a role in cartilage repair. Because the development of osteophytes is a major characteristic of OA and thought to be part of an attempted repair process, the purpose of this study was to determine whether HGF may be involved in osteophyte formation. HGF levels in synovial fluids from 41 patients assessed by enzyme immunosorbant assay were correlated with disease severity and osteophyte formation, evaluated by anteroposterior weight-bearing radiographs. Detection of HGF, c-Met, and CD68 in cartilage and synovial tissues was assessed by immunohistochemistry. Effects of HGF on the secretion of TGF-beta1 and BMP-2 by chondrocytes, fibroblast-like synovial cells (FLS), and macrophages as well as HGF-induced secretion of MCP-1 by FLS and chondrocytes were determined by ELISA. HGF was detected in all synovial fluids and concentrations correlated highly with disease severity and osteophyte formation (p < 0.001). Immunohistochemistry revealed weak synovial staining for HGF, whereas increasing numbers of HGF expressing chondrocytes were detected depending on disease severity. In addition, an increased number of macrophages in synovial specimens was observed, which was likewise severity dependent. In a series of subsequent in vitro studies, HGF remarkable induced MCP-1 secretion by FLS in a dose-dependent manner. No effect on TGF-beta1 and BMP-2 secretion by FLS and chondrocytes was evident upon HGF stimulation, whereas secretion of these growth factors by PMA-differentiated THP-1 cells was significantly increased by HGF. The results indicate that HGF may facilitate osteophyte development by promoting MCP-1-mediated entry of monocytes/macrophages into the OA-affected joint and/or by stimulating macrophage-derived growth factors.

  11. 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

  12. 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

  13. Caspase-12 mediates carbon tetrachloride-induced hepatocyte apoptosis in mice

    PubMed Central

    Liu, Hua; Wang, Zhe; Nowicki, Michael J

    2014-01-01

    AIM: To investigate the role of caspase-12 and its downstream targets in carbon tetrachloride (CCl4)-induced hepatocyte apoptosis. METHODS: The role of caspase-12 was determined by using caspase-12 knock-out (-/-) mice. CCl4 (300 μL/kg body weight) or vehicle (corn oil) was administered to caspase-12+/+ or caspase-12-/- mice as a single intraperitoneal injection. The animals were sacrificed 24 h after the CCl4 treatment. Blood was collected to evaluate liver function by the measurement of the activity of alanine aminotransferase. Liver samples were used for the measurements of reactive oxygen species using plasma malondialdehyde as biomarker, hepatocyte apoptosis was evaluated via terminal transferase-mediated dUTP nick-end labeling and controlled by morphologic study, and cytochrome C release and caspase activations were measured by Western blotting. RESULTS: Administration of a low dose of CCl4 resulted in hepatocyte apoptosis and acute liver injury in wild-type mice. CCl4 also induced the generation of reactive oxygen species and induction of endoplasmic reticulum stress in the liver followed by activations of caspase-12, -9 and -3 as well as release of small amounts of cytochrome C. However, in the CCl4-treated caspase-12-/- mice, activation of caspase-9 and -3 were significantly attenuated (P < 0.05); no effect was seen in cytochrome C release. CCl4-induced apoptosis and liver damage was markedly reduced in caspase-12-/- mice compared to caspase-12+/+ mice (P < 0.05). The active form of caspase-8 was not detected in either caspase-12+/+ or caspase-12-/- mice. There was no significant different in the formation of reactive oxygen species in the livers of caspase-12+/+ and caspase-12-/- mice treated with CCl4. CONCLUSION: Caspase-12 plays a pivotal role in CCl4-induced hepatic apoptosis through the activation of the downstream effector caspase-3 directly and/or indirectly via capase-9 activation. PMID:25561786

  14. 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

  15. 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.

  16. A plasma membrane protein is involved in cell contact-mediated regulation of tissue-specific genes in adult hepatocytes

    PubMed Central

    1991-01-01

    We have identified the liver-regulating protein (LRP), a cell surface protein involved in the maintenance of hepatocyte differentiation when cocultured with rat liver epithelial cells (RLEC). LRP was defined by immunoreactivity to a monoclonal antibody (mAb L8) prepared from RLEC. mAb L8 specifically detected two polypeptides of 85 and 73 kD in immunoprecipitation of both hepatocyte- and RLEC-iodinated plasma membranes. The involvement of these polypeptides, which are integral membrane proteins, in cell interaction-mediated regulation of hepatocytes was assessed by evaluating the perturbing effects of the antibody on cocultures with RLEC. Several parameters characteristic of differentiated hepatocytes were studied, such as liver-specific and house-keeping gene expression, cytoskeletal organization and deposition of extracellular matrix (ECM). An early cytoskeletal disturbance was evidenced and a marked alteration of hepatocyte functional capacity was observed in the presence of the antibody, together with a loss of ECM deposition. By contrast, cell-cell aggregation or cell adhesion to various extracellular matrix components were not affected. These findings suggest that LRP is distinct from an extracellular matrix receptor. The fact that early addition of mAb L8 during cell contact establishment was necessary to be effective may indicate that LRP is a novel plasma membrane protein that plays an early pivotal role in the coordinated metabolic changes which lead to the differentiated phenotype of mature hepatocytes. PMID:1918151

  17. Epithelial differentiation of metanephric mesenchymal cells after stimulation with hepatocyte growth factor or embryonic spinal cord.

    PubMed Central

    Karp, S L; Ortiz-Arduan, A; Li, S; Neilson, E G

    1994-01-01

    Mammalian kidney emerges from metanephric mesenchyme following the insertion of a migrating ureteric bud. The pattern morphology of mesenchymal specialization during tubular segmentation is remarkably complex, and the relative contribution of pattern gradients from the microenvironment versus the instructive role of individual cells is not known. We have started to examine the differentiation of metanephric mesenchyme using cultures of metanephric ridge (MMR) cells from day 13.5 mouse embryos to investigate the conversion of mesenchyme toward kidney epithelium in vitro. One of our mesenchymal clones, MMR1, expresses little Pax2, uvomorulin, or cytokeratin but does express neural cell adhesion molecule, bc12, and desmin; these are properties consistent with an early stem cell. Coculture of MMR1 cells with embryonic spinal cord leads to the induction of a more differentiated cell phenotype characterized by decreased expression of neural cell adhesion molecule, the appearance of uvomorulin, and the emergence of cytokeratin, all consistent with an evolution toward epithelium. We were also able to detect the hepatocyte growth factor receptor c-met on MMR1 cells by indirect immunofluorescence. When MMR1 cells were stimulated with hepatocyte growth factor, neural cell adhesion molecule expression decreased and uvomorulin appeared. This effect of hepatocyte growth factor, as a single cytokine, may be important in the early assemblage of kidney, since we were able to detect mRNA transcripts encoding c-met from mouse embryo metanephric kidneys. Images PMID:8202482

  18. Association of hepatocyte growth factor expression with salivary gland tumor differentiation.

    PubMed

    Tsukinoki, Keiichi; Yasuda, Masanori; Asano, Shigeyuki; Karakida, Kazunari; Ota, Yoshihide; Osamura, R Yoshiyuki; Watanabe, Yoshihisa

    2003-12-01

    To clarify the significance of hepatocyte growth factor (HGF) expression in salivary gland tumors, HGF distribution in tissue sections and HGF concentrations in saliva and serum were examined. Sixty salivary gland adenomas, 61 salivary gland carcinomas and three autopsy fetuses were studied. Hepatocyte growth factor expression was observed in the duct-type luminal cells by immunohistochemical staining and in situ hybridization. However, HGF failed to be expressed in acinar cells and myoepithelium of normal salivary gland tissue. Hepatocyte growth factor tended to be expressed more intensely in benign salivary gland tumors than in malignant salivary gland tumors (P < 0.0001). In highly malignant tumors, the expression was limited in some cases. Salivary and serological HGF concentrations of 18 patients, comprised of 12 benign cases and six malignant cases, were analyzed before and after operation by an ELISA system. The concentrations were distinctly elevated after operation, in both saliva and serum, compared to before operation (P < 0.0005). However, there were no significant relationships between HGF concentration and histology, age, gender, size or location. Our findings suggest that HGF may play an important role in the development of salivary ducts of normal salivary tissues and differentiation of ductal structures of their neoplasms, while HGF kinetics in saliva and serum would be less likely to reflect the neoplastic character, benign or malignant.

  19. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth

    PubMed Central

    1992-01-01

    Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit. HGF binding to endothelial cells identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190MET receptor. Sub- nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate. PMID:1383237

  20. 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

  1. Signal Transduction Mechanism for Serotonin 5-HT2B Receptor-Mediated DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes.

    PubMed

    Naito, Kota; Tanaka, Chizuru; Mitsuhashi, Manami; Moteki, Hajime; Kimura, Mitsutoshi; Natsume, Hideshi; Ogihara, Masahiko

    2016-01-01

    The involvement of serotonin (5-hydroxytryptamine; 5-HT) and the 5-HT2 receptor subtypes in the induction of DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes to elucidate the intracellular signal transduction mechanisms. Hepatocyte parenchymal cells maintained in a serum-free, defined medium, synthesized DNA and proliferated in the presence of 5-HT or a selective 5-HT2B receptor agonist, BW723C86, but not in the presence of 5-HT2A, or 5-HT2C receptor agonists (TCB-2 and CP809101, respectively), in a time- and dose-dependent manner. A selective 5-HT2B receptor antagonist, LY272015 (10(-7) M), and a specific phospholipase C (PLC) inhibitor, U-73122 (10(-6) M), as well as specific inhibitors of growth-related signal transducers-including AG1478, LY294002, PD98059, and rapamycin-completely inhibited 5-HT (10(-6) M)- or BW723C86 (10(-6) M)-induced hepatocyte DNA synthesis and proliferation. Both 5-HT and BW723C86 were shown to significantly stimulate the phosphorylation of epidermal growth factor (EGF)/transforming growth factor (TGF)-α receptor tyrosine kinase (p175 kDa) and extracellular signal-regulated kinase (ERK) 2 on Western blot analysis. These results suggest that the proliferative mechanism of activating 5-HT is mediated mainly through 5-HT2B receptor-stimulated Gq/PLC and EGF/TGF-α-receptor/phosphatidylinositol 3-kinase (PI3K)/ERK2/mammalian target of rapamycin (mTOR) signaling pathways in primary cultured hepatocytes.

  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. 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.

  4. Apoptosis signal-regulating kinase 1 mediates the inhibitory effect of hepatocyte nuclear factor-4α on hepatocellular carcinoma

    PubMed Central

    Xu, Wen-Ping; Shi, Bin; Zhang, Xin; Xie, Wei-Fen

    2016-01-01

    Previous studies provided substantial evidence of a striking suppressive effect of hepatocyte nuclear factor 4α (HNF4α) on hepatocellular carcinoma (HCC). Apoptosis signal-regulating kinase 1 (ASK1) is involved in death receptor-mediated apoptosis and may acts as a tumor suppressor in hepatocarcinogenesis. However, the status and function of ASK1 during HCC progression are unclear. In this study, we found that HNF4α increased ASK1 expression by directly binding to its promoter. ASK1 expression was dramatically suppressed and correlated with HNF4α levels in HCC tissues. Reduced ASK1 expression was associated with aggressive tumors and poor prognosis for human HCC. Moreover, ASK1 inhibited the malignant phenotype of HCC cells in vitro. Intratumoral ASK1 injection significantly suppressed the growth of subcutaneous HCC xenografts in nude mice. More interestingly, systemic ASK1 delivery strikingly inhibited the growth of orthotopic HCC nodules in NOD/SCID mice. In addition, inhibition of endogenous ASK1 partially reversed the suppressive effects of HNF4α on HCC. Collectively, this study highlights the suppressive effect of ASK1 on HCC and its biological significance in HCC development. These outcomes broaden the knowledge of ASK1 function in HCC progression, and provide a novel potential prognostic biomarker and therapeutic target for advanced HCC. PMID:27050273

  5. 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

  6. Echinomycin decreases induction of vascular endothelial growth factor and hepatocyte regeneration in acetaminophen toxicity in mice.

    PubMed

    Milesi-Hallé, Alessandra; McCullough, Sandra; Hinson, Jack A; Kurten, Richard C; Lamps, Laura W; Brown, Aliza; James, Laura P

    2012-04-01

    Up-regulation of vascular endothelial growth factor (VEGF) is important to hepatocyte regeneration in the late stages of acetaminophen (APAP) toxicity in the mouse. This study was conducted to examine the relationship of hypoxia-inducible factor 1α (HIF-1α) to VEGF and hepatocyte regeneration in APAP toxicity using an inhibitor of HIF-1α DNA-binding activity, echinomycin (EC). B6C3F1 male mice were treated with APAP (200 mg/kg IP), followed by EC (0.15 mg IP) and killed at 4 hr. Serum alanine aminotransferase (ALT), necrosis, hepatic glutathione (GSH) and APAP protein adducts were comparable in the APAP/EC and the APAP/veh mice at 4 hr. Additional studies showed that high dose EC (0.3 mg) reduced hepatic VEGF but also lowered hepatic GSH. Subsequent studies were performed using the 0.15-mg dose of EC. Although EC 0.15 mg had no effect on hepatic VEGF levels at 8 hr, by 24 hr VEGF levels were decreased by 40%. Toxicity (ALT and histopathology) was comparable in the APAP and APAP/EC groups at 24 and 48 hr. Proliferating cell nuclear antigen expression was reduced by both Western blot analysis and immunohistochemical staining in the APAP/EC mice at 48 hr. The data support the hypothesis that induction of HIF-1α, its binding to DNA and subsequent expression of VEGF are important factors in hepatocyte regeneration in APAP toxicity in the mouse.

  7. Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor beta 1.

    PubMed Central

    Oberhammer, F A; Pavelka, M; Sharma, S; Tiefenbacher, R; Purchio, A F; Bursch, W; Schulte-Hermann, R

    1992-01-01

    In previous studies hepatocytes undergoing cell death by apoptosis but not normal hepatocytes in rat liver showed immunostaining for transforming growth factor beta 1 (TGF-beta 1). Staining was much stronger with antibodies recognizing the pro-region of TGF-beta 1 than the mature peptide itself. Therefore we investigated the ability of both forms of TGF-beta 1 to induce apoptosis in primary cultures of rat hepatocytes. Mature TGF-beta 1 induced rounding up of the cells and fragmentation into multiple vesicles. As revealed by the DNA-specific stain H33258, the chromatin of these cells condensed and segregated into masses at the nuclear membrane; this was obviously followed by fragmentation of the nucleus. Ultrastructurally the cytoplasm was well preserved, as demonstrated by the presence of intact cell organelles. These features strongly suggest the occurrence of apoptosis. Quantification of nuclei with condensed chromatin revealed that mature TGF-beta 1 was 30-fold more effective than the TGF-beta 1 latency-associated protein complex. Finally, we administered TGF-beta 1 in vivo using an experimental model in which regression of rat liver was initiated by a short preceding treatment with the hepatomitogen cyproterone acetate. Two doses of TGF-beta 1, each 1 nM/kg, augmented the incidence of apoptotic hepatocytes 5-fold. Equimolar doses of TGF-beta 1 latency-associated protein complex were ineffective. These studies suggest that TGF-beta 1 is involved in the initiation of apoptosis in the liver and that the mature form of TGF-beta 1 is the active principle. Images PMID:1608949

  8. 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

  9. Ebola Virus Modulates Transforming Growth Factor β Signaling and Cellular Markers of Mesenchyme-Like Transition in Hepatocytes

    PubMed Central

    Wahl-Jensen, Victoria; Safronetz, David; Trost, Brett; Hoenen, Thomas; Arsenault, Ryan; Feldmann, Friederike; Traynor, Dawn; Postnikova, Elena; Kusalik, Anthony; Napper, Scott; Blaney, Joseph E.; Feldmann, Heinz; Jahrling, Peter B.

    2014-01-01

    ABSTRACT Ebola virus (EBOV) causes a severe hemorrhagic disease in humans and nonhuman primates, with a median case fatality rate of 78.4%. Although EBOV is considered a public health concern, there is a relative paucity of information regarding the modulation of the functional host response during infection. We employed temporal kinome analysis to investigate the relative early, intermediate, and late host kinome responses to EBOV infection in human hepatocytes. Pathway overrepresentation analysis and functional network analysis of kinome data revealed that transforming growth factor (TGF-β)-mediated signaling responses were temporally modulated in response to EBOV infection. Upregulation of TGF-β signaling in the kinome data sets correlated with the upregulation of TGF-β secretion from EBOV-infected cells. Kinase inhibitors targeting TGF-β signaling, or additional cell receptors and downstream signaling pathway intermediates identified from our kinome analysis, also inhibited EBOV replication. Further, the inhibition of select cell signaling intermediates identified from our kinome analysis provided partial protection in a lethal model of EBOV infection. To gain perspective on the cellular consequence of TGF-β signaling modulation during EBOV infection, we assessed cellular markers associated with upregulation of TGF-β signaling. We observed upregulation of matrix metalloproteinase 9, N-cadherin, and fibronectin expression with concomitant reductions in the expression of E-cadherin and claudin-1, responses that are standard characteristics of an epithelium-to-mesenchyme-like transition. Additionally, we identified phosphorylation events downstream of TGF-β that may contribute to this process. From these observations, we propose a model for a broader role of TGF-β-mediated signaling responses in the pathogenesis of Ebola virus disease. IMPORTANCE Ebola virus (EBOV), formerly Zaire ebolavirus, causes a severe hemorrhagic disease in humans and nonhuman

  10. 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.

  11. Identification of a novel variant hepatocyte growth factor secreted by spleen-derived stromal cells.

    PubMed

    Miau, L H; Jan, Y W; Shen, B J; Tsai, W H; Lee, H S; Lee, S C

    1996-06-25

    Stromal cells can interact with parenchymal cells by secreting various cytokines to affect the growth, differentiation or movement of the latter. Here we report the identification and characterization of a novel variant hepatocyte growth factor (HGF) from the conditioned medium of stromal cells derived from mouse spleen. Compared to human HGF, it has much lower heparin-binding activity and lacks the beta-chain. Its molecular weight, 70 kDa, is very close to that of the alpha-chain of HGF. Human HGF homologue was not found in the conditioned medium. The conditioned medium of stromal cells, like recombinant HGF, could inhibit the growth of rat hepatoma cells. The inhibitory activity was presumably attributed to this novel HGF because the inhibitory activity, as the existence of this novel HGF, was confined to the identical fractions after heparin-column chromatography. Furthermore, this activity could be specifically abrogated by neutralizing anti-HGF antibodies.

  12. 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

  13. Cytotoxic effect and role of exogenous antioxidants in carpet dust mediated toxicity in rat hepatocytes in vitro.

    PubMed

    Ameen, Mohamed; Ahmad, Iqbal; Musthapa, M Syed; Rahman, Qamar

    2004-08-01

    Carpet industries bear a great deal of economic and commercial significance in India. In order to safe guard the workers against the health hazards caused by dust in their occupational environment; it necessitates studying the biological importance of these dusts. The present study was designed to investigate the toxicity of carpet dust (knotted and tuffted) on isolated rat hepatocytes. The hepatocytes were isolated by collagenase perfusion method and cells were incubated with different concentration of carpet dust (100-5000 microg/10(6) cells) with various time (30-180 min) intervals. An exogenous antioxidant vitamin-E also used to find out the role of antioxidants and free radical production in carpet dust mediated toxicity. Cell viability by trypan blue exclusion and leakage of enzyme lactate dehydrogenase (LDH) were determined. Reduced glutathione (GSH), formation of thiobarbituric acid reactive substance (TBARS) were also measured. A significant decrease in the cell viability was observed after 60, 180 min upon incubation with tuffted carpet dust, while knotted carpet dust caused a significant decrease in the viability after 180 min. LDH leakage was parallel to the cell viability. Thiobarbituric acid reactive substance was significantly increased at 30 and 60 min with carpet dust treated hepatocytes. Dust at 1000 and 5000 microg dose level showed significantly increased formation of TBARS at 30 min incubation. However, when hepatocytes were co-incubated with carpet dust and Vit-E (10, 15 microM), a significant decrease in LDH release and TBARS production was observed while 15 microM Vit-E showed an enhanced protection than 10 microM Vit-E treated hepatocytes. The effect of carpet dust on cell viability, LDH leakage, TBARS production, GSH depletion was time and dose-dependent. Moreover, we observed that tuffted carpet dust causes greater effect than knotted one on the above mentioned parameters. Our studies also revealed that Vit-E in culture media diminishes

  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. Gene deletions and amplifications in human hepatocellular carcinomas: correlation with hepatocyte growth regulation.

    PubMed

    Nalesnik, Michael A; Tseng, George; Ding, Ying; Xiang, Guo-Sheng; Zheng, Zhong-liang; Yu, YanPing; Marsh, James W; Michalopoulos, George K; Luo, Jian-Hua

    2012-04-01

    Tissues from 98 human hepatocellular carcinomas (HCCs) obtained from hepatic resections were subjected to somatic copy number variation (CNV) analysis. Most of these HCCs were discovered in livers resected for orthotopic transplantation, although in a few cases, the tumors themselves were the reason for the hepatectomies. Genomic analysis revealed deletions and amplifications in several genes, and clustering analysis based on CNV revealed five clusters. The LSP1 gene had the most cases with CNV (46 deletions and 5 amplifications). High frequencies of CNV were also seen in PTPRD (21/98), GNB1L (18/98), KIAA1217 (18/98), RP1-1777G6.2 (17/98), ETS1 (11/98), RSU1 (10/98), TBC1D22A (10/98), BAHCC1 (9/98), MAML2 (9/98), RAB1B (9/98), and YIF1A (9/98). The existing literature regarding hepatocytes or other cell types has connected many of these genes to regulation of cytoskeletal architecture, signaling cascades related to growth regulation, and transcription factors directly interacting with nuclear signaling complexes. Correlations with existing literature indicate that genomic lesions associated with HCC at the level of resolution of CNV occur on many genes associated directly or indirectly with signaling pathways operating in liver regeneration and hepatocyte growth regulation.

  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. Human recombinant vascular endothelial growth factor reduces necrosis and enhances hepatocyte regeneration in a mouse model of acetaminophen toxicity.

    PubMed

    Donahower, Brian C; McCullough, Sandra S; Hennings, Leah; Simpson, Pippa M; Stowe, Cindy D; Saad, Ali G; Kurten, Richard C; Hinson, Jack A; James, Laura P

    2010-07-01

    We reported previously that vascular endothelial growth factor (VEGF) was increased in acetaminophen (APAP) toxicity in mice and treatment with a VEGF receptor inhibitor reduced hepatocyte regeneration. The effect of human recombinant VEGF (hrVEGF) on APAP toxicity in the mouse was examined. In early toxicity studies, B6C3F1 mice received hrVEGF (50 microg s.c.) or vehicle 30 min before receiving APAP (200 mg/kg i.p.) and were sacrificed at 2, 4, and 8 h. Toxicity was comparable at 2 and 4 h, but reduced in the APAP/hrVEGF mice at 8 h (p < 0.05) compared with the APAP/vehicle mice. Hepatic glutathione (GSH) and APAP protein adduct levels were comparable between the two groups of mice, with the exception that GSH was higher at 8 h in the hrVEGF-treated mice. Subsequently, mice received two doses (before and 10 h) or three doses (before and 10 and 24 h) of hrVEGF; alanine aminotransferase values and necrosis were reduced at 24 and 36 h, respectively, in the APAP/hrVEGF mice (p < 0.05) compared with the APAP/vehicle mice. Proliferating cell nuclear antigen expression was enhanced, and interleukin-6 expression was reduced in the mice that received hrVEGF (p < 0.05) compared with the APAP/vehicle mice. In addition, treatment with hrVEGF lowered plasma hyaluronic acid levels and neutrophil counts at 36 h. Cumulatively, the data show that treatment with hrVEGF reduced toxicity and increased hepatocyte regeneration in APAP toxicity in the mouse. Attenuation of sinusoidal cell endothelial dysfunction and changes in neutrophil dynamics may be operant mechanisms in the hepatoprotection mediated by hrVEGF in APAP toxicity.

  19. Electrochemical evidence for asialoglycoprotein receptor--mediated hepatocyte adhesion and proliferation in three dimensional tissue engineering scaffolds.

    PubMed

    Vasanthan, Kirthanashri S; Sethuraman, Swaminathan; Parthasarathy, Meera

    2015-08-26

    Asialoglycoprotein receptor (ASGPR) is one of the recognition motifs on the surface of hepatocytes, which promote their adhesion to extracellular matrix in liver tissue and appropriate artificial surfaces. ASGPR-mediated adhesion is expected to minimize trans-differentiation of hepatocytes in vitro that is generally observed in integrin-mediated adhesion. The aim of the present study is to verify the role of ASGPR in hepatocyte adhesion and proliferation in scaffolds for hepatic tissue engineering. Scanning Electrochemical Microscopy (SECM) is emerging as a suitable non-invasive analytical tool due to its high sensitivity and capability to correlate the morphology and activity of live cells. HepG2 cells and rat primary hepatocytes cultured in Polyvinyl alcohol (PVA)/Gelatin hydrogel scaffolds with and without galactose (a ligand for ASGPR) modification are studied using SECM. Systematic investigation of live cells cultured for different durations in scaffolds of different compositions (9:1 and 8:2 PVA:Gelatin with and without galactose) reveals significant improvement in cell-cell communication and proliferation on galactose incorporated scaffolds, thereby demonstrating the positive influence of ASGPR-mediated adhesion. In this work, we have also developed a methodology to quantify the respiratory activity and intracellular redox activity of live cells cultured in porous tissue engineering scaffolds. Using this methodology, SECM results are compared with routine cell culture assays viz., MTS ((1-Oxyl-2,2,5,5,-tetramethyl-Δ3-pyrroline-3-methyl) Methanethiosulfonate) and Albumin assays to demonstrate the better sensitivity of SECM. In addition, the present study demonstrates SECM as a reliable and sensitive tool to monitor the activity of live cells cultured in scaffolds for tissue engineering, which could be used on a routine basis.

  20. Electrochemical evidence for asialoglycoprotein receptor--mediated hepatocyte adhesion and proliferation in three dimensional tissue engineering scaffolds.

    PubMed

    Vasanthan, Kirthanashri S; Sethuraman, Swaminathan; Parthasarathy, Meera

    2015-08-26

    Asialoglycoprotein receptor (ASGPR) is one of the recognition motifs on the surface of hepatocytes, which promote their adhesion to extracellular matrix in liver tissue and appropriate artificial surfaces. ASGPR-mediated adhesion is expected to minimize trans-differentiation of hepatocytes in vitro that is generally observed in integrin-mediated adhesion. The aim of the present study is to verify the role of ASGPR in hepatocyte adhesion and proliferation in scaffolds for hepatic tissue engineering. Scanning Electrochemical Microscopy (SECM) is emerging as a suitable non-invasive analytical tool due to its high sensitivity and capability to correlate the morphology and activity of live cells. HepG2 cells and rat primary hepatocytes cultured in Polyvinyl alcohol (PVA)/Gelatin hydrogel scaffolds with and without galactose (a ligand for ASGPR) modification are studied using SECM. Systematic investigation of live cells cultured for different durations in scaffolds of different compositions (9:1 and 8:2 PVA:Gelatin with and without galactose) reveals significant improvement in cell-cell communication and proliferation on galactose incorporated scaffolds, thereby demonstrating the positive influence of ASGPR-mediated adhesion. In this work, we have also developed a methodology to quantify the respiratory activity and intracellular redox activity of live cells cultured in porous tissue engineering scaffolds. Using this methodology, SECM results are compared with routine cell culture assays viz., MTS ((1-Oxyl-2,2,5,5,-tetramethyl-Δ3-pyrroline-3-methyl) Methanethiosulfonate) and Albumin assays to demonstrate the better sensitivity of SECM. In addition, the present study demonstrates SECM as a reliable and sensitive tool to monitor the activity of live cells cultured in scaffolds for tissue engineering, which could be used on a routine basis. PMID:26347169

  1. Hepatocyte growth factor induces tubulogenesis of primary renal proximal tubular epithelial cells.

    PubMed

    Bowes, R C; Lightfoot, R T; Van De Water, B; Stevens, J L

    1999-07-01

    Hepatocyte growth factor (HGF)-induced tubulogenesis has been demonstrated with renal epithelial cell lines grown in collagen gels but not with primary cultured renal proximal tubular epithelial cells (RPTEs). We show that HGF selectively induces proliferation and branching morphogenesis of primary cultured rat RPTEs. Additional growth factors including fibroblast growth factor (FGF)-1, epidermal growth factor (EGF), FGF-7, or insulin-like growth factor-1 (IGF-1) did not selectively induce tubulogenesis. However, when administered in combination, these factors initiated branching morphogenesis comparable to HGF alone and greatly augmented HGF-induced proliferation and branching. Microscopic analysis revealed that branching RPTEs were undergoing tubulogenesis and formed a polarized epithelium. TGF-beta1 blocked HGF- or growth factor cocktail (GFC; HGF, FGF-1, EGF, IGF-1)-induced proliferation and branching morphogenesis. Adding TGF-beta1 after GFC-induced tubulogenesis had occurred caused a progressive regression of the tubular structures, a response associated with an increase in apoptosis of the RPTEs. Primary cultured RPTEs are capable of undergoing HGF-induced tubulogenesis. Unlike cell lines, combinations of growth factors differentially augment the response. PMID:10362020

  2. The Brain Hepatocyte Growth Factor/c-Met Receptor System: A New Target for the Treatment of Alzheimer's Disease.

    PubMed

    Wright, John W; Harding, Joseph W

    2015-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease increasing in frequency as life expectancy of the world's population increases. There are an estimated 5 million diagnosed AD patients in the U.S. and 16 million worldwide with no adequate treatment presently available. New therapeutic approaches are needed to slow, and hopefully reverse, disease progression. This review summarizes available information regarding an overlooked therapeutic target that may offer a treatment to slow and hopefully halt AD, namely the hepatocyte growth factor (HGF)/c-Met receptor system. Activation of the c-Met receptor stimulates mitogenesis, motogenesis, morphogenesis, the ability to mediate stem cell differentiation and neurogenesis, and protects against tissue insults in a wide range of cells including neurons. This growth factor system has recently been shown to induce dendritic arborization and synaptogenesis when stimulated by a newly developed angiotensin-based analogue, N-hexanoic-Tyr-Ile-(6) amino hexanoic amide (Dihexa). This small molecule was derived from the pre-prototype molecule Nle1-angiotensin IV and has shown promise in facilitating the formation of new functional synaptic connections and augmenting memory consolidation in animal models of AD. Dihexa is a first-in-class compound that is orally active, penetrates the blood-brain barrier, and facilitates memory consolidation and retrieval. This angiotensin-based small molecule may be efficacious as a treatment for AD.

  3. Biological effects of targeted inactivation of hepatocyte growth factor-like protein in mice.

    PubMed Central

    Bezerra, J A; Carrick, T L; Degen, J L; Witte, D; Degen, S J

    1998-01-01

    Hepatocyte growth factor-like protein (HGFL) is a liver-derived serum glycoprotein involved in cell proliferation and differentiation, and is proposed to have a fundamental role in embryogenesis, fertility, hematopoiesis, macrophage activation, and tissue repair. To assess the in vivo effects of total loss of HGFL, we generated mice with targeted disruption of the gene resulting in loss of the protein. Disruption of the HGFL gene allowed for normal embryogenesis, and followed a Mendelian pattern of genetic transmission. Mice homozygous for the targeted allele (HGFL-/- mice) are fertile, and grow to adulthood without obvious phenotypic abnormalities in unchallenged animals, except for development of lipid-containing cytoplasmic vacuoles in hepatocytes throughout the liver lobules. These histologic changes are not accompanied by discernible changes in synthetic or excretory hepatic functions. Hematopoiesis appears unaltered, and although macrophage activation is delayed in the absence of HGFL, migration to the peritoneal cavity upon challenge with thioglycollate was similar in HGFL-/- and wild-type mice. Challenged with incision to skin, HGFL-/- mice display normal wound healing. These data demonstrate that HGFL is not essential for embryogenesis, fertility, or wound healing. HGFL-deficient mice will provide a valuable means to assess the role of HGFL in hepatic and systemic responses to inflammatory and infectious stimuli in vivo. PMID:9486989

  4. Metabolic hormones regulate basal and growth hormone-dependent igf2 mRNA level in primary cultured coho salmon hepatocytes: effects of insulin, glucagon, dexamethasone, and triiodothyronine.

    PubMed

    Pierce, A L; Dickey, J T; Felli, L; Swanson, P; Dickhoff, W W

    2010-03-01

    Igf1 and Igf2 stimulate growth and development of vertebrates. Circulating Igfs are produced by the liver. In mammals, Igf1 mediates the postnatal growth-promoting effects of growth hormone (Gh), whereas Igf2 stimulates fetal and placental growth. Hepatic Igf2 production is not regulated by Gh in mammals. Little is known about the regulation of hepatic Igf2 production in nonmammalian vertebrates. We examined the regulation of igf2 mRNA level by metabolic hormones in primary cultured coho salmon hepatocytes. Gh, insulin, the glucocorticoid agonist dexamethasone (Dex), and glucagon increased igf2 mRNA levels, whereas triiodothyronine (T(3)) decreased igf2 mRNA levels. Gh stimulated igf2 mRNA at physiological concentrations (0.25x10(-9) M and above). Insulin strongly enhanced Gh stimulation of igf2 at low physiological concentrations (10(-11) M and above), and increased basal igf2 (10(-8) M and above). Dex stimulated basal igf2 at concentrations comparable to those of stressed circulating cortisol (10(-8) M and above). Glucagon stimulated basal and Gh-stimulated igf2 at supraphysiological concentrations (10(-7) M and above), whereas T(3) suppressed basal and Gh-stimulated igf2 at the single concentration tested (10(-7) M). These results show that igf2 mRNA level is highly regulated in salmon hepatocytes, suggesting that liver-derived Igf2 plays a significant role in salmon growth physiology. The synergistic regulation of igf2 by insulin and Gh in salmon hepatocytes is similar to the regulation of hepatic Igf1 production in mammals.

  5. Hepatitis A complicated with acute renal failure and high hepatocyte growth factor: A case report.

    PubMed

    Oe, Shinji; Shibata, Michihiko; Miyagawa, Koichiro; Honma, Yuichi; Hiura, Masaaki; Abe, Shintaro; Harada, Masaru

    2015-08-28

    A 58-year-old man was admitted to our hospital. Laboratory data showed severe liver injury and that the patient was positive for immunoglobulin M anti-hepatitis A virus (HAV) antibodies. He was also complicated with severe renal dysfunction and had an extremely high level of serum hepatocyte growth factor (HGF). Therefore, he was diagnosed with severe acute liver failure with acute renal failure (ARF) caused by HAV infection. Prognosis was expected to be poor because of complications by ARF and high serum HGF. However, liver and renal functions both improved rapidly without intensive treatment, and he was subsequently discharged from our hospital on the 21(st) hospital day. Although complication with ARF and high levels of serum HGF are both important factors predicting poor prognosis in acute liver failure patients, the present case achieved a favorable outcome. Endogenous HGF might play an important role as a regenerative effector in injured livers and kidneys.

  6. Tyrosine kinase inhibitor STI571 enhances thyroid cancer cell motile response to Hepatocyte Growth Factor.

    PubMed

    Frasca, F; Vigneri, P; Vella, V; Vigneri, R; Wang, J Y

    2001-06-28

    The Hepatocyte Growth Factor (HGF) and its receptor Met are physiological regulators of cell migration. HGF and Met have also been implicated in tumor progression and metastasis. We show here that the tyrosine kinase inhibitor STI571 has a stimulatory effect on HGF-induced migration and branching morphogenesis in thyroid cancer but not in primary or immortalized thyroid epithelial cells. These stimulatory effects of STI571 are observed at a concentration that is clinically relevant. The STI571-enhanced motile response can be correlated with an increase in the Met receptor tyrosine phosphorylation as well as ERK and Akt activation by HGF. Interestingly, one of the targets of STI571, namely the c-Abl tyrosine kinase, is activated by HGF and is recruited at the migrating edge of thyroid cancer cells. These data suggests that c-Abl and/or STI571-inhibited tyrosine kinases can negatively regulate the Met receptor to restrain the motile response in thyroid cancer cells.

  7. 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.

  8. Regulation of choline deficiency apoptosis by epidermal growth factor in CWSV-1 rat hepatocytes.

    PubMed

    Albright, Craig D; da Costa, Kerry-Ann; Craciunescu, Corneliu N; Klem, Erich; Mar, Mei-Heng; Zeisel, Steven H

    2005-01-01

    Previous studies show that acute choline deficiency (CD) triggers apoptosis in cultured rat hepatocytes (CWSV-1 cells). We demonstrate that prolonged EGF stimulation (10 ng/mL x 48 hrs) restores cell proliferation, as assessed by BrdU labeling, and protects cells from CD-induced apoptosis, as assessed by TUNEL labeling and cleavage of poly(ADP-ribose) polymerase. However, EGF rescue was not accompanied by restoration of depleted intracellular concentrations of choline, glycerphosphocholine, phosphocholine, or phosphatidylcholine. In contrast, we show that EGF stimulation blocks apoptosis by restoring mitochondrial membrane potential (Delta Psi(m)), as determined using the potential-sensitive dye chloromethyl-X-rosamine, and by preventing the release and nuclear localization of cytochrome c. We investigated whether EGF rescue involves EGF receptor phosphorylation and activation of the down-stream cell survival factor Akt. Compared to cells in control medium (CT, 70 micromol choline x 48 hrs), cells in CD medium (5 micromol choline) were less sensitive to EGF-induced (0-300 ng/mL x 5 min) receptor tyrosine phosphorylation. Compared to cells in CT medium, cells in CD medium treated with EGF (10 ng/mL x 5 min) exhibited higher levels of phosphatidylinositol 3-kinase (PI3K)-dependent phosphorylation of AktSer473. Inactivation of PI3K was sufficient to block EGF-stimulated activation of Akt, restoration of mitochondrial Delta Psi(m), and prevention of cytochrome c release. These studies indicate that stimulation with EGF activates a cell survival response against CD-apoptosis by restoring mitochondrial membrane potential and preventing cytochrome c release and nuclear translocation which are mediated by activation of Akt in hepatocytes. PMID:15665516

  9. 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

  10. 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

  11. ASGPR-Mediated Uptake of Multivalent Glycoconjugates for Drug Delivery in Hepatocytes.

    PubMed

    Monestier, Marie; Charbonnier, Peggy; Gateau, Christelle; Cuillel, Martine; Robert, Faustine; Lebrun, Colette; Mintz, Elisabeth; Renaudet, Olivier; Delangle, Pascale

    2016-04-01

    Liver cells are an essential target for drug delivery in many diseases. The hepatocytes express the asialoglycoprotein receptor (ASGPR), which promotes specific uptake by means of N-acetylgalactosamine (GalNAc) recognition. In this work, we designed two different chemical architectures to treat Wilson's disease by intracellular copper chelation. Two glycoconjugates functionalized with three or four GalNAc units each were shown to enter hepatic cells and chelate copper. Here, we studied two series of compounds derived from these glycoconjugates to find key parameters for the targeting of human hepatocytes. Efficient cellular uptake was demonstrated by flow cytometry using HepG2 human heptic cells that express the human oligomeric ASGPR. Dissociation constants in the nanomolar range showed efficient multivalent interactions with the receptor. Both architectures were therefore concluded to be able to compete with endogeneous asialoglycoproteins and serve as good vehicles for drug delivery in hepatocytes.

  12. ASGPR-Mediated Uptake of Multivalent Glycoconjugates for Drug Delivery in Hepatocytes.

    PubMed

    Monestier, Marie; Charbonnier, Peggy; Gateau, Christelle; Cuillel, Martine; Robert, Faustine; Lebrun, Colette; Mintz, Elisabeth; Renaudet, Olivier; Delangle, Pascale

    2016-04-01

    Liver cells are an essential target for drug delivery in many diseases. The hepatocytes express the asialoglycoprotein receptor (ASGPR), which promotes specific uptake by means of N-acetylgalactosamine (GalNAc) recognition. In this work, we designed two different chemical architectures to treat Wilson's disease by intracellular copper chelation. Two glycoconjugates functionalized with three or four GalNAc units each were shown to enter hepatic cells and chelate copper. Here, we studied two series of compounds derived from these glycoconjugates to find key parameters for the targeting of human hepatocytes. Efficient cellular uptake was demonstrated by flow cytometry using HepG2 human heptic cells that express the human oligomeric ASGPR. Dissociation constants in the nanomolar range showed efficient multivalent interactions with the receptor. Both architectures were therefore concluded to be able to compete with endogeneous asialoglycoproteins and serve as good vehicles for drug delivery in hepatocytes. PMID:26781030

  13. 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.

  14. 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.

  15. 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

  16. Invasive candidiasis stimulates hepatocyte and monocyte production of active transforming growth factor beta.

    PubMed

    Letterio, J J; Lehrnbecher, T; Pollack, G; Walsh, T J; Chanock, S J

    2001-08-01

    Candida albicans is an opportunistic fungal pathogen and a major cause of morbidity and mortality in patients with compromised immune function. The cytokine response to tissue invasion by C. albicans can influence the differentiation and function of lymphocytes and other mononuclear cells that are critical components of the host response. While the production of transforming growth factor beta (TGF-beta) has been documented in mice infected with C. albicans and is known to suppress phagocyte function, the cellular source and role of this cytokine in the pathogenesis of systemic candidiasis are not well understood. We have investigated the source of production of TGF-beta by immunohistochemical studies in tissue samples from patients with an uncommon complication of lymphoreticular malignancy, chronic disseminated candidiasis (CDC), and from a neutropenic-rabbit model of CDC. Liver biopsy specimens from patients with documented CDC demonstrated intense staining for extracellular matrix-associated TGF-beta1 within inflammatory granulomas, as well as staining for TGF-beta1 and TGF-beta3 within adjacent hepatocytes. These results correlate with the immunolocalization of TGF-beta observed in livers of infected neutropenic rabbits, using a neutralizing antibody that recognizes the mature TGF-beta protein. Human peripheral blood monocytes incubated with C. albicans in vitro release large amounts of biologically active TGF-beta1. The data demonstrate that local production of active TGF-betas by hepatocytes and by infected mononuclear cells is a component of the response to C. albicans infection that most probably contributes to disease progression in the immunocompromised host.

  17. 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

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

    PubMed

    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.

  19. 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.

  20. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    SciTech Connect

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. )

    1988-11-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

  1. MiR-19a regulates PTEN expression to mediate glycogen synthesis in hepatocytes.

    PubMed

    Dou, Lin; Meng, Xiangyu; Sui, Xiaofang; Wang, Shuyue; Shen, Tao; Huang, Xiuqing; Guo, Jun; Fang, Weiwei; Man, Yong; Xi, Jianzhong; Li, Jian

    2015-06-26

    MiR-19a, a member of mir-17-92 microRNA clusters, has been demonstrated to promote cell proliferation and angiogenesis via regulating the PI3K/AKT pathway, the major insulin signaling pathway. However, whether miR-19a plays an important role in glycogen synthesis in hepatocytes remains unknown. Here, we define the impact of miR-19a on glycogen synthesis and IL-6-induced reduced glycogenesis in hepatocytes and its underlying mechanisms. Our studies indicate that miR-19a was down-regulated in the livers of db/db mice and mice injected with IL-6, as well as mouse NCTC 1469 hepatocytes and HEP 1-6 hepatocytes treated by IL-6. We found that over-expression of miR-19a in NCTC 1469 cells and HEP 1-6 cells led to increased activation of the AKT/GSK pathway and synthesis of glycogen, whereas down-regulation of miR-19a impaired AKT/GSK phosphorylation and glycogenesis. Over-expression of miR-19a ameliorated IL-6-induced reduced glycogen synthesis in hepatocytes. Moreover, we identified PTEN as the target of miR-19a by a luciferase assay. Down-regulation of PTEN rescued the effects of miR-19a suppression on the activation of the AKT/GSK pathway and improved glycogenesis in NTC 1469 cells. These findings show for the first time that miR-19a might activate the AKT/GSK pathway and glycogenesis via down-regulation of PTEN expression.

  2. 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

  3. 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

  4. Hepatocyte growth factor levels in the saliva and gingival crevicular fluid in smokers with periodontitis.

    PubMed

    Anil, Sukumaran; Vellappally, Sajith; Preethanath, R S; Mokeem, Sameer A; AlMoharib, Hani S; Patil, Shankargouda; 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.

  5. Heparanase Plays a Dual Role in Driving Hepatocyte Growth Factor (HGF) Signaling by Enhancing HGF Expression and Activity*♦

    PubMed Central

    Ramani, Vishnu C.; Yang, Yang; Ren, Yongsheng; Nan, Li; Sanderson, Ralph D.

    2011-01-01

    Hepatocyte growth factor (HGF) is a heparin-binding cytokine that enhances growth, motility, and angiogenesis of many tumor types, including multiple myeloma where it is often highly expressed. However, little is known regarding what controls HGF level and activity in these tumors. Evaluation of bone marrow biopsies from myeloma patients revealed a strong positive correlation between the levels of HGF and heparanase, an endoglucuronidase known to promote aggressive tumor behavior. In vitro, addition of recombinant heparanase to myeloma cells or transfection of myeloma cell lines with the cDNA for heparanase significantly increased tumor cell expression and secretion of biologically active HGF. Shed syndecan-1, whose levels in myeloma are also enhanced by heparanase expression, binds to secreted HGF. This syndecan-1-HGF complex is active as shown by its ability to stimulate paracrine signaling via c-Met, the cell surface receptor for HGF. Surprisingly, heparanase enzyme activity was not required for up-regulation of HGF expression by the tumor cells. This is in contrast to the heparanase-mediated enhanced syndecan-1 shedding, which does require activity of the enzyme. This suggests that two different functional domains within the heparanase enzyme (the enzyme active site and a separate site) contribute to events leading to enhanced HGF signaling. These findings demonstrate a novel mechanism driving the HGF pathway whereby heparanase stimulates an increase in both HGF expression and syndecan-1 shedding to enhance HGF signaling. This work also provides further mechanistic insight into the dynamic role of heparanase in driving aggressive tumor progression. PMID:21131364

  6. Induction of hepatocyte growth factor production in human dermal fibroblasts by caffeic acid derivatives.

    PubMed

    Kurisu, Manami; Nakasone, Rie; Miyamae, Yusaku; Matsuura, Daisuke; Kanatani, Hirotoshi; Yano, Shingo; Shigemori, Hideyuki

    2013-01-01

    Hepatocyte growth factor (HGF) has mitogenic, motogenic, and morphogenic activities in epithelial cells. Induction of HGF production may be involved in organ regeneration, wound healing and embryogenesis. In this study, we examined the effects of caffeic acid derivatives including 4,5-di-O-caffeoylquinic acid (1) and acteoside (2) on HGF production in Neonatal Normal Human Dermal Fibroblasts (NHDF). Both 4,5-di-O-caffeoylquinic acid (1) and acteoside (2) significantly induced HGF production dose-dependent manner. To know the important substructure for HGF production activity, we next investigated the effect of the partial structure of these caffeic acid derivatives. From the results, caffeic acid (3) showed strong activity on the promotion of HGF production, while hydroxytyrosol (4) and quinic acid (5) didn't show any activity. Our findings suggest that the caffeoyl moiety of caffeic acid derivatives is essential for accelerated production of HGF. The compound which has the caffeoyl moiety may be useful for the treatment of some intractable organ disease.

  7. Hepatocyte growth factor is a mouse fetal Leydig cell terminal differentiation factor.

    PubMed

    Ricci, Giulia; Guglielmo, Maria Cristina; Caruso, Maria; Ferranti, Francesca; Canipari, Rita; Galdieri, Michela; Catizone, Angela

    2012-06-01

    The hepatocyte growth factor (HGF) is a pleiotropic cytokine and a well-known regulator of mouse embryonic organogenesis. In previous papers, we have shown the expression pattern of HGF and its receptor, C-MET, during the different stages of testis prenatal development. We demonstrated that C-MET is expressed in fetal Leydig cells (FLCs) and that HGF stimulates testosterone secretion in organ culture of late fetal testes. In the present study, we analyzed the proliferation rate, apoptotic index, and differentiation of FLCs in testicular organ culture of 17.5 days postcoitum (17.5 dpc) embryos to clarify the physiological role of HGF in late testis organogenesis. Based on our data, we conclude the following: 1) HGF acts as an antiapoptotic factor that is able to reduce the number of apoptotic FLCs and testicular caspase-3 active fragment; 2) HGF does not affect FLC proliferation; 3) HGF significantly increases expression of insulin-like 3 (INSL3), a marker of Leydig cell terminal differentiation, without affecting 3beta-hydroxysteroid dehydrogenase (3betaHSD) expression; 4) HGF significantly decreases the expression of nestin, a marker of Leydig cell progenitors; and 5) HGF significantly increases the number of fully developed FLCs. Taken together, these observations demonstrate that HGF is able to act in vitro as a survival and differentiation factor in FLC population.

  8. Uptake and distribution of hepatocyte growth factor in normal and regenerating adult rat liver.

    PubMed Central

    Liu, M. L.; Mars, W. M.; Zarnegar, R.; Michalopoulos, G. K.

    1994-01-01

    We have previously shown that systemically injected hepatocyte growth factor (HGF) is primarily taken up by the liver. The present study shows that HGF injected systemically or through the portal circulation is retained primarily at periportal sites. The periportal retention of HGF seems to persist longer in regenerating liver. The percentage of the total HGF injected that was retained within the liver at 1 minute after injection varied with the dose. A maximal amount of 0.157 +/- 0.012 microgram of HGF per gram liver tissue is retained by normal liver. Analysis of the circulating form of HGF in the plasma showed a relative enrichment with time for the heterodimeric form of HGF. A portion of portally injected HGF, composed of both single chain and two chain (heterodimeric) form was excreted intact in the bile. This was found in both normal and regenerating liver. These studies show that the liver can sequester large amounts of HGF and that the sequestration occurs primarily at periportal sites. Our studies support the hypothesis that a nonlysosomal processing pathway for HGF is present in the liver. Images Figure 1 Figure 3 Figure 4 Figure 6 PMID:8291602

  9. 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.

  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. 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.

  12. 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.

  13. 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

  14. Hepatocyte Growth Factor from a Clinical Perspective: A Pancreatic Cancer Challenge

    PubMed Central

    Rizwani, Wasia; Allen, Amanda E.; Trevino, Jose G.

    2015-01-01

    Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States and incidence rates are rising. Both detection and treatment options for pancreatic cancer are limited, providing a less than 5% five-year survival advantage. The need for new biomarkers for early detection and treatment of pancreatic cancer demands the efficient translation of bench knowledge to provide clinical benefit. One source of therapeutic resistance is the pancreatic tumor microenvironment, which is characterized by desmoplasia and hypoxia making it less conducive to current therapies. A major factor regulating desmoplasia and subsequently promoting chemoresistance in pancreatic cancer is hepatocyte growth factor (HGF), the sole ligand for c-MET (mesenchymal-epithelial transition), an epithelial tyrosine kinase receptor. Binding of HGF to c-MET leads to receptor dimerization and autophosphorylation resulting in the activation of multiple cellular processes that support cancer progression. Inhibiting activation of c-MET in cancer cells, in combination with other approaches for reducing desmoplasia in the tumor microenvironment, might significantly improve the success of chemotherapy. Therefore, HGF makes a potent novel target for developing therapeutic strategies in combination with existing drugs for treating pancreatic adenocarcinoma. This review provides a comprehensive analysis of HGF and its promising potential as a chemotherapeutic target for pancreatic cancer. PMID:26404380

  15. 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.

  16. 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.

  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

    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.

  19. 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

  20. 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

  1. The acute transcriptomic and proteomic response of HC-04 hepatoma cells to hepatocyte growth factor and its implications for Plasmodium falciparum sporozoite invasion.

    PubMed

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

    2014-05-01

    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.

  2. 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

  3. 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.

  4. Glycogen synthase kinase 3 regulates IL-1β mediated iNOS expression in hepatocytes by down-regulating c-Jun.

    PubMed

    Lakshmanan, Jaganathan; Zhang, Baochun; Nweze, Ikenna C; Du, Yibo; Harbrecht, Brian G

    2015-01-01

    Excessive nitric oxide from the inducible nitric oxide synthase (iNOS) increases shock-induced hepatic injury, hepatic dysfunction, inflammation, and mortality in animal models. Cytokines increase the expression of iNOS in hepatocytes, but the signaling mechanisms involved are not completely understood. We have previously demonstrated that Akt mediates the inhibitory effect of cAMP and insulin on cytokine-induced hepatocyte iNOS expression. We hypothesized that glycogen synthase kinase 3 (GSK3), a target of Akt phosphorylation, would regulate hepatocyte iNOS expression. In cultured rat hepatocytes, GSK3 inhibitors decreased IL-1β mediated nitric oxide (NO) production and iNOS protein expression, while the phosphatidylinositol 3-kinase (PI3K)/Akt pathway inhibitor LY294002 increased the cytokine-mediated NO production and iNOS expression. Over-expression of the constitutively active form of GSK3β enhanced IL-1β-mediated iNOS expression. GSK3 catalyzes the phosphorylation of c-Jun at the c-terminal Thr239 that facilitates c-Jun degradation. Inhibition of GSK3 with SB216763 and lithium chloride significantly reduced, whereas blocking PI3K/Akt increased phosphorylation of c-Jun at Thr239. The levels of total-c-Jun and c-Jun phosphorylated at Ser63 inversely correlated with c-Jun phosphorylated at Thr239, GSK3 activation and iNOS expression. Over-expression of a dominant negative c-Jun not only caused an increase in IL-1β-mediated iNOS promoter activity and iNOS protein expression but was also able to reverse the SB216763-mediated suppression of iNOS. These results demonstrate that GSK3, a downstream target of Akt, regulates IL-1β-stimulated iNOS expression in hepatocytes by directly phosphorylating c-Jun in an inhibitory manner. PMID:25160751

  5. Hepatitis B virus (HBV) X protein-mediated regulation of hepatocyte metabolic pathways affects viral replication.

    PubMed

    Bagga, Sumedha; Rawat, Siddhartha; Ajenjo, Marcia; Bouchard, Michael J

    2016-11-01

    Chronic HBV infection is a risk factor for hepatocellular carcinoma (HCC). The HBV HBx protein stimulates HBV replication and likely influences the development of HBV-associated HCC. Whether HBx affects regulators of metabolism in normal hepatocytes has not been addressed. We used an ex vivo, cultured primary rat hepatocyte system to assess the interplay between HBV replication and mechanistic target of rapamycin complex 1 (mTORC1) signaling. HBx activated mTORC1 signaling; however, inhibition of mTORC1 enhanced HBV replication. HBx also decreased ATP levels and activated the energy-sensing factor AMP-activated protein kinase (AMPK). Inhibition of AMPK decreased HBV replication. Inhibition of AMPK activates mTORC1, and we showed that activated mTORC1 is one factor that reduces HBV replication when AMPK is inhibited. HBx activation of both AMPK and mTORC1 suggests that these activities could provide a balancing mechanism to facilitate persistent HBV replication. HBx activation of mTORC1 and AMPK could also influence HCC development.

  6. Novel role for endogenous hepatocyte growth factor in the pathogenesis of intracranial aneurysms.

    PubMed

    Peña-Silva, Ricardo A; Chalouhi, Nohra; Wegman-Points, Lauren; Ali, Muhammad; Mitchell, Ian; Pierce, Gary L; Chu, Yi; Ballas, Zuhair K; Heistad, Donald; Hasan, David

    2015-03-01

    Inflammation plays a key role in formation and rupture of intracranial aneurysms. Because hepatocyte growth factor (HGF) protects against vascular inflammation, we sought to assess the role of endogenous HGF in the pathogenesis of intracranial aneurysms. Circulating HGF concentrations in blood samples drawn from the lumen of human intracranial aneurysms or femoral arteries were compared in 16 patients. Tissue from superficial temporal arteries and ruptured or unruptured intracranial aneurysms collected from patients undergoing clipping (n=10) were immunostained with antibodies to HGF and its receptor c-Met. Intracranial aneurysms were induced in mice treated with PF-04217903 (a c-Met antagonist) or vehicle. Expression of inflammatory molecules was also measured in cultured human endothelial, smooth muscle cells and monocytes treated with lipopolysaccharides in presence or absence of HGF and PF-04217903. We found that HGF concentrations were significantly higher in blood collected from human intracranial aneurysms (1076±656 pg/mL) than in femoral arteries (196±436 pg/mL; P<0.001). HGF and c-Met were detected by immunostaining in superficial temporal arteries and in both ruptured and unruptured human intracranial aneurysms. A c-Met antagonist did not alter the formation of intracranial aneurysms (P>0.05), but significantly increased the prevalence of subarachnoid hemorrhage and decreased survival in mice (P<0.05). HGF attenuated expression of vascular cell adhesion molecule-1 (P<0.05) and E-Selectin (P<0.05) in human aortic endothelial cells. In conclusion, plasma HGF concentrations are elevated in intracranial aneurysms. HGF and c-Met are expressed in superficial temporal arteries and in intracranial aneurysms. HGF signaling through c-Met may decrease inflammation in endothelial cells and protect against intracranial aneurysm rupture. PMID:25510828

  7. Hepatocyte growth factor is involved in the morphogenesis of tooth germ in murine molars.

    PubMed

    Tabata, M J; Kim, K; Liu, J G; Yamashita, K; Matsumura, T; Kato, J; Iwamoto, M; Wakisaka, S; Matsumoto, K; Nakamura, T; Kumegawa, M; Kurisu, K

    1996-04-01

    The patterns of gene expression for hepatocyte growth factor (HGF) and its receptor, c-Met, were revealed in the tooth germ of rat mandibular molars using RT-PCR. In situ hybridization demonstrated that the HGF gene was expressed only in the cells of the dental papilla of the tooth germ in vivo. The characteristic temporospatial distribution of HGF and c-Met during germ development was revealed using immunohistochemical studies in vivo. In order to demonstrate the functional role played by HGF in tooth development, HGF translation arrest by antisense phosphorothioate oligodeoxynucleotide (ODN) was carried out in vitro. In the control experiment, explants of tooth germs from embryonic 14 day mice were cultured in a modification of Trowell's system under serum-free and chemically defined conditions for two weeks. Other explants were cultured with 15mer antisense or sense ODN targeted to the HGF mRNA. Both the control and the sense-treated explants showed normal histological structure, as observed in vivo. On the other hand, antisense-treated explants exhibited an abnormal structure in which the enamel organs were surrounded by a thin layer of dentin and dental papilla, appearing 'inside-out' compared to the control and sense-treated explants, although the cytodifferentiation of ameloblasts and odontoblasts was not inhibited. The explants treated with recombinant human HGF combined with antisense ODN showed normal development, indicating that exogenous HGF rescued the explants from the abnormal structure caused by antisense ODN. The findings of a BrdU incorporation experiment suggested that the imbalance between the proliferation activity of the inner enamel epithelium and that of the dental papilla caused by HGF translation arrest results in the abnormal structure of the tooth germ. These results indicate that HGF is involved in the morphogenesis of the murine molar. PMID:8620851

  8. Regulation of Hepatocyte Growth Factor in Mice with Pneumonia by Peptidases and Trans-Alveolar Flux

    PubMed Central

    Raymond, Wilfred W.; Xu, Xiang; Nimishakavi, Shilpa; Le, Catherine; McDonald, Donald M.; Caughey, George H.

    2015-01-01

    Hepatocyte growth factor (HGF) promotes lung epithelial repair after injury. Because prior studies established that human neutrophil proteases inactivate HGF in vitro, we predicted that HGF levels decrease in lungs infiltrated with neutrophils and that injury is less severe in lungs lacking HGF-inactivating proteases. After establishing that mouse neutrophil elastase cleaves mouse HGF in vitro, we tested our predictions in vivo by examining lung pathology and HGF in mice infected with Mycoplasma pulmonis, which causes neutrophilic tracheobronchitis and pneumonia. Unexpectedly, pneumonia severity was similar in wild type and dipeptidylpeptidase I-deficient (Dppi-/-) mice lacking neutrophil serine protease activity. To assess how this finding related to our prediction that Dppi-activated proteases regulate HGF levels, we measured HGF in serum, bronchoalveolar lavage fluid, and lung tissue from Dppi+/+ and Dppi-/- mice. Contrary to prediction, HGF levels were higher in lavage fluid from infected mice. However, serum and tissue concentrations were not different in infected and uninfected mice, and HGF lung transcript levels did not change. Increased HGF correlated with increased albumin in lavage fluid from infected mice, and immunostaining failed to detect increased lung tissue expression of HGF in infected mice. These findings are consistent with trans-alveolar flux rather than local production as the source of increased HGF in lavage fluid. However, levels of intact HGF from infected mice, normalized for albumin concentration, were two-fold higher in Dppi-/- versus Dppi+/+ lavage fluid, suggesting regulation by Dppi-activated proteases. Consistent with the presence of active HGF, increased expression of activated receptor c-Met was observed in infected tissues. These data suggest that HGF entering alveoli from the bloodstream during pneumonia compensates for destruction by Dppi-activated inflammatory proteases to allow HGF to contribute to epithelial repair. PMID

  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. 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

  11. Overexpression of MACC1 and the association with hepatocyte growth factor/c-Met in epithelial ovarian cancer

    PubMed Central

    LI, HONGYU; ZHANG, HUI; ZHAO, SHUJUN; SHI, YUN; YAO, JUNGE; ZHANG, YANYAN; GUO, HUANHUAN; LIU, XINGSUO

    2015-01-01

    Metastasis-associated in colon cancer-1 (MACC1) is a gene that has been newly identified by a genome-wide search for differentially expressed genes in human colon cancer tissues, metastases and normal tissues. MACC1 exerts an important role in colon cancer metastasis through upregulation of the c-Met proto-oncogene. The tyrosine kinase receptor encoded by the c-Met oncogene exhibits the unusual property of mediating the invasive growth of epithelial cells upon binding with the hepatocyte growth factor (HGF). MACC1 has been investigated with regard to colon carcinoma and MACC1 expression is associated with metastasis in various types of human cancer. However, the value of MACC1 as a potential biomarker for ovarian cancer remains unknown, although the c-Met/HGF receptor has been shown to be overexpressed in epithelial ovarian cancer tissues. To investigate the role of MACC1 in epithelial ovarian tumors, the expression levels of MACC1 mRNA in ovarian tumor specimens were analyzed together with the prognostic significance. MACC1 protein expression was also detected in the epithelial ovarian tissue specimens, and the effects of MACC1 overexpression on ovarian cancer migration, invasion and prognosis were evaluated. Due to the close association between MACC1 and c-Met expression levels in colon cancer, the expression levels of HGF/c-Met in the ovarian specimens were also examined to determine whether such a correlation is also present in epithelial ovarian cancer. A total of 92 epithelial ovarian tissue samples were used to assess the expression levels of MACC1 mRNA and protein using reverse transcription-polymerase chain reaction and immunohistochemical methods, respectively. The serum levels of MACC1 protein expression in patients with epithelial ovarian cancer were detected by enzyme-linked immunosorbent assay. The results indicated that MACC1 may be important in the malignant progression of epithelial ovarian tumors, in particular for early stage patients. Thus, MACC

  12. The endocannabinoid N-arachidonoyl dopamine (NADA) selectively induces oxidative stress-mediated cell death in hepatic stellate cells but not in hepatocytes.

    PubMed

    Wojtalla, Alexandra; Herweck, Frank; Granzow, Michaela; Klein, Sabine; Trebicka, Jonel; Huss, Sebastian; Lerner, Raissa; Lutz, Beat; Schildberg, Frank Alexander; Knolle, Percy Alexander; Sauerbruch, Tilman; Singer, Manfred Vincenz; Zimmer, Andreas; Siegmund, Sören Volker

    2012-04-15

    The endocannabinoid system is a crucial regulator of hepatic fibrogenesis. We have previously shown that the endocannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces death in hepatic stellate cells (HSCs), the main fibrogenic cell type in the liver, but not in hepatocytes. However, the effects of other endocannabinoids such as N-arachidonoyl dopamine (NADA) have not yet been investigated. The NADA-synthesizing enzyme tyrosine hydroxylase was mainly expressed in sympathetic neurons in portal tracts. Its expression pattern stayed unchanged in normal or fibrotic liver. NADA dose dependently induced cell death in culture-activated primary murine or human HSCs after 2-4 h, starting from 5 μM. Despite caspase 3 cleavage, NADA-mediated cell death showed typical features of necrosis, including ATP depletion. Although the cannabinoid receptors CB1, CB2, or transient receptor potential cation channel subfamily V, member 1 were expressed in HSCs, their pharmacological or genetic blockade failed to inhibit NADA-mediated death, indicating a cannabinoid-receptor-independent mechanism. Interestingly, membrane cholesterol depletion with methyl-β-cyclodextrin inhibited AEA- but not NADA-induced death. NADA significantly induced reactive oxygen species formation in HSCs. The antioxidant glutathione (GSH) significantly decreased NADA-induced cell death. Similar to AEA, primary hepatocytes were highly resistant against NADA-induced death. Resistance to NADA in hepatocytes was due to high levels of GSH, since GSH depletion significantly increased NADA-induced death. Moreover, high expression of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in hepatocytes also conferred resistance towards NADA-induced death, since pharmacological or genetic FAAH inhibition significantly augmented hepatocyte death. Thus the selective induction of cell death in HSCs proposes NADA as a novel antifibrogenic mediator.

  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. 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

  15. 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.

  16. 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

  17. Cr(VI) induces premature senescence through ROS-mediated p53 pathway in L-02 hepatocytes

    PubMed Central

    Zhang, Yujing; Zhang, Yiyuan; Zhong, Caigao; Xiao, Fang

    2016-01-01

    Hexavalent Chromium [Cr(VI)], which can be found of various uses in industries such as metallurgy and textile dying, can cause a number of human disease including inflammation and cancer. Unlike previous research that focused on Cr(VI)-induced oxidative damage and apoptosis, this study placed emphasis on premature senescence that can be induced by low-dose and long-term Cr(VI) exposure. We found Cr(VI) induced premature senescence in L-02 hepatocytes, as confirmed by increase in senescence associated-β-galactosidase (SA-β-Gal) activity. Cr(VI) stabilized p53 through phosphorylation at Ser15 and increased expression of p53-transcriptional target p21. Mechanism study revealed Cr(VI) targeted and inhibited mitochondrial respiratory chain complex (MRCC) I and II to enhance reactive oxygen species (ROS) production. By applying antioxidant Trolox, we also confirmed that ROS mediated p53 activation. A tetracycline-inducible lentiviral expression system containing shRNA to p53 was used to knockout p53. We found p53 could inhibit pro-survival genes B-cell lymphoma-2 (Bcl-2), myeloid leukemia-1 (Mcl-1) and S phase related cell cycle proteins cyclin-dependent kinase 2 (CDK2), Cyclin E to induce premature senescence, and the functional role of ROS in Cr(VI)-induced premature senescence is depend on p53. The results suggest that Cr(VI) has a role in premature senescence by promoting ROS-dependent p53 activation in L-02 hepatocytes. PMID:27698449

  18. Hepatocyte growth factor-transfected skeletal myoblasts to limit the development of postinfarction heart failure.

    PubMed

    Poppe, Annika; Golsong, Peter; Blumenthal, Britta; von Wattenwyl, Robert; Blanke, Philipp; Beyersdorf, Friedhelm; Schlensak, Christian; Siepe, Matthias

    2012-03-01

    Stem cells transplanted to an injured heart affect the host myocardium indirectly. The cytokine hepatocyte growth factor (HGF) may play a key role in this paracrine activity. We hypothesized that HGF-overexpressing stem cells would restore cardiac function after myocardial infarction (MI). Because there is a high rate of cell death when injecting the cells intramyocardially, we used scaffold-based cell transfer. Skeletal myoblasts (SkMs) were isolated and expanded from newborn Lewis rats. Cells were transfected with pcDNA3-huHGF and seeded on polyurethane (PU) scaffolds or diluted in medium for cell injection. The seeded scaffolds were transplanted in rats two weeks after MI (group: PU-HGF-SkM) or the infection solution was intramyocardially injected (group: Inj-HGF-SkM). Two groups (Inj-SkM and PU-SkM) have been prepared with untransfected cells and sham group without any cell therapy served as control (n = 10 each group). At the beginning of treatment (baseline) and six weeks later, hemodynamic parameters were assessed. At the end of the study, histological analysis was employed. In sham animals we detected a decrease in systolic and diastolic function during the observation time. Treatment with untransfected myoblasts did not lead to any significant changes in hemodynamic parameters between the intervention and six weeks later. In group PU-HGF-SkM, systolic parameters like dP/dt(max), dP/dt(min) and isovolumic contraction improved significantly from baseline to study end. Some diastolic parameters were inferior as compared to baseline (SB-Ked, pressure half time [PHT], Tau). In group Inj-HGF-SkM, only PHT was impaired as compared to preinterventional values. Histological analysis showed significantly more capillaries in the infarction border zone in groups PU-HGF-SkM than in sham and Inj-SkM group. The infarction size was not affected by the therapy. Transplanting HGF-transfected myoblasts after MI can limit the development of ventricular dysfunction

  19. Identification of the source of elevated hepatocyte growth factor levels in multiple myeloma patients

    PubMed Central

    2014-01-01

    Background Hepatocyte growth factor (HGF) is a pleiotropic cytokine which can lead to cancer cell proliferation, migration and metastasis. In multiple myeloma (MM) patients it is an abundant component of the bone marrow. HGF levels are elevated in 50% of patients and associated with poor prognosis. Here we aim to investigate its source in myeloma. Methods HGF mRNA levels in bone marrow core biopsies from healthy individuals and myeloma patients were quantified by real-time PCR. HGF gene expression profiling in CD138+ cells isolated from bone marrow aspirates of healthy individuals and MM patients was performed by microarray analysis. HGF protein concentrations present in peripheral blood of MM patients were measured by enzyme-linked immunosorbent assay (ELISA). Cytogenetic status of CD138+ cells was determined by fluorescence in situ hybridization (FISH) and DNA sequencing of the HGF gene promoter. HGF secretion in co-cultures of human myeloma cell lines and bone marrow stromal cells was measured by ELISA. Results HGF gene expression profiling in both bone marrow core biopsies and CD138+ cells showed elevated HGF mRNA levels in myeloma patients. HGF mRNA levels in biopsies and in myeloma cells correlated. Quantification of HGF protein levels in serum also correlated with HGF mRNA levels in CD138+ cells from corresponding patients. Cytogenetic analysis showed myeloma cell clones with HGF copy numbers between 1 and 3 copies. There was no correlation between HGF copy number and HGF mRNA levels. Co-cultivation of the human myeloma cell lines ANBL-6 and JJN3 with bone marrow stromal cells or the HS-5 cell line resulted in a significant increase in secreted HGF. Conclusions We here show that in myeloma patients HGF is primarily produced by malignant plasma cells, and that HGF production by these cells might be supported by the bone marrow microenvironment. Considering the fact that elevated HGF serum and plasma levels predict poor prognosis, these findings are of

  20. Gene transfer of human hepatocyte growth factor by the use of nanosecond pulsed laser-induced stress waves

    NASA Astrophysics Data System (ADS)

    Terakawa, Mitsuhiro; Sato, Shunichi; Saitoh, Daizoh; Hasegawa, Makoto; Ashida, Hiroshi; Okano, Hideyuki; Obara, Minoru

    2006-05-01

    We successfully delivered a therapeutic vector construct, which carries hepatocyte growth factor (HGF) gene, to rat skin in vivo. After HGF expression vector had been intradermally injected to rat skin, LISWs were generated by irradiating the laser target put on the rat skin with nanosecond pulses from the second harmonics (532 nm) of a Q-switched Nd:YAG laser. Concentration of HGF protein increased by a factor of four by the application of LISWs when compared with that of control samples without LISW application. We also investigated the effects of LISWs on the integrity of plasmid DNA.

  1. Lipoteichoic acid and interleukin 1 stimulate synergistically production of hepatocyte growth factor (scatter factor) in human gingival fibroblasts in culture.

    PubMed Central

    Sugiyama, A; Arakaki, R; Ohnishi, T; Arakaki, N; Daikuhara, Y; Takada, H

    1996-01-01

    Lipoteichoic acids (LTA) from various gram-positive bacteria, including oral streptococci such as Streptococcus sanguis, enhanced the production of hepatocyte growth factor (HGF) (scatter factor) by human gingival fibroblasts in culture, whereas lipopolysaccharides (LPS) from various gram-negative bacteria did not. In contrast, LPS induced interleukin 1 activity in human gingival epithelial cells in culture, while LTA had little effect. LTA and recombinant human interleukin 1 alpha enhanced synergistically the production of HGF/SF in human gingival fibroblast cultures. Recombinant human HGF, in turn, enhanced the proliferation of human gingival epithelial cells in culture. PMID:8606111

  2. 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.

  3. 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

  4. 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

  5. Efficient large-scale generation of functional hepatocytes from mouse embryonic stem cells grown in a rotating bioreactor with exogenous growth factors and hormones

    PubMed Central

    2013-01-01

    Introduction Embryonic stem (ES) cells are considered a potentially advantageous source of hepatocytes for both transplantation and the development of bioartificial livers. However, the efficient large-scale generation of functional hepatocytes from ES cells remains a major challenge, especially for those methods compatible with clinical applications. Methods In this study, we investigated whether a large number of functional hepatocytes can be differentiated from mouse ES (mES) cells using a simulated microgravity bioreactor. mES cells were cultured in a rotating bioreactor in the presence of exogenous growth factors and hormones to form embryoid bodies (EBs), which then differentiated into hepatocytes. Results During the rotating culture, most of the EB-derived cells gradually showed the histologic characteristics of normal hepatocytes. More specifically, the expression of hepatic genes and proteins was detected at a higher level in the differentiated cells from the bioreactor culture than in cells from a static culture. On further growing, the EBs on tissue-culture plates, most of the EB-derived cells were found to display the morphologic features of hepatocytes, as well as albumin synthesis. In addition, the EB-derived cells grown in the rotating bioreactor exhibited higher levels of liver-specific functions, such as glycogen storage, cytochrome P450 activity, low-density lipoprotein, and indocyanine green uptake, than did differentiated cells grown in static culture. When the EB-derived cells from day-14 EBs and the cells’ culture supernatant were injected into nude mice, the transplanted cells were engrafted into the recipient livers. Conclusions Large quantities of high-quality hepatocytes can be generated from mES cells in a rotating bioreactor via EB formation. This system may be useful in the large-scale generation of hepatocytes for both cell transplantation and the development of bioartificial livers. PMID:24294908

  6. Kinetics of myc-max-mad gene expression during hepatocyte proliferation in vivo: Differential regulation of mad family and stress-mediated induction of c-myc.

    PubMed

    Mauleon, Itsaso; Lombard, Marie-Noëlle; Muñoz-Alonso, Maria J; Cañelles, Matilde; Leon, Javier

    2004-02-01

    Mad proteins (Mad1, Mxi1, Mad3, Mad4, Mnt/Rox) are biochemical and biological antagonists of c-Myc oncoprotein. Mad-Max dimers repress the transcription of the same target genes activated by Myc-Max dimers. Despite the critical role of Max and Mad proteins as modulators of c-Myc functions, there are no comparative data on their regulation in vivo. We carried out a systematic analysis of c-myc, max, and mad family expression in a model of synchronized cell proliferation in vivo in adult tissues, that is, rat hepatocytes after partial hepatectomy. We confirmed the previously reported early peak of c-myc expression after hepatectomy but we show that it did not correlate with hepatocyte proliferation as it also occurred in sham-operated animals as a result of surgical stresses. A second peak of c-myc expression was observed later, at the time of the wave of DNA synthesis. No such expression was detected in sham-operated rat quiescent hepatocytes. max expression increased around 4-16 h after hepatectomy, before the peaks of c-myc and DNA synthesis. mxi1 and mad4 were slightly downregulated during liver regeneration. mnt/rox expression did not change. These expression patterns suggest a role of Myc-Max for efficient mitogenic response of hepatocytes. We also analyzed the effects of Myc and Max ectopic expression on the clonogenic growth of the rat hepatoma cells. Expression of c-Myc and Max increased clonogenic growth, whereas the reduction of c-Myc levels by an antisense vector decreased growth. The results suggest nonredundant roles for mad genes in hepatocyte proliferation and point to c-Myc as a putative target for anticancer therapy of liver cancer.

  7. 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.

  8. 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

  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.

  10. Metabolic danger signals, uric acid and ATP, mediate inflammatory cross-talk between hepatocytes and immune cells in alcoholic liver disease.

    PubMed

    Petrasek, Jan; Iracheta-Vellve, Arvin; Saha, Banishree; Satishchandran, Abhishek; Kodys, Karen; Fitzgerald, Katherine A; Kurt-Jones, Evelyn A; Szabo, Gyongyi

    2015-08-01

    Inflammation defines the progression of ALD from reversible to advanced stages. Translocation of bacterial LPS to the liver from the gut is necessary for alcohol-induced liver inflammation. However, it is not known whether endogenous, metabolic danger signals are required for inflammation in ALD. Uric acid and ATP, 2 major proinflammatory danger signals, were evaluated in the serum of human volunteers exposed to a single dose of ethanol or in supernatants of primary human hepatocytes exposed to ethanol. In vitro studies were used to evaluate the role of uric acid and ATP in inflammatory cross-talk between hepatocytes and immune cells. The significance of signaling downstream of uric acid and ATP in the liver was evaluated in NLRP3-deficient mice fed a Lieber-DeCarli ethanol diet. Exposure of healthy human volunteers to a single dose of ethanol resulted in increased serum levels of uric acid and ATP. In vitro, we identified hepatocytes as a significant source of these endogenous inflammatory signals. Uric acid and ATP mediated a paracrine inflammatory cross-talk between damaged hepatocytes and immune cells and significantly increased the expression of LPS-inducible cytokines, IL-1β and TNF-α, by immune cells. Deficiency of NLRP3, a ligand-sensing component of the inflammasome recognizing uric acid and ATP, prevented the development of alcohol-induced liver inflammation in mice and significantly ameliorated liver damage and steatosis. Endogenous metabolic danger signals, uric acid, and ATP are involved in inflammatory cross-talk between hepatocytes and immune cells and play a crucial role in alcohol-induced liver inflammation.

  11. 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

  12. 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

  13. 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

  14. Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma.

    PubMed

    Słowińska, Mariola; Bukowska, Joanna; Hejmej, Anna; Bilińska, Barbara; Kozłowski, Krzysztof; Jankowski, Jan; Ciereszko, Andrzej

    2015-08-01

    A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases. It is of interest if the single-domain Kazal-type inhibitor controls the activity of turkey seminal plasma proteinases. Pure preparations of the Kazal-type inhibitor and anti-Kazal-type inhibitor monospecific immunoglobulin Gs were used as ligands in affinity chromatography for proteinase isolation from turkey seminal plasma. Gene expression and the immunohistochemical detection of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. The hepatocyte growth factor activator (HGFA) was identified in the binding fraction in affinity chromatography. Hepatocyte growth factor activator activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner. This protease was a primary physiological target for the single-domain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma, and phosphorylation was the primary posttranslational modification of HGFA. In addition to HGFA, acrosin was a target proteinase for the single-domain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and was not present as a free enzyme. The single-domain Kazal-type inhibitor was specific for the reproductive tract. The germ cell-specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis; secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in the changes in sperm membranes during maturation and in the maintenance of the microenvironment in which sperm maturation occurred and sperm was stored. The role of HGFA in these processes remains to be established.

  15. Implication of CpG-ODN and reactive oxygen species in the inhibition of intracellular growth of Salmonella typhimurium in hepatocytes.

    PubMed

    Sanchez-Campillo, María; Chicano, Antonio; Torío, Alberto; Martín-Orozco, Elena; Gámiz, Pilar; Hernández-Caselles, Trinidad; García-Peñarrubia, Pilar

    2004-07-01

    Bacterial DNA acts as an alert signal for eukaryotic cells through immunostimulatory CpG motifs. These sequences have therapeutic properties promoting protective immune TH1 responses and are recognized by a membrane protein belonging to the Toll-like receptor (TLR) family, named TLR-9. The aim of this study was to test the capability of murine hepatocytes to sense bacterial DNA and to develop antibacterial mechanisms against Salmonella typhimurium. We show that hepatocyte cell lines and mRNA extracts from murine liver constitutively express TLR-9, which is down-regulated by LPS and the mix of IFNgamma, IL-1beta and LPS. Also, we have found that hepatocyte cell lines can sense the presence of bacterial DNA and respond to it by increasing the pool of intracellular peroxides. This results in inhibition of intracellular growth of S. typhimurium when infected cells were incubated in the presence of CpG synthetic oligonucleotides (CpG-ODN). Expression of hepatocyte Mn-SOD is also induced by stimulation with CpG-oligodeoxynucleotides, LPS, and the mix of IFNgamma, IL-1beta and LPS. These results reinforce the prominent role of hepatocytes as a microbial product-responsive cell and the capabilities of CpG-ODN sequences as potent inducers of the innate immune response through the activation of a broad range of cell types.

  16. Effects of insulin-like growth factor-1 on the assembly and secretion of very low-density lipoproteins in cow hepatocytes in vitro.

    PubMed

    Li, Xinwei; Guan, Yuan; Li, Ying; Wu, Dianjun; Liu, Lei; Deng, Qinghua; Li, Xiaobing; Wang, Zhe; Liu, Guowen

    2016-01-15

    Fatty liver is a major metabolic disorder of dairy cows. One important reason is that hepatic very low-density lipoproteins (VLDL) assembly was significant decreased in dairy cows with fatty liver. In addition, the impairment of insulin-like growth factor (IGF)-1 synthesis was involved in the development of fatty liver. Therefore, the objective of this study was to investigate the effects of IGF-1 on the VLDL assembly in cow hepatocytes. In this study, cow hepatocytes were cultured and then transfected with Ad-GFP-IGF-1 (inhibited the IGF-1 expression) and Ad-GFP (negative control), and treated with different concentrations of IGF-1, respectively. The results showed that IGF-1 increased the mRNA abundance of apolipoprotein B100 (ApoB100), apolipoprotein E (ApoE), microsomal triglyceride transfer protein (MTTP), and low-density lipoprotein receptor (LDLR) and then increased the VLDL assembly in cow hepatocytes. Nevertheless, impairment of IGF-1 expression by Ad-GFP-IGF-1 could inhibit above genes expression and VLDL assembly in hepatocytes. Taken together, these results indicate that IGF-1 increases the VLDL assembly and impairment of IGF-1 expression decreases the VLDL assembly in cow hepatocytes.

  17. 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.

  18. Hepatocyte growth factor/c-met promotes proliferation, suppresses apoptosis, and improves matrix metabolism in rabbit nucleus pulposus cells in vitro.

    PubMed

    Ishibashi, Hidenobu; Tonomura, Hitoshi; Ikeda, Takumi; Nagae, Masateru; Sakata, Munehiro; Fujiwara, Hiroyoshi; Tanida, Takashi; Mastuda, Ken-Ichi; Kawata, Mitsuhiro; Kubo, Toshikazu

    2016-04-01

    The etiology of intervertebral disc (IVD) degeneration is closely related to apoptosis and extracellular matrix degradation in nucleus pulposus (NP) cells. These defects in NP cells are induced by excessive external stressors such as reactive oxygen species (ROS) and inflammatory cytokines. Recently, hepatocyte growth factor (HGF) has been shown to repair damage in various diseases through anti-apoptotic and anti-inflammatory activity. In this study, we investigated the effects of HGF on NP cell abnormality caused by ROS and inflammatory cytokines by using primary NP cells isolated from rabbit IVD. HGF significantly enhanced the proliferation of NP cells. Apoptosis of NP cells induced by H2 O2 or TNF-α was significantly inhibited by HGF. Induction of mRNA expression of the inflammation mediators cyclooxygenase-2 and matrix metalloproteinase-3 and -9 by TNF-α was significantly suppressed by HGF treatment. Expression of c-Met, a specific receptor for HGF, was confirmed in NP cells and was increased by TNF-α, suggesting that inflammatory cytokines increase sensitivity to HGF. These findings demonstrate that activation of HGF/c-Met signaling suppresses damage caused by ROS and inflammation in NP cells through multiple pathways. We further suggest the clinical potential of HGF for counteracting IVD degradation involved in NP cell abnormalities.

  19. 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

  20. In vitro and in vivo gene delivery mediated by Lactosylated dendrimer/alpha-cyclodextrin conjugates (G2) into hepatocytes.

    PubMed

    Arima, Hidetoshi; Yamashita, Shogo; Mori, Yoshimasa; Hayashi, Yuya; Motoyama, Keiichi; Hattori, Kenjiro; Takeuchi, Tomoko; Jono, Hirofumi; Ando, Yukio; Hirayama, Fumitoshi; Uekama, Kaneto

    2010-08-17

    The purpose of this study is to evaluate in vitro and in vivo gene delivery efficiency of polyamidoamine (PAMAM) starburst dendrimer (generation 2, G2) conjugates with alpha-cyclodextrin (alpha-CDE (G2)) bearing lactose (Lac-alpha-CDE) with various degrees of substitution of the lactose moiety (DSL) as a novel hepatocyte-selective carrier in hepatocytes. Lac-alpha-CDE (DSL 2.6) was found to have much higher gene transfer activity than dendrimer, alpha-CDE, Lac-alpha-CDE (DSL 1.2, 4.6, 6.2 and 10.2) and lactosylated dendrimer (Lac-dendrimer, DSL 2.4) in HepG2 cells, which are dependent on the expression of cell-surface asialoglycoprotein receptor (ASGP-R), reflecting the cellular association of the plasmid DNA (pDNA) complexes. The physicochemical properties of pDNA complex with Lac-alpha-CDE (DSL 2.6) were almost comparable to that with alpha-CDE. Lac-alpha-CDE (DSL 2.6) provided negligible cytotoxicity up to a charge ratio of 150 in HepG2 cells. Lac-alpha-CDE (DSL 2.6) provided gene transfer activity higher than jetPEI-Hepatocyte to hepatocytes with much less changes of blood chemistry values 12h after intravenous administration in mice. These results suggest the potential use of Lac-alpha-CDE (DSL 2.6) as a non-viral vector for gene delivery toward hepatocytes.

  1. The sympathetic nervous system promotes carbon tetrachloride-induced liver cirrhosis in rats by suppressing apoptosis and enhancing the growth kinetics of regenerating hepatocytes.

    PubMed

    Hamasaki, K; Nakashima, M; Naito, S; Akiyama, Y; Ohtsuru, A; Hamanaka, Y; Hsu, C T; Ito, M; Sekine, I

    2001-02-01

    Norepinephrine is considered to possess potent anti-apoptotic action in regenerating hepatocytes. To clarify the role of the sympathetic nervous system in apoptosis that occurs in chronic liver damage and following the promotion of liver cirrhosis, we studied a carbon tetrachloride (CCl4)-induced liver injury model, using spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and chemically sympathectomized WKY. At 24 h after CCl4 administration. acute damage, characterized by vacuolated hepatocytes in the centrilobular zone, was greater in SHR than in WKY. This vacuolated change in WKY hepatocytes was significantly reduced by chemical sympathectomy with 6-hydroxydopamine (6-OHDA). After 48 h, the acute damage was dramatically improved in each animal, without significant differences between the three groups. In chronic damage after weekly repetition of CCl4 treatment for 4 weeks, fibrosis was evident in SHR, while in the other groups there was only scant fibrosis in the centrilobular zone. After 8 weeks' repetition of CCl4, liver cirrhosis was seen only in SHR. The incidence of apoptotic cells in areas of both acute and chronic damage in WKY, detected by terminal deoxynucleotidyl transferase-dUTP nick end labeling, was significantly increased in comparison with that in SHR, and was further increased by 6-OHDA pretreatment. In contrast, there was significantly greater enhancement of the growth of hepatocytes in SHR than in WKY in both acute and chronic damage. Moreover. hepatocyte growth kinetics in WKY was significantly inhibited after sympathectomy in acute injury, as evidenced by immunohistochemistry for proliferating cell nuclear antigen (PCNA). In vitro, the amount of hepatocellular apoptosis induced by transforming growth factor-beta1 was significantly decreased by incubation with norepinephrine. These findings suggest that the anti-apoptotic effect of the sympathetic nervous system increases cell growth kinetics and promotes liver cirrhosis in this

  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. A novel mechanism in maggot debridement therapy: protease in excretion/secretion promotes hepatocyte growth factor production.

    PubMed

    Honda, Kenjiro; Okamoto, Koji; Mochida, Yasuhiro; Ishioka, Kunihiro; Oka, Machiko; Maesato, Kyoko; Ikee, Ryota; Moriya, Hidekazu; Hidaka, Sumi; Ohtake, Takayasu; Doi, Kent; Fujita, Toshiro; Kobayashi, Shuzo; Noiri, Eisei

    2011-12-01

    Maggot debridement therapy (MDT) is effective for treating intractable wounds, but its precise molecular mechanism, including the association between MDT and growth factors, remains unknown. We administered MDT to nine patients (66.3 ± 11.8 yr, 5 male and 4 female) with intractable wounds of lower extremities because they did not respond to conventional therapies. Significant increases of hepatocyte growth factor (HGF) levels were observed in femoral vein blood during 48 h of MDT (P < 0.05), but no significant change was found for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-β1 (TGF-β1), or tumor necrosis factor-α (TNF-α). We conducted NIH-3T3 cell stimulation assay to evaluate the relation between HGF and protease activity in excretion/secretion (ES) derived from maggots. Compared with the control group, HGF was significantly higher in the 0.05 μg/ml ES group (P < 0.01). Furthermore, protease inhibitors suppressed the increase of HGF (P < 0.05). The HGF expression was increased in proportion to the ES protein concentration of 0.025 to 0.5 μg/ml. In fact, ES showed stronger capability of promoting HGF production and less cytotoxicity than chymotrypsin or bromelain. HGF is an important factor involved in cutaneous wound healing. Therefore, these results suggest that formation of healthy granulation tissue observed during MDT results from the increased HGF. Further investigation to identify molecules enhancing HGF expression by MDT will contribute greatly to drug target discovery for intractable wound healing therapy.

  5. The current status of primary hepatocyte culture

    PubMed Central

    Mitaka, Toshihiro

    1998-01-01

    Recently, there have been significant advances toward the development of culture conditions that promote proliferation of primary rodent hepatocytes. There are two major methods for the multiplication of hepatocytes in vitro: one is the use of nicotinamide, the other is the use of a nutrient-rich medium. In the medium containing a high concentration of nicotinamide and a growth factor, primary hepatocytes can proliferate well. In this culture condition small mononucleate cells, which are named small hepatocytes, appear and form colonies. Small hepatocytes have a high potential to proliferate while maintaining hepatic characteristics, and can differentiate into mature ones. On the other hand, combining the nutrient-rich medium with 2% DMSO, the proliferated hepatocytes can recover the hepatic differentiated functions and maintain them for a long time. In this review I describe the culture conditions for the proliferation and differentiation of primary hepatocytes and discuss the small hepatocytes, especially their roles in liver growth. PMID:10319020

  6. 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.

  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. 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.

  9. 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'...

  10. Identification of cytochrome CYP2E1 as critical mediator of synergistic effects of alcohol and cellular lipid accumulation in hepatocytes in vitro

    PubMed Central

    Mahli, Abdo; Thasler, Wolfgang E.; Patsenker, Eleonora; Müller, Sebastian; Stickel, Felix; Müller, Martina; Seitz, Helmut K.; Cederbaum, Arthur I.; Hellerbrand, Claus

    2015-01-01

    Clinical studies propose a causative link between the consumption of alcohol and the development and progression of liver disease in obese individuals. However, it is incompletely understood how alcohol and obesity interact and whether the combined effects are additive or synergistic. In this study, we developed an in vitro model to address this question. Lipid accumulation in primary human hepatocytes was induced by incubation with oleic acid. Subsequently, steatotic and control hepatocytes were incubated with up to 50 mM alcohol. This alcohol concentration on its own revealed only minimal effects but significantly enhanced oleate-induced lipogenesis and cellular triglyceride content compared to control cells. Similarly, lipid peroxidation, oxidative stress and pro-inflammatory gene expression as well as CYP2E1 levels and activity were synergistically induced by alcohol and steatosis. CYP2E1 inhibition blunted these synergistic pathological effects. Notably, alcohol and cellular steatosis also induced autophagy in a synergistic manner, and also this was mediated via CYP2E1. Further induction of autophagy ameliorated the joint effects of alcohol and oleic acid on hepatocellular lipid accumulation and inflammatory gene expression while inhibition of autophagy further enhanced the dual pathological effects. Further analyses revealed that the joint synergistic effect of alcohol and steatosis on autophagy was mediated via activation of the JNK-pathway. In summary, our data indicate that alcohol induces not only pathological but also protective mechanisms in steatotic hepatocytes via CYP2E1. These findings may have important implications on the prognosis and treatment of alcoholic liver disease particularly in obese individuals. PMID:26497211

  11. Dissociation of c-Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species.

    PubMed

    Ikebuchi, Fumie; Oka, Kiyomasa; Mizuno, Shinya; Fukuta, Kazuhiro; Hayata, Daichika; Ohnishi, Hiroyuki; Nakamura, Toshikazu

    2013-06-01

    Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c-Met. We previously demonstrated that HGF α-chain hairpin-loop, K1 domain and β-chain are required for c-Met signalling. The sequential phosphorylation of tyrosine residues, from c-Met kinase domain to multidocking regions, is required for HGF-signalling transduction. Herein, we provide evidence that the disconcerted activation of c-Met tyrosine regions fails to induce biological functions. When human cells were incubated with 'mouse HGF', kinase domain activation (i.e. phospho-Tyr-1230/34/35) became evident, but the multidocking site (i.e. Tyr-1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α-chain, or of β-chain, to human c-Met was lower than that of human HGF, as evidenced by HGF-chimera assay. Notably, only four amino acid positions in HGF α-chain hairpin-loop and K1 domain and six positions in β-chain differed between human HGF and mouse HGF. The human-specific amino acids (such as Gln-95 in hairpin-loop, Arg-134 in K1 domain and Cys-561 in β-chain) may be important for accurate c-Met assembly and signalling transduction.

  12. 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.

  13. The hepatocyte growth factor receptor (MET) gene is not associated with refractive error and ocular biometrics in a Caucasian population

    PubMed Central

    Schache, M.; Chen, C.Y.; Dirani, M.; Baird, P.N.

    2009-01-01

    Purpose The purpose of this study was to determine if genetic variants in the hepatocyte growth factor receptor (MET) gene are associated with refractive error and ocular biometric measures in a Caucasian cohort. Methods A case-control association study using 818 Caucasian adults (37.2% male, 62.8% female; average age: 51.21±17.17 years) was undertaken. All individuals were genotyped for 16 tag single nucleotide polymorphisms (tSNPs) across the MET gene region. Myopia was defined as –0.5 DS or worse in both eyes and divided into high myopia (≤–6.0 DS) and low/moderate myopia (–0.5 DS to –5.99 DS). Hypermetropia was defined as at least +1.0 DS in both eyes. Genotyping results were analyzed using PLINK, comparing cases (all myopia, high myopia, low/moderate myopia, and hypermetropia) to controls (emmetropia). Association tests were also performed using the quantitative traits of refraction, axial length, anterior chamber depth, and corneal curvature. Results No statistically significant genetic associations were detected for any of the 16 tSNPs with refractive error (myopia and hypermetropia) or ocular biometric measures. Conclusions These data indicate there is likely no genetic association of the MET gene with myopia, axial length, anterior chamber depth, and corneal curvature in this cohort. PMID:20011629

  14. 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

  15. Correlation Between Hepatocyte Growth Factor (HGF) and Gamma-Aminobutyric Acid (GABA) Plasma Levels in Autistic Children.

    PubMed

    Russo, Anthony J

    2013-01-01

    There is much support for the role of Gamma-Aminobutyric acid (GABA) in the etiology of autism. Recent research has shown that hepatocyte growth factor (HGF) modulates GABAergic inhibition and seizure susceptibility. This study was designed to determine and correlate plasma levels of HGF, GABA, as well as symptom severity, in autistic children and neurotypical controls. Plasma from 48 autistic children and 29 neurotypical controls was assessed for HGF and GABA concentration using ELISAs. Symptom severity was assessed in these autistic individuals and compared to HGF and GABA concentrations. We previously reported that autistic children had significantly decreased levels of HGF. In this study, the same autistic children had significantly increased plasma levels of GABA (P = 0.002) and decreased HGF levels correlated with these increased GABA levels (r = 0.3; P = 0.05). High GABA levels correlated with increasing hyperactivity (r = 0.6; P = 0.0007) and impulsivity severity (r = 0.5; P = 0.007), tip toeing severity (r = 0.35; P = 0.03), light sensitivity (r = 0.4; P = 0.02), and tactile sensitivity (r = 0.4; P = 0.01). HGF levels did not correlate significantly with any symptom severity. These results suggest an association between HGF and GABA levels and suggest that plasma GABA levels are related to symptom severity in autistic children.

  16. Hepatocyte growth factor triggers distinct mechanisms of Asef and Tiam1 activation to induce endothelial barrier enhancement.

    PubMed

    Higginbotham, Katherine; Tian, Yufeng; Gawlak, Grzegorz; Moldobaeva, Nurgul; Shah, Alok; Birukova, Anna A

    2014-11-01

    Previous reports described an important role of hepatocyte growth factor (HGF) in mitigation of pulmonary endothelial barrier dysfunction and cell injury induced by pathologic agonists and mechanical forces. HGF protective effects have been associated with Rac-GTPase signaling pathway activated by Rac-specific guanine nucleotide exchange factor Tiam1 and leading to enhancement of intercellular adherens junctions. This study tested involvement of a novel Rac-specific activator, Asef, in endothelial barrier enhancement by HGF and investigated a mechanism of HGF-induced Asef activation. Si-RNA-based knockdown of Tiam1 and Asef had an additive effect on attenuation of HGF-induced Rac activation and endothelial cell (EC) barrier enhancement. Tiam1 and Asef activation was abolished by pharmacologic inhibitors of HGF receptor and PI3-kinase. In contrast to Tiam1, Asef interacted with APC and associated with microtubule fraction upon HGF stimulation. EC treatment by low dose nocodazole to inhibit peripheral microtubule dynamics partially attenuated HGF-induced Asef peripheral translocation, but had negligible effect on Tiam1 translocation. These effects were associated with attenuation of HGF-induced barrier enhancement in EC pretreated with low ND dose and activation of Rac and its cytoskeletal effectors PAK1 and cortactin. These data demonstrate, that in addition to microtubule-independent Tiam1 activation, HGF engages additional microtubule- and APC-dependent pathway of Asef activation. These mechanisms may complement each other to provide the fine tuning of Rac signaling and endothelial barrier enhancement in response to various agonists.

  17. A comparison of uptake of metformin and phenformin mediated by hOCT1 in human hepatocytes.

    PubMed

    Sogame, Yoshihisa; Kitamura, Atsushi; Yabuki, Masashi; Komuro, Setsuko

    2009-11-01

    Metformin, a biguanide that has been used to treat type 2 diabetes mellitus, is reportedly transported into human hepatocytes by human organic cation transporter 1 (hOCT1). The objective of this study was to investigate differences in the hepatic uptake of metformin and phenformin, a biguanide derivative similar to metformin. Special focus was on the role of active transport into cells. Experiments were therefore performed using human cryopreserved hepatocytes and hOCT1 expressing oocytes. Both biguanides proved to be good substrates for hOCT1. However, phenformin exhibited a much higher affinity and transport activity, with a marked difference in uptake kinetics compared with metformin. Both biguanides were transported actively by hOCT1, with the active transport components much greater than passive transport components in both cases, suggesting that functional changes in hOCT1 might affect the transport of both compounds to the same degree. This study for the first time produced detailed comparative findings for uptake profiles of metformin and phenformin in human hepatocytes and hOCT1 expressing oocytes. It is considered that hOCT1 may not be the only key factor that determines the frequency of metformin and phenformin toxicity, considering the major contribution of this transporter to the total hepatic uptake and comparable width of their therapeutic concentrations.

  18. Hepatocyte growth factor inhibits hypoxia/reoxygenation-induced activation of xanthine oxidase in endothelial cells through the JAK2 signaling pathway

    PubMed Central

    Zhang, Ying Qian; Hu, Shun Ying; Chen, Yun Dai; Guo, Ming Zhou; Wang, Shan

    2016-01-01

    Vascular endothelial cells (ECs) appear to be one of the primary targets of hypoxia/reoxygenation (H/R) injury. In our previous study, we demonstrated that hepatocyte growth factor (HGF) exhibited a protective effect in cardiac microvascular endothelial cells (CMECs) subjected to H/R by inhibiting xanthine oxidase (XO) by reducing the cytosolic Ca2+ concentration increased in response to H/R. The precise mechanisms through which HGF inhibits XO activation remain to be determined. In the present study, we examined the signaling pathway through which HGF regulates Ca2+ concentrations and the activation of XO during H/R in primary cultured rat CMECs. CMECs were exposed to 4 h of hypoxia and 1 h of reoxygenation. The protein expression of XO and the activation of the phosphoinositide 3-kinase (PI3K), janus kinase 2 (JAK2) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways were detected by western blot analysis. Cytosolic calcium (Ca2+) concentrations and reactive oxygen species (ROS) levels were measured by flow cytometry. The small interfering RNA (siRNA)-mediated knockdown of XO inhibited the increase in ROS production induced by H/R. LY294002 and AG490 inhibited the H/R-induced increase in the production and activation of XO. The PI3K and JAK2 signaling pathways were activated by H/R. The siRNA-mediated knockdown of PI3K and JAK2 also inhibited the increase in the production of XO protein. HGF inhibited JAK2 activation whereas it had no effect on PI3K activation. The siRNA-mediated knockdown of JAK2 prevented the increase in cytosolic Ca2+ induced by H/R. Taken together, these findings suggest that H/R induces the production and activation of XO through the JAK2 and PI3K signaling pathways. Furthermore, HGF prevents XO activation following H/R primarily by inhibiting the JAK2 signaling pathway and in turn, inhibiting the increase in cytosolic Ca2+. PMID:27573711

  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. 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

  1. Rapid growth of invasive metastatic melanoma in carcinogen-treated hepatocyte growth factor/scatter factor-transgenic mice carrying an oncogenic CDK4 mutation.

    PubMed

    Tormo, Damia; Ferrer, Aleix; Gaffal, Evelyn; Wenzel, Jörg; Basner-Tschakarjan, Etiena; Steitz, Julia; Heukamp, Lukas C; Gütgemann, Ines; Buettner, Reinhard; Malumbres, Marcos; Barbacid, Mariano; Merlino, Glenn; Tüting, Thomas

    2006-08-01

    Currently, novel mouse models of melanoma are being generated that recapitulate the histopathology and molecular pathogenesis observed in human disease. Impaired cell-cycle control, which is a hallmark of both familial and sporadic melanoma, promotes slowly growing carcinogen-induced melanomas in the skin of mice carrying a mutated cyclin-dependent kinase 4 (CDK4(R24C)). Deregulated receptor tyrosine kinase signaling, which is another important feature of human melanoma, leads to spontaneous development of metastatic melanoma after a long latency period in mice overexpressing hepatocyte growth factor/scatter factor (HGF/SF mice). Here we report that treatment with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate induced metastatic melanomas in all HGF/SF mice on the C57BL/6 background, which histologically resemble human melanoma. Importantly, mutant CDK4 dramatically increased the number and the growth kinetics of carcinogen-induced primary melanomas in the skin and promoted the growth of spontaneous metastases in lymph nodes and lungs in all HGF/SF mice within the first 3 months of life. Apart from very few skin papillomas, we did not observe tumors of other histology in carcinogen-treated HGF/SF x CDK4(R24C) mice. This new experimental mouse model can now be exploited to study further the biology of melanoma and evaluate new treatment modalities.

  2. 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.

  3. 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.

  4. Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros.

    PubMed

    Woolf, A S; Kolatsi-Joannou, M; Hardman, P; Andermarcher, E; Moorby, C; Fine, L G; Jat, P S; Noble, M D; Gherardi, E

    1995-01-01

    Several lines of evidence suggest that hepatocyte growth factor/scatter factor (HGF/SF), a soluble protein secreted by embryo fibroblasts and several fibroblast lines, may elicit morphogenesis in adjacent epithelial cells. We investigated the role of HGF/SF and its membrane receptor, the product of the c-met protooncogene, in the early development of the metanephric kidney. At the inception of the mouse metanephros at embryonic day 11, HGF/SF was expressed in the mesenchyme, while met was expressed in both the ureteric bud and the mesenchyme, as assessed by reverse transcription PCR, in situ hybridization, and immunohistochemistry. To further investigate the expression of met in renal mesenchyme, we isolated 13 conditionally immortal clonal cell lines from transgenic mice expressing a temperature-sensitive mutant of the SV-40 large T antigen. Five had the HGF/SF+/met+ phenotype and eight had the HGF/SF-/met+ phenotype. None had the HGF/SF+/met- nor the HGF/SF-/met- phenotypes. Thus the renal mesenchyme contains cells that express HGF/SF and met or met alone. When metanephric rudiments were grown in serum-free organ culture, anti-HGF/SF antibodies (a) inhibited the differentiation of metanephric mesenchymal cells into the epithelial precursors of the nephron; (b) increased cell death within the renal mesenchyme; and (c) perturbed branching morphogenesis of the ureteric bud. These data provide the first demonstration for coexpression of the HGF/SF and met genes in mesenchymal cells during embryonic development and also imply an autocrine and/or paracrine role for HGF/SF and met in the survival of the renal mesenchyme and in the mesenchymal-epithelial transition that occurs during nephrogenesis. They also confirm the postulated paracrine role of HGF/SF in the branching of the ureteric bud.

  5. 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.

  6. Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte growth factor.

    PubMed

    Tatsumi, Ryuichi; Liu, Xiaosong; Pulido, Antonio; Morales, Mark; Sakata, Tomowa; Dial, Sharon; Hattori, Akihito; Ikeuchi, Yoshihide; Allen, Ronald E

    2006-06-01

    In the present study, we examined the roles of hepatocyte growth factor (HGF) and nitric oxide (NO) in the activation of satellite cells in passively stretched rat skeletal muscle. A hindlimb suspension model was developed in which the vastus, adductor, and gracilis muscles were subjected to stretch for 1 h. Satellite cells were activated by stretch determined on the basis of 5-bromo-2'-deoxyuridine (BrdU) incorporation in vivo. Extracts from stretched muscles stimulated BrdU incorporation in freshly isolated control rat satellite cells in a concentration-dependent manner. Extracts from stretched muscles contained the active form of HGF, and the satellite cell-activating activity could be neutralized by incubation with anti-HGF antibody. The involvement of NO was investigated by administering nitro-L-arginine methyl ester (L-NAME) or the inactive enantiomer N(G)-nitro-D-arginine methyl ester HCl (D-NAME) before stretch treatment. In vivo activation of satellite cells in stretched muscle was not inhibited by D-NAME but was inhibited by L-NAME. The activity of stretched muscle extract was abolished by L-NAME treatment but could be restored by the addition of HGF, indicating that the extract was not inhibitory. Finally, NO synthase activity in stretched and unstretched muscles was assayed in muscle extracts immediately after 2-h stretch treatment and was found to be elevated in stretched muscle but not in stretched muscle from L-NAME-treated rats. The results of these experiments demonstrate that stretching muscle liberates HGF in a NO-dependent manner, which can activate satellite cells.

  7. 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

  8. Increased Epidermal Growth Factor Receptor (EGFR) Associated with Hepatocyte Growth Factor (HGF) and Symptom Severity in Children with Autism Spectrum Disorders (ASDs)

    PubMed Central

    Russo, Anthony J

    2014-01-01

    BACKGROUND One in 88 children in the US is thought to have one of the autism spectrum disorders (ASDs). ASDs are characterized by social impairments and communication problems. Growth factors and their receptors may play a role in the etiology of ASDs. Research has shown that epidermal growth factor receptor (EGFR) activation is associated with nerve cell development and repair. This study was designed to measure plasma levels of EGFR in autistic children and correlate these levels with its ligand, epidermal growth factor, other related putative biomarkers such as hepatocyte growth factor (HGF), the ligand for MET (MNNG HOS transforming gene) receptor, as well as the symptom severity of 19 different behavioral symptoms. SUBJECTS AND METHODS Plasma EGFR concentration was measured in 33 autistic children and 34 age- and gender-similar neurotypical controls, using an enzyme-linked immunosorbent assay. Plasma EGFR levels were compared to putative biomarkers known to be associated with EGFR and MET and severity levels of 19 autism-related symptoms. RESULTS We found plasma EGFR levels significantly higher in autistic children, when compared to neurotypical controls. EGFR levels correlated with HGF and high-mobility group protein B1 (HMGB1) levels, but not other tested putative biomarkers, and EGFR levels correlated significantly with severity of expressive language, conversational language, focus/attention, hyperactivity, eye contact, and sound sensitivity deficiencies. CONCLUSIONS These results suggest a relationship between increased plasma EGFR levels and designated symptom severity in autistic children. A strong correlation between plasma EGFR and HGF and HMGB1 suggests that increased EGFR levels may be associated with the HGF/Met signaling pathway, as well as inflammation. PMID:25249767

  9. Chronic lymphocytic leukemia nurse-like cells express hepatocyte growth factor receptor (c-MET) and indoleamine 2,3-dioxygenase and display features of immunosuppressive type 2 skewed macrophages.

    PubMed

    Giannoni, Paolo; Pietra, Gabriella; Travaini, Giorgia; Quarto, Rodolfo; Shyti, Genti; Benelli, Roberto; Ottaggio, Laura; Mingari, Maria Cristina; Zupo, Simona; Cutrona, Giovanna; Pierri, Ivana; Balleari, Enrico; Pattarozzi, Alessandra; Calvaruso, Marco; Tripodo, Claudio; Ferrarini, Manlio; de Totero, Daniela

    2014-06-01

    Hepatocyte growth factor, produced by stromal and follicular dendritic cells, and present at high concentrations in the sera of patients with chronic lymphocytic leukemia, prolongs the survival of leukemic B cells by interacting with their receptor, c-MET. It is, however, unknown whether hepatocyte growth factor influences microenvironmental cells, such as nurse-like cells, which deliver survival signals to the leukemic clone. We evaluated the expression of c-MET on nurse-like cells and monocytes from patients with chronic lymphocytic leukemia and searched for phenotypic/functional features supposed to be influenced by the hepatocyte growth factor/c-MET interaction. c-MET is expressed at high levels on nurse-like cells and at significantly higher levels than normal on monocytes from patients. Moreover, the hepatocyte growth factor/c-MET interaction activates STAT3(TYR705) phosphorylation in nurse-like cells. Indoleamine 2,3-dioxygenase, an enzyme modulating T-cell proliferation and induced on normal monocytes after hepatocyte growth factor treatment, was detected together with interleukin-10 on nurse-like cells, and on freshly-prepared patients' monocytes. Immunohistochemical/immunostaining analyses demonstrated the presence of c-MET(+) and indoleamine 2,3-dioxygenase(+) cells in lymph node biopsies, co-expressed with CD68 and vimentin. Furthermore nurse-like cells and chronic lymphocytic monocytes significantly inhibited T-cell proliferation, prevented by anti-transforming growth factor beta and interleukin-10 antibodies and indoleamine 2,3-dioxygenase inhibitors, and supported CD4(+)CD25(high+)/FOXP3(+) T regulatory cell expansion. We suggest that nurse-like cells display features of immunosuppressive type 2 macrophages: higher hepatocyte growth factor levels, produced by leukemic or other microenvironmental surrounding cells, may cooperate to induce M2 polarization. Hepatocyte growth factor may thus have a dual pathophysiological role: directly through

  10. Chronic lymphocytic leukemia nurse-like cells express hepatocyte growth factor receptor (c-MET) and indoleamine 2,3-dioxygenase and display features of immunosuppressive type 2 skewed macrophages

    PubMed Central

    Giannoni, Paolo; Pietra, Gabriella; Travaini, Giorgia; Quarto, Rodolfo; Shyti, Genti; Benelli, Roberto; Ottaggio, Laura; Mingari, Maria Cristina; Zupo, Simona; Cutrona, Giovanna; Pierri, Ivana; Balleari, Enrico; Pattarozzi, Alessandra; Calvaruso, Marco; Tripodo, Claudio; Ferrarini, Manlio; de Totero, Daniela

    2014-01-01

    Hepatocyte growth factor, produced by stromal and follicular dendritic cells, and present at high concentrations in the sera of patients with chronic lymphocytic leukemia, prolongs the survival of leukemic B cells by interacting with their receptor, c-MET. It is, however, unknown whether hepatocyte growth factor influences microenvironmental cells, such as nurse-like cells, which deliver survival signals to the leukemic clone. We evaluated the expression of c-MET on nurse-like cells and monocytes from patients with chronic lymphocytic leukemia and searched for phenotypic/functional features supposed to be influenced by the hepatocyte growth factor/c-MET interaction. c-MET is expressed at high levels on nurse-like cells and at significantly higher levels than normal on monocytes from patients. Moreover, the hepatocyte growth factor/c-MET interaction activates STAT3TYR705 phosphorylation in nurse-like cells. Indoleamine 2,3-dioxygenase, an enzyme modulating T-cell proliferation and induced on normal monocytes after hepatocyte growth factor treatment, was detected together with interleukin-10 on nurse-like cells, and on freshly-prepared patients’ monocytes. Immunohistochemical/immunostaining analyses demonstrated the presence of c-MET+ and indoleamine 2,3-dioxygenase+ cells in lymph node biopsies, co-expressed with CD68 and vimentin. Furthermore nurse-like cells and chronic lymphocytic monocytes significantly inhibited T-cell proliferation, prevented by anti-transforming growth factor beta and interleukin-10 antibodies and indoleamine 2,3-dioxygenase inhibitors, and supported CD4+CD25high+/FOXP3+ T regulatory cell expansion. We suggest that nurse-like cells display features of immunosuppressive type 2 macrophages: higher hepatocyte growth factor levels, produced by leukemic or other microenvironmental surrounding cells, may cooperate to induce M2 polarization. Hepatocyte growth factor may thus have a dual pathophysiological role: directly through enhancement of

  11. 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.

  12. 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.

  13. Effects of hepatocyte growth factor/scatter factor on the invasion of colorectal cancer cells in vitro

    PubMed Central

    Li, Hong-Wu; Shan, Ji-Xian

    2005-01-01

    AIM: Hepatocyte growth factor (HGF) is a multifunctional growth factor which has pleiotrophic biological effects on epithelial cells, such as proliferation, motogenesis, invas-iveness and morphogenesis. There are few reports about the role of HGF played in the colorectal cancer invasion. In the present study, we tried to investigate the possible mechanism of HGF involved in the invasion of colorectal cancer cells in vitro. METHODS: Matrigel migration assay was used to analyze the migrational ability of Caco-2 and Colo320 in vitro. We detected the mRNA expressive levels of MMP-2, MMP-9 and their natural inhibitors TIMP-1, TIMP-2 in Caco-2 cells by reverse-transcription polymerase chain reaction (PCR) technique. RESULTS: After 48 h incubation, there were notable differences when we compared the migrational numbers of Caco-2 cells in the group of HGF and PD98059 (the inhibitor of p42/p44MAPK) with the control (104.40 ± 4.77 vs 126.80 ± 5.40, t = 7.17, P = 0.002 < 0.01; 104.40 ± 4.77 vs 82.80 ± 4.15, t = 7.96, P = 0.001 < 0.01). The deviation between the HGF and PD98059 was significant (P < 0.01). Compared with controls, MMP-2 and MMP-9 mRNA expres-sions were up-regulated by HGF (0.997 ± 0.011 vs 1.207 ± 0.003, t = 35.002,P = 0.001 < 0.01; 0.387 ± 0.128 vs 0.971 ± 0.147, t = 106.036, P = 0.0000 < 0.01, respectively); compared with controls, TIMP-1, TIMP-2 mRNA expressions were increased by PD98059 (1.344 ± 0.007 vs 1.905 ± 0.049, t = 17.541, P = 0.003 < 0.01; 1.286 ± 0.020 vs 1.887 ± 0.022, t = 24.623, P = 0.002 < 0.01, respectively). CONCLUSION: HGF promoted Caco-2 migration mainly by p42/p44MAPK pathway; HGF/SF stimulated the expression of MMP-2, MMP-9 in Caco-2 and enabled tumoral cells to damage the ECM and reach the distant organ and develop metastasis; HGF played the function of promoted-invasion and promoted-metastasis, in which cellular selection was possible. PMID:15991286

  14. 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

  15. Identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse hepatocyte growth factor gene.

    PubMed Central

    Liu, Y; Beedle, A B; Lin, L; Bell, A W; Zarnegar, R

    1994-01-01

    Hepatocyte growth factor (HGF), a cytokine with multiple functions, exhibits cell-type-specific as well as cytokine- and steroid hormone-regulated expression. The HGF gene is known to be expressed predominately in mesenchymal but not in epithelial cells. In this study, we report the identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse HGF gene, which is evidently responsible for the suppression of HGF expression in epithelial cells. Gel mobility shift assays and DNase I footprinting studies revealed that a 27-bp element (-16 to +11) around the transcription initiation site is responsible for the binding of a nuclear protein which is present in epithelial but not in mesenchymally derived cells. Further analysis of the binding activity of the DNA region with nuclear protein revealed that an approximately 19-bp sequence containing a unique palindromic structure (5'-AACCGACCGGTT-3') overlapped by a CAP box is essential for binding. Substitution of a single base (the contact site) within this region by site-directed mutagenesis resulted in total abrogation of the binding of the nuclear protein and a concomitant increase in the transcriptional activity of various lengths of HGF-chloramphenicol acetyltransferase fused genes when transfected into the epithelial cell line RL95-2 but not the mesenchymal cell line NIH 3T3. Southwestern (DNA-protein) analyses revealed that the nuclear protein which binds to this repressor element is a single polypeptide of approximately 70 kDa. Analysis of the nuclear extract prepared from regenerating mouse liver at various times after two-thirds partial hepatectomy by gel mobility shift assay revealed a substantial reduction (more than 75% within 3 h) in the binding of the repressor to its cognate binding site. Our results suggest that a cis-acting transcriptional repressor in the promoter region of the mouse HGF gene is involved in cell-type-specific regulation through binding to its cognate

  16. 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

  17. Elimination of HCV via a non-ISG-mediated mechanism by vaniprevir and BMS-788329 combination therapy in human hepatocyte chimeric mice.

    PubMed

    Uchida, Takuro; Hiraga, Nobuhiko; Imamura, Michio; Yoshimi, Satoshi; Kan, Hiromi; Miyaki, Eisuke; Tsuge, Masataka; Abe, Hiromi; Hayes, C Nelson; Aikata, Hiroshi; Ishida, Yuji; Tateno, Chise; Ellis, Joan D; Chayama, Kazuaki

    2016-02-01

    We previously reported that interferon (IFN)-free direct-acting antiviral combination treatment succeeded in eradicating genotype 1b hepatitis C virus (HCV) in human hepatocyte chimeric mice. In this study, we examined the effect of vaniprevir (MK7009, NS3/4A protease inhibitor) and BMS-788329 (NS5A inhibitor) combination treatment on HCV genotype 1b and the expression of IFN-stimulated genes (ISGs) using a subgenomic replicon system and the same animal model. Combination treatment with vaniprevir and BMS-788329 significantly reduced HCV replication compared to vaniprevir monotherapy in HCV replicon cells (Huh7/Rep-Feo cells). HCV genotype 1b-infected human hepatocyte chimeric mice were treated with vaniprevir alone or in combination with BMS-788329 for four weeks. Vaniprevir monotherapy reduced serum HCV RNA titers in mice, but viral breakthrough was observed in mice with high HCV titers. Ultra-deep sequence analysis revealed a predominant replacement by drug-resistant substitutions at 168 in HCV NS3 region in these mice. Conversely, in mice with low HCV titers, HCV was eradicated by vaniprevir monotherapy without viral breakthrough. In contrast to monotherapy, combination treatment with vaniprevir and BMS-788329 succeeded in completely eradicating HCV regardless of serum viral titer. IFN-alpha treatment significantly increased ISG expression; however, vaniprevir and BMS-788329 combination treatment caused no increase in ISG expression both in cultured cells and in mouse livers. Therefore, combination treatment with vaniprevir and BMS-788329 eliminated HCV via a non-ISG-mediated mechanism. This oral treatment might offer an alternative DAA combination therapy for patients with chronic hepatitis C. PMID:26569595

  18. 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

  19. P2Y2 purinergic receptor activation is essential for efficient hepatocyte proliferation in response to partial hepatectomy

    PubMed Central

    Tackett, Bryan C.; Sun, Hongdan; Mei, Yu; Maynard, Janielle P.; Cheruvu, Sayuri; Mani, Arunmani; Hernandez-Garcia, Andres; Vigneswaran, Nadarajah; Karpen, Saul J.

    2014-01-01

    Extracellular nucleotides via activation of P2 purinergic receptors influence hepatocyte proliferation and liver regeneration in response to 70% partial hepatectomy (PH). Adult hepatocytes express multiple P2Y (G protein-coupled) and P2X (ligand-gated ion channels) purinergic receptor subtypes. However, the identity of key receptor subtype(s) important for efficient hepatocyte proliferation in regenerating livers remains unknown. To evaluate the impact of P2Y2 purinergic receptor-mediated signaling on hepatocyte proliferation in regenerating livers, wild-type (WT) and P2Y2 purinergic receptor knockout (P2Y2−/−) mice were subjected to 70% PH. Liver tissues were analyzed for activation of early events critical for hepatocyte priming and subsequent cell cycle progression. Our findings suggest that early activation of p42/44 ERK MAPK (5 min), early growth response-1 (Egr-1) and activator protein-1 (AP-1) DNA-binding activity (30 min), and subsequent hepatocyte proliferation (24–72 h) in response to 70% PH were impaired in P2Y2−/− mice. Interestingly, early induction of cytokines (TNF-α, IL-6) and cytokine-mediated signaling (NF-κB, STAT-3) were intact in P2Y2−/− remnant livers, uncovering the importance of cytokine-independent and nucleotide-dependent early priming events critical for subsequent hepatocyte proliferation in regenerating livers. Hepatocytes isolated from the WT and P2Y2−/− mice were treated with ATP or ATPγS for 5–120 min and 12–24 h. Extracellular ATP alone, via activation of P2Y2 purinergic receptors, was sufficient to induce ERK phosphorylation, Egr-1 protein expression, and key cyclins and cell cycle progression of hepatocytes in vitro. Collectively, these findings highlight the functional significance of P2Y2 purinergic receptor activation for efficient hepatocyte priming and proliferation in response to PH. PMID:25301185

  20. The Pore-Forming Toxin Listeriolysin O Mediates a Novel Entry Pathway of L. monocytogenes into Human Hepatocytes

    PubMed Central

    Vadia, Stephen; Arnett, Eusondia; Haghighat, Anne-Cécile; Wilson-Kubalek, Elisabeth M.; Tweten, Rodney K.; Seveau, Stephanie

    2011-01-01

    Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell. PMID:22072970

  1. 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

  2. Activation of nitrosamines and other carcinogens by mouse-liver S9, mouse hepatocytes and in the host-mediated assay produces different mutagenic responses in Salmonella TA1535.

    PubMed

    Kerklaan, P; Bouter, S; Mohn, G

    1983-01-01

    5 indirect alkylating carcinogens, namely, dimethylnitrosamine (DMNA), methylethylnitrosamine (MENA), diethylnitrosamine (DENA), 1,2-dimethylhydrazine (DMH) and cyclophosphamide (CP), were tested in liquid incubation assays for their mutagenic activity towards Salmonella TA1535 in the presence of mouse-liver homogenate (S9) or freshly isolated, single liver-cell preparations. The capacity of these mouse-liver preparations to activate the compounds to mutagens for TA1535 was compared with the mutagenic effect of low doses of the carcinogens in intrasanguineous host-mediated assays, with the same strain of mice as host. Although the mouse hepatocytes retained their activating capacity longer than S9 preparations did during incubation at 37 degrees C, the latter gave much higher yields of mutants with 10 mM (DMNA, MENA, DMH) and 5 mM (CP) of 4 out of the 5 compounds. DENA was not mutagenic in either assay. These differences between whole cell and disrupted cell preparations were reduced or absent when the concentrations of the test compounds were reduced by a factor of 10. It was concluded that hepatocytes at the maximal concentration of cells have a limited capacity to metabolize the mutagens. On the basis of protein concentration, hepatocytes are more effective (nitrosamines) or equally effective (CP and DMH) in activating the compounds. Compared with the host-mediated assays, both liver fractions have only a marginal potential to activate equal low amounts of the carcinogens. The present results do not indicate that hepatocytes take an 'intermediate' position between existing in vitro and in vivo activation systems, although they do suggest that these mouse hepatocyte preparations activate the nitrosamines DMNA and MENA in a quantitatively or qualitatively different way than do mouse-liver homogenates.

  3. Messenger RNA transcripts of the hepatocyte nuclear factor-1alpha gene containing premature termination codons are subject to nonsense-mediated decay.

    PubMed

    Harries, Lorna W; Hattersley, Andrew T; Ellard, Sian

    2004-02-01

    Mutations in the hepatocyte nuclear factor-1alpha (HNF-1a) gene cause maturity-onset diabetes of the young (MODY). Approximately 30% of these mutations generate mRNA transcripts harboring premature termination codons (PTCs). Degradation of such transcripts by the nonsense-mediated decay (NMD) pathway has been reported for many genes. To determine whether PTC mutant transcripts of the HNF-1alpha gene elicit NMD, we have developed a novel quantitative RT-PCR assay. We performed quantification of ectopically expressed mutant transcripts relative to normal transcripts in lymphoblastoid cell lines using a coding single nucleotide polymorphism (cSNP) as a marker. The nonsense mutations R171X, I414G415ATCG-->CCA, and P291fsinsC showed reduced mutant mRNA expression to 40% (P = 0.009), <0.01% (P

  4. Daylength mediated control of seasonal growth patterns in perennial trees.

    PubMed

    Petterle, Anna; Karlberg, Anna; Bhalerao, Rishikesh P

    2013-06-01

    Daylength is a key regulator of seasonal growth patterns in perennial trees in temperate regions. Cessation of growth is induced by short day signal in these trees before the advent of winter and constitutes a major adaptive developmental program. In this review, we report on the recent progress made in identifying the molecular mechanisms that underlie the daylength mediated control of seasonal growth in perennial trees. A major finding that has emerged from the analysis of this process is that the regulation of growth cessation in perennial trees and flowering time by daylength in annuals such as Arabidopsis thaliana involves identical signalling components.

  5. Surfactant mediated growth of Ti/Ni multilayers

    SciTech Connect

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

    2011-03-07

    The surfactant mediated growth of Ti/Ni multilayers is studied. They were prepared using ion beam sputtering at different adatom energies. It was found that the interface roughness decreased significantly when the multilayers were sputtered with Ag as surfactant at an ion energy of 0.75 keV. On the other hand, when the ion energy was increased to 1 keV, it resulted in enhanced intermixing at the interfaces and no appreciable effect of Ag surfactant could be observed. On the basis of the obtained results, the influence of adatom energy on the surfactant mediated growth mechanism is discussed.

  6. 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.

  7. 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…

  8. 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.

  9. 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

  10. 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.

  11. Cancer Associated Fibroblast-Derived Hepatocyte Growth Factor Inhibits the Paclitaxel-Induced Apoptosis of Lung Cancer A549 Cells by Up-Regulating the PI3K/Akt and GRP78 Signaling on a Microfluidic Platform.

    PubMed

    Ying, Li; Zhu, Ziwei; Xu, Zhiyun; He, Tianrui; Li, Encheng; Guo, Zhe; Liu, Fen; Jiang, Chunmeng; Wang, Qi

    2015-01-01

    Tumor stroma and growth factors provide a survival environment to tumor cells and can modulate their chemoresistance by dysregulating several signal pathways. In this study, we fabricated a three-dimensional (3D) microfluidic chip using polydimethylsiloxane (PDMS) to investigate the impact of hepatocyte growth factor (HGF) from cancer-associated fibroblasts (CAF) on the Met/PI3K/AKT activation, glucose regulatory protein (GRP78) expression and the paclitaxel-induced A549 cell apoptosis. With a concentration gradient generator, the assembled chip was able to reconstruct a tumor microenvironment in vitro. We found high levels of HGF in the supernatants of CAF and the CAF matrix from the supernatants of activated HFL1 fibroblasts or HGF enhanced the levels of Met, PI3K and AKT phosphorylation and GRP78 expression in A549 cells cultured in a 3D cell chamber, which was abrogated by anti-HGF. Inhibition of Met attenuated the CAF matrix-enhanced PI3K/AKT phosphorylation and GRP78 expression while inhibition of PI3K reduced GRP78 expression, but not Met phosphorylation in A549 cells. Inhibition of GRP78 failed to modulate the CAF matrix-enhanced Met/PI3K/AKT phosphorylation in A549 cells. Furthermore, inhibition of PI3K or GRP78 enhanced spontaneous and paclitaxel-induced A549 cell apoptosis. Moreover, treatment with the CAF matrix inhibited spontaneous and medium or high dose of paclitaxel-induced A549 cell apoptosis. Inhibition of PI3K or GRP78 attenuated the CAF matrix-mediated inhibition on paclitaxel-induced A549 cell apoptosis. Our data indicated that HGF in the CAF matrix activated the Met/PI3K/AKT and up-regulated GRP78 expression, promoting chemoresistance to paclitaxel-mediated apoptosis in A549 cells. Our findings suggest that the microfluidic system may represent an ideal platform for signaling research and drug screening. PMID:26115510

  12. Cancer Associated Fibroblast-Derived Hepatocyte Growth Factor Inhibits the Paclitaxel-Induced Apoptosis of Lung Cancer A549 Cells by Up-Regulating the PI3K/Akt and GRP78 Signaling on a Microfluidic Platform

    PubMed Central

    Xu, Zhiyun; He, Tianrui; Li, Encheng; Guo, Zhe; Liu, Fen; Jiang, Chunmeng; Wang, Qi

    2015-01-01

    Tumor stroma and growth factors provide a survival environment to tumor cells and can modulate their chemoresistance by dysregulating several signal pathways. In this study, we fabricated a three-dimensional (3D) microfluidic chip using polydimethylsiloxane (PDMS) to investigate the impact of hepatocyte growth factor (HGF) from cancer-associated fibroblasts (CAF) on the Met/PI3K/AKT activation, glucose regulatory protein (GRP78) expression and the paclitaxel-induced A549 cell apoptosis. With a concentration gradient generator, the assembled chip was able to reconstruct a tumor microenvironment in vitro. We found high levels of HGF in the supernatants of CAF and the CAF matrix from the supernatants of activated HFL1 fibroblasts or HGF enhanced the levels of Met, PI3K and AKT phosphorylation and GRP78 expression in A549 cells cultured in a 3D cell chamber, which was abrogated by anti-HGF. Inhibition of Met attenuated the CAF matrix-enhanced PI3K/AKT phosphorylation and GRP78 expression while inhibition of PI3K reduced GRP78 expression, but not Met phosphorylation in A549 cells. Inhibition of GRP78 failed to modulate the CAF matrix-enhanced Met/PI3K/AKT phosphorylation in A549 cells. Furthermore, inhibition of PI3K or GRP78 enhanced spontaneous and paclitaxel-induced A549 cell apoptosis. Moreover, treatment with the CAF matrix inhibited spontaneous and medium or high dose of paclitaxel-induced A549 cell apoptosis. Inhibition of PI3K or GRP78 attenuated the CAF matrix-mediated inhibition on paclitaxel-induced A549 cell apoptosis. Our data indicated that HGF in the CAF matrix activated the Met/PI3K/AKT and up-regulated GRP78 expression, promoting chemoresistance to paclitaxel-mediated apoptosis in A549 cells. Our findings suggest that the microfluidic system may represent an ideal platform for signaling research and drug screening. PMID:26115510

  13. 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

  14. 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

  15. 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.

  16. Hepatocyte Death: A Clear and Present Danger

    PubMed Central

    MALHI, HARMEET; GUICCIARDI, MARIA EUGENIA; GORES, GREGORY J.

    2010-01-01

    The hepatocyte is especially vulnerable to injury due to its central role in xenobiotic metabolism including drugs and alcohol, participation in lipid and fatty acid metabolism, its unique role in the enterohepatic circulation of bile acids, the widespread prevalence of hepatotropic viruses, and its existence within a milieu of innate immune responding cells. Apoptosis and necrosis are the most widely recognized forms of hepatocyte cell death. The hepatocyte displays many unique features regarding cell death by apoptosis. It is quite susceptible to death receptor-mediated injury, and its death receptor signaling pathways involve the mitochondrial pathway for efficient cell killing. Also, death receptors can trigger lysosomal disruption in hepatocytes which further promote cell and tissue injury. Interestingly, hepatocytes are protected from cell death by only two anti-apoptotic proteins, Bcl-xL and Mcl-1, which have nonredundant functions. Endoplasmic reticulum stress or the unfolded protein response contributes to hepatocyte cell death during alterations of lipid and fatty acid metabolism. Finally, the current information implicating RIP kinases in necrosis provides an approach to more fully address this mode of cell death in hepatocyte injury. All of these processes contributing to hepatocyte injury are discussed in the context of potential therapeutic strategies. PMID:20664081

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Apoptosis is induced by N-myc expression in hepatocytes, a frequent event in hepadnavirus oncogenesis, and is blocked by insulin-like growth factor II.

    PubMed Central

    Ueda, K; Ganem, D

    1996-01-01

    Induction of hepatocellular carcinoma in woodchucks by woodchuck hepatitis virus is associated with the activation of N-myc gene expression, usually by viral DNA integration in cis to the N-myc locus. We have examined the consequences of N-myc up-regulation in rodent hepatic cells in culture. Mouse alpha ML hepatocytes infected with a retroviral vector overexpressing the woodchuck N-myc2 gene display a higher proliferation rate than parental alpha ML cells but are morphologically unchanged and do not form colonies in soft agar. However, they display an increased propensity to undergo apoptosis, an effect that is markedly augmented by serum deprivation. Expression of the woodchuck hepatitis virus X gene in alpha ML cells does not alter the growth phenotype of the cells and has no effect upon N-myc-dependent apoptosis. However, apoptosis in N-myc2-expressing alpha ML cells is strongly inhibited by insulin-like growth factor II (IGF II). IGF II gene expression is also strongly up-regulated during hepatic carcinogenesis in vivo in virally infected animals and has been speculated to be part of an autocrine growth-stimulatory pathway. Our results suggest that IGF II may play another role in the development of virus-induced hepatoma: the prevention of programmed cell death triggered by deregulated N-myc expression. PMID:8627653

  2. 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

  3. 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

  4. 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

  5. Lipid rafts mediate chemotropic guidance of nerve growth cones.

    PubMed

    Guirland, Carmine; Suzuki, Shingo; Kojima, Masami; Lu, Bai; Zheng, James Q

    2004-04-01

    Axon guidance requires signal transduction of extracellular cues through the plasma membrane for directional motility. Here we present evidence that cholesterol- and sphingolipid-enriched membrane microdomains (lipid rafts) mediate specific guidance responses of nerve growth cones. Disruption of lipid rafts by various approaches targeting cholesterol or gangliosides selectively abolished growth cone attraction and repulsion in BDNF and netrin-1 gradients, respectively, without affecting glutamate-induced attraction. Interestingly, local raft disruption on one side of the growth cone in bath BDNF or netrin-1 produced opposite turning responses to that induced by the gradients. Raft manipulation also blocked Semaphorin 3A-induced growth cone repulsion, inhibition, and collapse. Finally, guidance responses appeared to involve raft-dependent activation of p42/p44 MAPK and ligand-induced receptor recruitment to lipid rafts. Together with the observation of asymmetric receptor-raft associations at the growth cone in guidance gradients, our findings indicate that localized signaling through membrane rafts plays a role in mediating guidance actions of extracellular cues on developing axons. PMID:15066264

  6. 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

  7. 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.

  8. 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

  9. Thyroid hormone mediates otolith growth and development during flatfish metamorphosis.

    PubMed

    Schreiber, A M; Wang, X; Tan, Y; Sievers, Q; Sievers, B; Lee, M; Burrall, K

    2010-11-01

    Flatfish begin life as bilaterally symmetrical larvae that swim up-right, then abruptly metamorphose into asymmetrically shaped juveniles with lateralized swimming postures. Flatfish metamorphosis is mediated entirely by thyroid hormone (TH). Changes in flatfish swim posture are thought to be regulated via vestibular remodeling, although the influence of TH on teleost inner ear development remains unclear. This study addresses the role of TH on the development of the three otolith end-organs (sacculus, utricle, and lagena) during southern flounder (Paralichthys lethostigma) metamorphosis. Compared with pre-metamorphosis, growth rates of the sacculus and utricle otoliths increase dramatically during metamorphosis in a manner that is uncoupled from general somatic growth. Treatment of P. lethostigma larvae with methimazol (a pharmacological inhibitor of endogenous TH production) inhibits growth of the sacculus and utricle, whereas treatment with TH dramatically accelerates their growth. In contrast with the sacculus and utricle otoliths that begin to form and mineralize during embryogenesis, a non-mineralized lagena otolith is first visible 10-12 days after hatching. The lagena grows during pre- and pro-metamorphosis, then abruptly mineralizes during metamorphic climax. Mineralization of the lagena, but not growth, can be induced with TH treatment, whereas treatment with methimazol completely inhibits lagena mineralization without inhibiting its growth. These findings suggest that during southern flounder metamorphosis TH exerts differential effects on growth and development among the three types of otolith.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. The unique Polarity Phenotype of Hepatocytes

    PubMed Central

    Müsch, Anne

    2014-01-01

    Hepatocytes, the main epithelial cell type of the liver, function like all epithelial cells to mediate the vectorial flow of macromolecules into and out of the organ they encompass. They do so by establishing polarized surface domains and by restricting paracellular flow via their tight junctions and cell-cell adhesion. Yet, the cell and tissue organization of hepatocytes differs profoundly from that of most other epithelia, including those of the digestive and urinary tracts, the lung or the breast. The latter form monolayered tissues in which the apical domains of individual cells align around a central continuous luminal cavity that constitutes the tubules and acini characteristic of these organs. Hepatocytes, by contrast, form capillary-sized lumina with multiple neighbors resulting in a branched, tree-like bile canaliculi network that spreads across the liver parenchyme. I will discuss some of the key molecular features that distinguish the hepatocyte polarity phenotype from that of monopolar, columnar epithelia. PMID:24956563

  16. 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.

  17. 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

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

    PubMed

    Armentano, D; Thompson, A R; Darlington, G; Woo, S L

    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. We 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. The Procognitive and Synaptogenic Effects of Angiotensin IV–Derived Peptides Are Dependent on Activation of the Hepatocyte Growth Factor/c-Met System

    PubMed Central

    Benoist, Caroline C.; Kawas, Leen H.; Zhu, Mingyan; Tyson, Katherine A.; Stillmaker, Lori; Appleyard, Suzanne M.; Wright, John W.

    2014-01-01

    A subset of angiotensin IV (AngIV)–related molecules are known to possess procognitive/antidementia properties and have been considered as templates for potential therapeutics. However, this potential has not been realized because of two factors: 1) a lack of blood-brain barrier–penetrant analogs, and 2) the absence of a validated mechanism of action. The pharmacokinetic barrier has recently been overcome with the synthesis of the orally active, blood-brain barrier–permeable analog N-hexanoic-tyrosine-isoleucine-(6) aminohexanoic amide (dihexa). Therefore, the goal of this study was to elucidate the mechanism that underlies dihexa’s procognitive activity. Here, we demonstrate that dihexa binds with high affinity to hepatocyte growth factor (HGF) and both dihexa and its parent compound Norleucine 1-AngIV (Nle1-AngIV) induce c-Met phosphorylation in the presence of subthreshold concentrations of HGF and augment HGF-dependent cell scattering. Further, dihexa and Nle1-AngIV induce hippocampal spinogenesis and synaptogenesis similar to HGF itself. These actions were inhibited by an HGF antagonist and a short hairpin RNA directed at c-Met. Most importantly, the procognitive/antidementia capacity of orally delivered dihexa was blocked by an HGF antagonist delivered intracerebroventricularly as measured using the Morris water maze task of spatial learning. PMID:25187433

  7. Sustained release of hepatocyte growth factor by cationic self-assembling peptide/heparin hybrid hydrogel improves β-cell survival and function through modulating inflammatory response

    PubMed Central

    Liu, Shuyun; Zhang, Lanlan; Cheng, Jingqiu; Lu, Yanrong; Liu, Jingping

    2016-01-01

    Inflammatory response is a major cause of grafts dysfunction in islet transplantation. Hepatocyte growth factor (HGF) had shown anti-inflammatory activity in multiple diseases. In this study, we aim to deliver HGF by self-assembling peptide/heparin (SAP/Hep) hybrid gel to protect β-cell from inflammatory injury. The morphological and slow release properties of SAPs were analyzed. Rat INS-1 β-cell line was treated with tumor necrosis factor α in vitro and transplanted into rat kidney capsule in vivo, and the viability, apoptosis, function, and inflammation of β-cells were evaluated. Cationic KLD1R and KLD2R self-assembled to nanofiber hydrogel, which showed higher binding affinity for Hep and HGF because of electrostatic interaction. Slow release of HGF from cationic SAP/Hep gel is a two-step mechanism involving binding affinity with Hep and molecular diffusion. In vitro and in vivo results showed that HGF-loaded KLD2R/Hep gel promoted β-cell survival and insulin secretion, and inhibited cell apoptosis, cytokine release, T-cell infiltration, and activation of NFκB/p38 MAPK pathways in β-cells. This study suggested that SAP/Hep gel is a promising carrier for local delivery of bioactive proteins in islet transplantation. PMID:27729786

  8. Epidermal growth factor-induced selective phosphorylation of cultured rat hepatocyte 55-kD cytokeratin before filament reorganization and DNA synthesis

    PubMed Central

    1989-01-01

    We have reported previously that the addition of dexamethasone to cultured quiescent suckling rat hepatocytes in the presence of insulin, a culture condition which does not cause growth activation, induces a selective increase in the synthesis of the 49-kD/55-kD cytokeratin (CK49/CK55) pair over a 24-h period. This increased synthesis coincides with the formation of dense filament networks reminiscent of those observed in situ at the cell periphery (Marceau, N., H. Baribault, and I. Leroux-Nicollet. 1985. Can. J. Biochem. Cell Biol. 63:448-457). We show here for the first time that when EGF is added 48 h after insulin and dexamethasone, there is an early preferential phosphorylation of the CK55 of the CK49/CK55 pair, an induced filament rearrangement from the cell periphery to the cytoplasm, and a subsequent entry into S phase and mitosis after a lag period of 8 h. Indirect immunofluorescence microscopy with monoclonal antibodies to CK49 and CK55 indicate that, while before EGF treatment the cytokeratin filaments were mainly distributed near the cell periphery, the addition of EGF resulted in their reorganization to a predominantly cytoplasmic localization within less than 3 h. Antitubulin and anti-actin antibodies showed no detectable alteration in the distribution of microtubules and microfilaments. Pulse-chase measurements with [35S]methionine showed no apparent change in the turnover of either CK49 or CK55 during the period that precedes the initiation of DNA synthesis. 32P-labeling in vivo followed by SDS-PAGE demonstrated that CK55 was phosphorylated at a much higher level than CK49 in nonstimulated hepatocytes, and that the addition of EGF resulted in a selective stimulation of 32P-CK55 labeling within less than 30 min. Comparative analyses by two-dimensional PAGE of [35S]methionine and 32P- labeled cytokeratins at various times after EGF stimulation demonstrated a rapid increase in a first phosphorylated form of CK55 and the appearance of a second

  9. Growth hormone STAT5-mediated signaling and its modulation in mice liver during the growth period.

    PubMed

    Martinez, Carolina S; Piazza, Verónica G; Ratner, Laura D; Matos, Marina N; González, Lorena; Rulli, Susana B; Miquet, Johanna G; Sotelo, Ana I

    2013-01-01

    Postnatal growth exhibits two instances of rapid growth in mice: the first is perinatal and independent of growth hormone (GH), the second is peripuberal and GH-dependent. Signal transducer and activator of transcription 5b (STAT5b) is the main GH-signaling mediator and it is related to IGF1 synthesis and somatic growth. The aim of this work was to assess differential STAT5 sensitivity to GH during the growth period in mouse liver of both sexes. Three representative ages were selected: 1-week-old animals, in the GH-independent phase of growth; 2.5-week-old mice, at the onset of the GH-dependent phase of growth; and 9-week-old young adults. GH-signaling mediators were assessed by immunoblotting, quantitative RT-PCR and immunohistochemistry. GH-induced STAT5 phosphorylation is low at one-week and maximal at 2.5-weeks of age when compared to young adults, accompanied by higher protein content at the onset of growth. Suppressor CIS and phosphatase PTP1B exhibit high levels in one-week animals, which gradually decline, while SOCS2 and SOCS3 display higher levels at adulthood. Nuclear phosphorylated STAT5 is low in one-week animals while in 2.5-week animals it is similar to 9-week control; expression of SOCS3, an early response GH-target gene, mimics this pattern. STAT5 coactivators glucocorticoid receptor (GR) and hepatic nuclear factor 1 (HNF1) abundance is higher in adulthood. Therefore, GH-induced STAT5 signaling presents age-dependent activity in liver, with its maximum coinciding with the onset of GH-dependent phase of growth, accompanied by an age-dependent variation of modulating factors. This work contributes to elucidate the molecular mechanisms implicated in GH responsiveness during growth.

  10. 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

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

    PubMed Central

    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

  12. 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

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

    PubMed

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

    2015-09-15

    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.

  14. Conjugation of mono and di-GalNAc sugars enhances the potency of antisense oligonucleotides via ASGR mediated delivery to hepatocytes.

    PubMed

    Kinberger, Garth A; Prakash, Thazha P; Yu, Jinghua; Vasquez, Guillermo; Low, Audrey; Chappell, Alfred; Schmidt, Karsten; Murray, Heather M; Gaus, Hans; Swayze, Eric E; Seth, Punit P

    2016-08-01

    Antisense oligonucleotides (ASOs) conjugated to trivalent GalNAc ligands show 10-fold enhanced potency for suppressing gene targets expressed in hepatocytes. Trivalent GalNAc is a high affinity ligand for the asialoglycoprotein receptor (ASGR)-a C-type lectin expressed almost exclusively on hepatocytes in the liver. In this communication, we show that conjugation of two and even one GalNAc sugar to single stranded chemically modified ASOs can enhance potency 5-10 fold in mice. Evaluation of the mono- and di-GalNAc ASO conjugates in an ASGR binding assay suggested that chemical features of the ASO enhance binding to the receptor and provide a rationale for the enhanced potency.

  15. Sulforaphane- and phenethyl isothiocyanate-induced inhibition of aflatoxin B1-mediated genotoxicity in human hepatocytes: role of GSTM1 genotype and CYP3A4 gene expression.

    PubMed

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

    2010-08-01

    Primary cultures of human hepatocytes were used to investigate whether the dietary isothiocyanates, sulforaphane (SFN), and phenethyl isothiocyanate (PEITC) can reduce DNA adduct formation of the hepatocarcinogen aflatoxin B(1) (AFB). Following 48 h of pretreatment, 10 and 50 microM SFN greatly decreased AFB-DNA adduct levels, whereas 25muM PEITC decreased AFB-DNA adducts in some but not all hepatocyte preparations. Microarray and quantitative reverse transcriptase (RT)-PCR analyses of gene expression in SFN and PEITC-treated hepatocytes demonstrated that SFN greatly decreased cytochrome P450 (CYP) 3A4 mRNA but did not induce the expression of either glutathione S-transferase (GST) M1 or GSTT1. The protective effects of SFN required pretreatment; cotreatment of hepatocytes with SFN and AFB in the absence of pretreatment had no effect on AFB-DNA adduct formation. When AFB-DNA adduct formation was evaluated by GST genotype, the presence of one or two functional alleles of GSTM1 was associated with a 75% reduction in AFB-DNA adducts, compared with GSTM1 null. In conclusion, these results demonstrate that the inhibition of AFB-DNA adduct formation by SFN is dependent on changes in gene expression rather than direct inhibition of catalytic activity. Transcriptional repression of genes involved in AFB bioactivation (CYP3A4 and CYP1A2), but not transcriptional activation of GSTs, may be responsible for the protective effects of SFN. Although GSTM1 expression was not induced by SFN, the presence of a functional GSTM1 allele can afford substantial protection against AFB-DNA damage in human liver. The downregulation of CYP3A4 by SFN may have important implications for drug interactions. PMID:20442190

  16. Predictions of cytochrome P450-mediated drug-drug interactions using cryopreserved human hepatocytes: comparison of plasma and protein-free media incubation conditions.

    PubMed

    Mao, Jialin; Mohutsky, Michael A; Harrelson, John P; Wrighton, Steven A; Hall, Stephen D

    2012-04-01

    Cryopreserved human hepatocytes suspended in human plasma (HHSHP) have previously provided accurate CYP3A drug-drug interaction (DDI) predictions from a single IC(50) that captures both reversible and time-dependent inhibition. The goal of this study was to compare the accuracy of DDI predictions by a protein-free human hepatocyte system combined with the fraction unbound in plasma for inhibitor(s) with those obtained with protein-containing incubations. Seventeen CYP3A, CYP2C9, or CYP2D6 inhibitors were incubated with hepatocytes in human plasma or hepatocyte maintenance medium (HMM) for 20 min over a range of concentrations after which midazolam 1'-hydroxylation, diclofenac 4'-hydroxylation or (R)-bufuralol 1'-hydroxylation were used to quantify the corresponding cytochrome P450 (P450) catalytic activities. Two methods were used to predict the human exposure ratio of the victim drug in the presence and absence of inhibitor. The HMM K(i, app) values were combined with the free average systemic plasma concentration ("free [I] with HMM K(i, app)") and the plasma K(i, app) values were combined with the total average systemic plasma concentration ("total [I] with plasma K(i, app)"). Of 63 clinical DDI studies, the total [I] with plasma K(i, app) method predicted 89% of cases within 2-fold of the reported interaction whereas the free [I] with HMM K(i, app) method predicted only 59%. There was a general underprediction by the free [I] with HMM K(i, app) method, which is consistent with an underestimation of in vitro inhibition potency in this system. In conclusion, the HHSHP system proved to be a simple, accurate predictor of DDIs for three major P450s and superior to the protein-free approach. PMID:22228749

  17. Insulin-like 3-induced rat preantral follicular growth is mediated by growth differentiation factor 9.

    PubMed

    Xue, Kai; Kim, Ji Young; Liu, Jia-yin; Tsang, Benjamin K

    2014-01-01

    The communication of somatic cells and oocytes by intrafollicular paracrine factors is essential for follicular growth in the ovary. Insulin-like 3 (INSL3) is a theca cell-secreted paracrine factor. Androgens and growth differentiation factor 9 (GDF9), an oocyte-derived growth factor, are essential for follicular development. Using a rat preantral follicle culture model, we examined in the present study the influence of INSL3 on preantral follicular growth and the molecular mechanisms involved. We have observed that the receptor for INSL3, relaxin/insulin-like family peptide receptor 2 (RXFP2), was exclusively expressed in oocytes. Recombinant INSL3 stimulated Gdf9 expression, preantral follicular growth, and testosterone synthesis in vitro. Inhibition of the cAMP/protein kinase A signaling pathway (with cAMP antagonist, 8-bromoadenosine 3',5'-cyclic monophosphorothioate, Rp-isomer) attenuated INSL3-induced Gdf9 expression and preantral follicular growth. Moreover, knocking down Gdf9 expression (with small interfering RNA) or inhibiting GDF9 signaling (with SB431542, an activin receptor-like kinase receptor 5 inhibitor, or specific inhibitor of mothers against decapentaplegic homolog 3) or androgen action (with flutamide, an androgen receptor antagonist) suppressed INSL3-induced preantral follicular growth. In addition, LH and DHT regulated the expression of Insl3 mRNA in preantral follicles. These observations suggest that INSL3 is a key theca cell-derived growth factor for preantral follicle and that its action is mediated by GDF9.

  18. Multiple regulatory mechanisms of hepatocyte growth factor expression in malignant cells with a short poly(dA) sequence in the HGF gene promoter.

    PubMed

    Sakai, Kazuko; Takeda, Masayuki; Okamoto, Isamu; Nakagawa, Kazuhiko; Nishio, Kazuto

    2015-01-01

    Hepatocyte growth factor (HGF) expression is a poor prognostic factor in various types of cancer. Expression levels of HGF have been reported to be regulated by shorter poly(dA) sequences in the promoter region. In the present study, the poly(dA) mononucleotide tract in various types of human cancer cell lines was examined and compared with the HGF expression levels in those cells. Short deoxyadenosine repeat sequences were detected in five of the 55 cell lines used in the present study. The H69, IM95, CCK-81, Sui73 and H28 cells exhibited a truncated poly(dA) sequence in which the number of poly(dA) repeats was reduced by ≥5 bp. Two of the cell lines exhibited high HGF expression, determined by reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The CCK-81, Sui73 and H28 cells with shorter poly(dA) sequences exhibited low HGF expression. The cause of the suppression of HGF expression in the CCK-81, Sui73 and H28 cells was clarified by two approaches, suppression by methylation and single nucleotide polymorphisms in the HGF gene. Exposure to 5-Aza-dC, an inhibitor of DNA methyltransferase 1, induced an increased expression of HGF in the CCK-81 cells, but not in the other cells. Single-nucleotide polymorphism (SNP) rs72525097 in intron 1 was detected in the Sui73 and H28 cells. Taken together, it was found that the defect of poly(dA) in the HGF promoter was present in various types of cancer, including lung, stomach, colorectal, pancreas and mesothelioma. The present study proposes the negative regulation mechanisms by methylation and SNP in intron 1 of HGF for HGF expression in cancer cells with short poly(dA).

  19. 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

  20. Importance of osteoprotegrin and receptor activator of nuclear factor κB in breast cancer response to hepatocyte growth factor and the bone microenvironment in vitro.

    PubMed

    Owen, Sioned; Sanders, Andrew J; Mason, Malcolm D; Jiang, Wen G

    2016-03-01

    Osteoprotegrin (OPG), receptor activator of nuclear factor κB (RANK) and RANK ligand (RANKL) are signal transducers which have pleiotropic actions. Each tumour necrosis factor receptor superfamily member has unique structural attributes which directly couples them to signalling pathways involved in cell proliferation, differentiation and survival. Previous studies have clinically linked OPG, RANK and RANKL to increasing tumour burden, metastatic bone involvement and estrogen status. This study aimed to establish the potential implications of targeting endogenously produced OPG and RANK in the osteotropic breast cancer cell line MDA-MB‑231 in vitro. Subsequently this study also aimed to explore the potential links between these molecules with regards to hepatocyte growth factor (HGF) signalling and extracted bone proteins (BME). OPG and RANK expression was successfully suppressed using hammerhead ribozyme technology. Subsequently effects were explored in MDA-MB‑231 cell proliferation, matrix adhesion, migration and invasion in vitro function assays. Reduced OPG expression resulted in increased breast cancer cell migration and invasion. These increases, particularly invasion, appeared to however be reduced under the influence of the exogenous stimuli (HGF and BME). In contrast, suppression of RANK in MDA-MB‑231 breast cancer cells resulted in decreased cancer cell proliferation, matrix-adhesion, motility and invasion with little cumulative effect being noted after the addition of exogenous stimuli. The complexity of the bone environment underpins the vast number of soluble factors and signalling pathways which can influence osteotropic cancer behaviour and progression. Further work into elucidating all the pathways affected could potentially lead to better identification of those patients most at risk. PMID:26781475

  1. Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

    PubMed

    Fang, Jung-Da; Lee, Sheau-Ling

    2014-07-01

    Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

  2. Involvement of an SHP-2-Rho small G protein pathway in hepatocyte growth factor/scatter factor-induced cell scattering.

    PubMed

    Kodama, A; Matozaki, T; Fukuhara, A; Kikyo, M; Ichihashi, M; Takai, Y

    2000-08-01

    Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase-type HGF/SF receptor c-Met. We have previously shown that Rho small G protein (Rho) is involved in the HGF/SF-induced scattering of Madin-Darby canine kidney (MDCK) cells by regulating at least the assembly and disassembly of stress fibers and focal adhesions, but it remains unknown how c-Met regulates Rho activity. We have found here a novel signaling pathway of c-Met consisting of SHP-2-Rho that regulates the assembly and disassembly of stress fibers and focal adhesions in MDCK cells. SHP-2 is a protein-tyrosine phosphatase that contains src homology-2 domains. Expression of a dominant negative mutant of SHP-2 (SHP-2-C/S) markedly increased the formation of stress fibers and focal adhesions in MDCK cells and inhibited their scattering. C3, a Clostridium botulinum ADP-ribosyltransferase, and Y-27632, a specific inhibitor for ROCK, reversed the stimulatory effect of SHP-2-C/S on stress fiber formation and the inhibitory effect on cell scattering. Vav2 is a GDP/GTP exchange protein for Rho. Expression of a dominant negative mutant of Vav2 blocked the stimulatory effect of SHP-2-C/S on stress fiber formation. Conversely, expression of mutants of Vav2 that increased stress fiber formation inhibited HGF/SF-induced cell scattering. These results indicate that SHP-2 physiologically modulates the activity of Rho to form stress fibers and focal adhesions and thereby regulates HGF/SF-induced cell scattering. In addition, Vav2 may be involved in the SHP-2-Rho pathway.

  3. A functional domain in the heavy chain of scatter factor/hepatocyte growth factor binds the c-Met receptor and induces cell dissociation but not mitogenesis.

    PubMed Central

    Hartmann, G; Naldini, L; Weidner, K M; Sachs, M; Vigna, E; Comoglio, P M; Birchmeier, W

    1992-01-01

    We recently found that scatter factor (SF), a cell motility factor with a multimodular structure, is identical to hepatocyte growth factor (HGF), a potent mitogen of various cell types. SF/HGF is the ligand of the c-Met receptor tyrosine kinase. Here we used transient expression of naturally occurring and in vitro mutagenized cDNAs of SF/HGF to delineate the protein domains necessary for biological activity and binding to the c-Met receptor. (i) A single-chain SF/HGF resulting from the destruction of the protease cleavage site between heavy and light chain (Arg-494--> Gln) was largely inactive, indicating that proteolytic cleavage is essential for acquisition of the biologically active conformation. (ii) A SF/HGF splice variant encoding a protein with a 5-amino acid deletion in the first kringle domain was as highly active as the wild-type molecule. (iii) The separately expressed light chain (with serine protease homology) was inactive in all assays tested. (iv) The separate heavy chain as well as a naturally occurring splice variant consisting of the N terminus and the first two kringle domains bound the c-Met receptor, stimulated tyrosine auto-phosphorylation, and induced scattering of epithelial cells but not mitogenesis. These data indicate that a functional domain in the N terminus/first two kringle regions of SF/HGF is sufficient for binding to the Met receptor and that this leads to the activation of the downstream signal cascade involved in the motility response. However, the complete SF/HGF protein seems to be required for mitogenic activity. Images PMID:1280830

  4. 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.

  5. 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.

  6. Polymer-mediated growth of crystals and mesocrystals.

    PubMed

    Cölfen, Helmut

    2013-01-01

    Polymers are important additives for the control of mineralization reactions in both biological and bioinspired mineralization. The reason is that they allow for a number of interactions with the growing crystals and even amorphous minerals. These can substantially influence the way the mineral grows on several levels. Already in the prenucleation phase, polymers can control the formation of prenucleation clusters and subsequently the nucleation event. Also, polymers can control whether the further crystallization follows a classical or nonclassical particle-mediated growth path. In this chapter, the main ways in which polymers can be used to control a crystallization reaction will be highlighted. In addition, polymers that are useful for this purpose and the experimental conditions suitable for directing a crystallization reaction into the desired direction through the use of polymers will be described.

  7. 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.

  8. PLGA microsphere-mediated growth hormone release hormone expression induces intergenerational growth.

    PubMed

    Ren, Xiao-Hui; Zhang, Yong-Liang; Luo, Hu-Ying; Li, Hong-Yi; Liu, Song-Cai; Zhang, Ming-Jun; Ouyang, Song-Ying; Xi, Qian-Yun; Jiang, Qing-Yan

    2009-01-01

    To improve animal growth, growth hormone-releasing hormone (GHRH) expression vectors that maintain constant GHRH expression can be directly injected into muscles. To deliver the GHRH expression vectors, biodegradable microspheres have been used as a sustained release system. Although administering GHRH through microspheres is a common practice, the intergenerational effects of this delivery system are unknown. To investigate the intergenerational effects of polylactic-co-glycolic acid (PLGA) encapsulated plasmid-mediated GHRH supplements, pCMV-Rep-GHRH microspheres were injected into pregnant mice. Growth and expression of GHRH were measured in the offspring. RT-PCR and immunohistochemistry reveal GHRH expression 3-21 days post-injection. The proportion of GH-positive cells in the GHRH treated offspring was 48.2% higher than in the control group (P < 0.01). The GHRH treated offspring were 6.15% (P < 0.05) larger than the control offspring. At day 49 post-injection, IGF-I serum levels were significantly higher in the treatment group than in the control group. This study confirms that intramuscular expression of GHRH mediated by PLGA microspheres significantly enhances intergenerational growth.

  9. A Natural Hepatocyte Growth Factor/Scatter Factor Autocrine Loop in Myoblast Cells and the Effect of the Constitutive Met Kinase Activation on Myogenic Differentiation

    PubMed Central

    Anastasi, Sergio; Giordano, Silvia; Sthandier, Olga; Gambarotta, Giovanna; Maione, Rossella; Comoglio, Paolo; Amati, Paolo

    1997-01-01

    As a rule, hepatocyte growth factor/scatter factor (HGF/SF) is produced by mesenchymal cells, while its receptor, the tyrosine kinase encoded by the met proto-oncogene, is expressed by the neighboring epithelial cells in a canonical paracrine fashion. In the present work we show that both HGF/SF and met are coexpressed by undifferentiated C2 mouse myoblasts. In growing cells, the autocrine loop is active as the receptor exhibits a constitutive phosphorylation on tyrosine that can be abrogated by exogenously added anti-HGF/SF neutralizing antibodies. The transcription of HGF/SF and met genes is downregulated when myoblasts stop proliferating and differentiate. The coexpression of HGF/SF and met genes is not exclusive to C2 cells since it has been assessed also in other myogenic cell lines and in mouse primary satellite cells, suggesting that HGF/SF could play a role in muscle development through an autocrine way. To analyze the biological effects of HGF/SF receptor activation, we stably expressed the constitutively activated receptor catalytic domain (p65tpr-met) in C2 cells. This active kinase determined profound changes in cell shape and inhibited myogenesis at both morphological and biochemical levels. Notably, a complete absence of muscle regulatory markers such as MyoD and myogenin was observed in p65tpr-met highly expressing C2 clones. We also studied the effects of the ectopic expression of human isoforms of met receptor (h-met) and of HGF/SF (h-HGF/SF) in stable transfected C2 cells. Single constitutive expression of h-met or h-HGF/SF does not alter substantially the growth and differentiation properties of the myoblast cells, probably because of a species-specific ligand–receptor interaction. A C2 clone expressing simultaneously both h-met and h-HGF/SF is able to grow in soft agar and shows a decrease in myogenic potential comparable to that promoted by p65tpr-met kinase. These data indicate that a met kinase signal released from differentiation

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Role of hepatocyte nuclear factor 4α (HNF4α) in cell proliferation and cancer.

    PubMed

    Walesky, Chad; Apte, Udayan

    2015-01-01

    Hepatocyte nuclear factor 4α (HNF4α) is an orphan nuclear receptor commonly known as the master regulator of hepatic differentiation, owing to the large number of hepatocyte-specific genes it regulates. Whereas the role of HNF4α in hepatocyte differentiation is well recognized and extensively studied, its role in regulation of cell proliferation is relatively less known. Recent studies have revealed that HNF4α inhibits proliferation not only of hepatocytes but also cells in colon and kidney. Further, a growing number of studies have demonstrated that inhibition or loss of HNF4α promotes tumorigenesis in the liver and colon, and reexpression of HNF4α results in decreased cancer growth. Studies using tissue-specific conditional knockout mice, knock-in studies, and combinatorial bioinformatics of RNA/ChIP-sequencing data indicate that the mechanisms of HNF4α-mediated inhibition of cell proliferation are multifold, involving epigenetic repression of promitogenic genes, significant cross talk with other cell cycle regulators including c-Myc and cyclin D1, and regulation of miRNAs. Furthermore, studies indicate that posttranslational modifications of HNF4α may change its activity and may be at the core of its dual role as a differentiation factor and repressor of proliferation. This review summarizes recent findings on the role of HNF4α in cell proliferation and highlights the newly understood function of this old receptor.

  15. 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

  16. 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

  17. 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

  18. A Fusion Protein of Hepatocyte Growth Factor Enhances Reconstruction of Myocardium in a Cardiac Patch Derived from Porcine Urinary Bladder Matrix

    PubMed Central

    Ota, Takeyoshi; Gilbert, Thomas W.; Schwartzman, David; McTiernan, Charles F.; Kitajima, Takashi; Ito, Yoshihiro; Sawa, Yoshiki; Badylak, Stephen F.; Zenati, Marco A.

    2008-01-01

    Objectives We sought to promote myocardial repair using urinary bladder matrix (UBM) incorporated with a fusion protein which combined hepatocyte growth factor (HGF) and fibronectin collagen-binding-domain (CBD) in a porcine model. CBD acted as an intermediary to promote HGF binding and enhance HGF stability within UBM. Methods UBM incorporated with CBD-HGF was implanted into the porcine right ventricular wall (F-group) to repair a surgically created defect. Untreated UBM patches (U-group) and Dacron patches (D-group) served as controls (N=5/group). Electromechanical mapping was performed at 60-days after surgery. Linear local shortening (LLS) was used to assess regional contractility, and electrical activity was recorded. Results The LLS was significantly improved in the F group compared with controls (F:0.51 ± 1.57%*, U:− 1.06 ± 1.84%, D: −2.72 ± 2.59%; *p<0.05), while they were inferior to the normal myocardium(13.7 ± 4.3%)*. Mean electrical activity was 1.49±0.82mV in the F group, which was statistically greater than control groups (U:0.93 ± 0.71mV; D:0.30 ± 0.22mV)* and less than the normal myocardium (8.24±2.49mV)*. Histological examination showed predominant α-smooth muscle actin positive cells with the F group showing the thickest layer and the D group the thinnest layer, with an endocardial endothelial monolayer. Scattered isolated islands of α-actinin positive cells were observed only in the F group, but not in the controls, suggesting the presence of cardiomyocytes. Conclusions The CBD-HGF-UBM patch demonstrated increased contractility and electrical activity compared to UBM alone or Dacron, and facilitated a homogeneous repopulation of host cells. UBM incorporated with CBD-HGF may contribute to constructive myocardial remodeling. PMID:19026821

  19. 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

  20. 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

  1. 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.

  2. Hepatocyte growth factor is a preferred in vitro substrate for human hepsin, a membrane-anchored serine protease implicated in prostate and ovarian cancers

    PubMed Central

    Herter, Sylvia; Piper, Derek E.; Aaron, Wade; Gabriele, Timothy; Cutler, Gene; Cao, Ping; Bhatt, Ami S.; Choe, Youngchool; Craik, Charles S.; Walker, Nigel; Meininger, David; Hoey, Timothy; Austin, Richard J.

    2005-01-01

    Hepsin is a membrane-anchored, trypsin-like serine protease with prominent expression in the human liver and tumours of the prostate and ovaries. To better understand the biological functions of hepsin, we identified macromolecular substrates employing a tetrapeptide PS-SCL (positional scanning-synthetic combinatorial library) screen that rapidly determines the P1–P4 substrate specificity. Hepsin exhibited strong preference at the P1 position for arginine over lysine, and favoured threonine, leucine or asparagine at the P2, glutamine or lysine at the P3, and proline or lysine at the P4 position. The relative activity of hepsin toward individual AMC (7-amino-4-methylcoumarin)-tetrapeptides was generally consistent with the overall peptide profiling results derived from the PC-SCL screen. The most active tetrapeptide substrate Ac (acetyl)-KQLR-AMC matched with the activation cleavage site of the hepatocyte growth factor precursor sc-HGF (single-chain HGF), KQLR↓VVNG (where ↓ denotes the cleavage site), as identified by a database analysis of trypsin-like precursors. X-ray crystallographic studies with KQLR chloromethylketone showed that the KQLR peptide fits well into the substrate-binding cleft of hepsin. This hepsin-processed HGF induced c-Met receptor tyrosine phosphorylation in SKOV-3 ovarian cancer cells, indicating that the hepsin-cleaved HGF is biologically active. Activation cleavage site mutants of sc-HGF with predicted non-preferred sequences, DPGR↓VVNG or KQLQ↓VVNG, were not processed, illustrating that the P4–P1 residues can be important determinants for substrate specificity. In addition to finding macromolecular hepsin substrates, the extracellular inhibitors of the HGF activator, HAI-1 and HAI-2, were potent inhibitors of hepsin activity (IC50 4±0.2 nM and 12±0.5 nM respectively). Together, our findings suggest that the HGF precursor is a potential in vivo substrate for hepsin in tumours, where hepsin expression is dysregulated and may

  3. Mechanisms of the statins cytotoxicity in freshly isolated rat hepatocytes.

    PubMed

    Abdoli, Narges; Heidari, Reza; Azarmi, Yadollah; Eghbal, Mohammad Ali

    2013-06-01

    Statins are potent drugs, used as lipid-lowering agents in cardiovascular diseases. Hepatotoxicity is one of the serious adverse effects of statins, and the exact mechanism of hepatotoxicity is not yet clear. In this study, the cytotoxic effects of the most commonly used statins, that is, atorvastatin, lovastatin, and simvastatin toward isolated rat hepatocytes, were evaluated. Markers, such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and the amount of reduced and oxidized glutathione in the statin-treated hepatocytes, were investigated. It was found that the statins caused cytotoxicity toward rat hepatocytes dose dependently. An elevation in ROS formation, accompanied by a significant amount of lipid peroxidation and mitochondrial depolarization, was observed. Cellular glutathione reservoirs were decreased, and a significant amount of oxidized glutathione was formed. This study suggests that the adverse effect of statins toward hepatocytes is mediated through oxidative stress and the hepatocytes mitochondria play an important role in the statin-induced toxicity.

  4. Minireview: mechanisms of growth hormone-mediated gene regulation.

    PubMed

    Chia, Dennis J

    2014-07-01

    GH exerts a diverse array of physiological actions that include prominent roles in growth and metabolism, with a major contribution via stimulating IGF-1 synthesis. GH achieves its effects by influencing gene expression profiles, and Igf1 is a key transcriptional target of GH signaling in liver and other tissues. This review examines the mechanisms of GH-mediated gene regulation that begin with signal transduction pathways activated downstream of the GH receptor and continue with chromatin events at target genes and additionally encompasses the topics of negative regulation and cross talk with other cellular inputs. The transcription factor, signal transducer and activator of transcription 5b, is regarded as the major signaling pathway by which GH achieves its physiological effects, including in stimulating Igf1 gene transcription in liver. Recent studies exploring the mechanisms of how activated signal transducer and activator of transcription 5b accomplishes this are highlighted, which begin to characterize epigenetic features at regulatory domains of the Igf1 locus. Further research in this field offers promise to better understand the GH-IGF-1 axis in normal physiology and disease and to identify strategies to manipulate the axis to improve human health.

  5. The effects of human umbilical cord perivascular cells on rat hepatocyte structure and functional polarity.

    PubMed

    Gómez-Aristizábal, Alejandro; Davies, John Edward

    2013-06-01

    Hepatocyte culture is a useful tool for the study of their biology and the development of bioartificial livers. However, many challenges have to be overcome since hepatocytes rapidly lose their normal phenotype in vitro. We have recently demonstrated that human umbilical cord perivascular cells (HUCPVCs) are able to provide support to hepatocytes. In the present study we go further into exploring the effects that HUCPVCs have in the functional polarization, and both the internal and external organization, of hepatocytes. Also, we investigate HUCPVC-hepatocyte crosstalk by tracking both the effects of HUCPVCs on hepatocyte transcription factors and those of hepatocytes on the expression of hepatotrophic factors in HUCPVCs. Our results show that HUCPVCs maintain the functional polarity of hepatocytes ex vivo, as judged by the secretion of fluorescein into bile canaliculi, for at least 40 days. Transmission electron microscopy revealed that hepatocytes in coculture organize in an organoid-like structure embedded in extracellular matrix surrounded by HUCPVCs. In coculture, hepatocytes displayed a higher expression of C/EBPα, implicated in maintenance of the mature hepatocyte phenotype, and HUCPVCs upregulated hepatocyte growth factor and Jagged1 indicating that these genes may play important roles in HUCPVC-hepatocyte interactions.

  6. Type III interferons are expressed by Coxsackievirus-infected human primary hepatocytes and regulate hepatocyte permissiveness to infection

    PubMed Central

    Lind, K; Svedin, E; Utorova, R; Stone, V M; Flodström-Tullberg, M

    2014-01-01

    Hepatitis is a common and potentially fatal manifestation of severe Coxsackievirus infections, particularly in newborn children. Little is known of the immune-mediated mechanisms regulating permissiveness to liver infection. It is well established that type I interferons (IFNs) play an important role in the host innate immune response to Coxsackievirus infections. Recent studies have highlighted a role for another IFN family, the type III IFNs (also called IFN-λ), in anti-viral defence. Whether type III IFNs are produced by hepatocytes during a Coxsackievirus infection remains unknown. Moreover, whether or not type III IFNs protects hepatocytes from a Coxsackievirus infection has not been addressed. In this study, we show that primary human hepatocytes respond to a Coxsackievirus B3 (CVB3) infection by up-regulating the expression of type III IFNs. We also demonstrate that type III IFNs induce an anti-viral state in hepatocytes characterized by the up-regulated expression of IFN-stimulated genes, including IFN-stimulated gene (ISG15), 2′-5′-oligoadenylate synthetase 2 (OAS2), protein kinase regulated by dsRNA (PKR) and myxovirus resistance protein 1 (Mx1). Furthermore, our study reveals that type III IFNs attenuate CVB3 replication both in hepatocyte cell lines and primary human hepatocytes. Our studies suggest that human hepatocytes express type III IFNs in response to a Coxsackievirus infection and highlight a novel role for type III IFNs in regulating hepatocyte permissiveness to this clinically relevant type of virus. PMID:24773058

  7. Autophagy Mediates HBx-Induced Nuclear Factor-κB Activation and Release of IL-6, IL-8, and CXCL2 in Hepatocytes.

    PubMed

    Luo, Millore X M; Wong, Sunny H; Chan, Matthew T V; Yu, Le; Yu, Sidney S B; Wu, Feng; Xiao, Zhangang; Wang, Xiaojuan; Zhang, Lin; Cheng, Alfred S L; Ng, Simon S M; Chan, Francis K L; Cho, Chi H; Yu, Jun; Sung, Joseph J Y; Wu, William K K

    2015-10-01

    Hepatitis B virus (HBV) and one of its encoded proteins, HBV X protein (HBx), have been shown to induce autophagy in hepatoma cells. Substantial evidence indicates that autophagy is a potent suppressor of inflammation. However, sporadic reports suggest that autophagy could promote pro-inflammatory cytokine expression and inflammation in some biological contexts. Here, we show that overexpression of HBx induces LC3B-positive autophagosome formation, increases autophagic flux and enhances the expression of ATG5, ATG7, and LC3B-II in normal hepatocytes. Abrogation of autophagy by small interfering RNA against ATG5 and ATG7 prevents HBx-induced formation of autophagosomes. Autophagy inhibition also abrogates HBx-induced activation of nuclear factor-κB (NF-κB) and production of interleukin-6 (IL-6), IL-8, and CXCL2. These findings suggest that autophagy is required for HBx-induced NF-κB activation and pro-inflammatory cytokine production and could shed new light on the complex role of autophagy in the modulation of inflammation.

  8. Epidermal growth factor receptor distribution in burn wounds. Implications for growth factor-mediated repair.

    PubMed Central

    Wenczak, B A; Lynch, J B; Nanney, L B

    1992-01-01

    Epidermal growth factor (EGF) along with several related peptide growth factors has been shown both in vivo and in vitro to accelerate events associated with epidermal wound repair. EGF and transforming growth factor alpha act by binding to a common EGF receptor tyrosine kinase thereby initiating a series of events which ultimately regulate cell proliferation. This study examined the immunohistochemical localization of EGF receptor (EGF-R) in burn wound margins, adjacent proliferating epithelium, and closely associated sweat ducts, sebaceous glands, and hair follicles. Tissue specimens removed during surgical debridement were obtained from full and partial thickness burn wounds in 32 patients with total body surface area burns ranging from 2 to 88%. In the early postburn period (days 2-4), prominent staining for EGF-R was found in undifferentiated, marginal keratinocytes, adjacent proliferating, hypertrophic epithelium, and both marginal and nonmarginal hair follicles, sweat ducts, and sebaceous glands. During the late postburn period (days 5-16), EGF-R was depleted along leading epithelial margins; however, immunoreactive EGF-R remained intensely positive in the hypertrophic epithelium and all skin appendages. Increased detection of immunoreactive EGF-R and the presence of [125I]EGF binding in the hypertrophic epithelium correlated positively with proliferating cell nuclear antigen distributions. Thus, the presence of EGF-R in the appropriate keratinocyte populations suggests a functional role for this receptor during wound repair. Dynamic modulation in EGF receptor distribution during the temporal sequence of repair provides further evidence that an EGF/transforming growth factor alpha/EGF-R-mediated pathway is activated during human wound repair. Images PMID:1361495

  9. 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.

  10. ψ-Bufarenogin, a novel anti-tumor compound, suppresses liver cancer growth by inhibiting receptor tyrosine kinase-mediated signaling.

    PubMed

    Ding, Jin; Wen, Wen; Xiang, Daimin; Yin, Peipei; Liu, Yanfang; Liu, Chang; He, Guoping; Cheng, Zhuo; Yin, Jianpeng; Sheng, Chunquan; Zhang, Wen; Nan, Fajun; Ye, Wencai; Zhang, Xiuli; Wang, Hongyang

    2015-05-10

    Resistance of hepatocellular carcinoma (HCC) to existing chemotherapeutic agents largely contributes to the poor prognosis of patients, and discovery of novel anti-HCC drug is in an urgent need. Herein we report ψ-Bufarenogin, a novel active compound that we isolated from the extract of toad skin, exhibited potent therapeutic effect in xenografted human hepatoma without notable side effects. In vitro, ψ-Bufarenogin suppressed HCC cells proliferation through impeding cell cycle progression, and it facilitated cell apoptosis by downregulating Mcl-1 expression. Moreover, ψ-Bufarenogin decreased the number of hepatoma stem cells through Sox2 depression and exhibited synergistic effect with conventional chemotherapeutics. Mechanistic study revealed that ψ-Bufarenogin impaired the activation of MEK/ERK pathway, which is essential in the proliferation of hepatoma cells. ψ-Bufarenogin notably suppressed PI3-K/Akt cascade, which was required in ψ-Bufarenogin-mediated reduction of Mcl-1 and Sox2. ψ-Bufarenogin inhibited the auto-phosphorylation and activation of epithelial growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met), thereafter suppressed their primary downstream cascades Raf/MEK/ERK and PI3-K/Akt signaling. Taken together, ψ-Bufarenogin suppressed HCC growth via inhibiting, at least partially, receptor tyrosine kinases-regulated signaling, suggesting that ψ-Bufarenogin could be a novel lead compound for anti-HCC drug.

  11. Strigolactones as mediators of plant growth responses to environmental conditions.

    PubMed

    Koltai, Hinanit; Kapulnik, Yoram

    2011-01-01

    Strigolactones (SLs) have been recently identified as a new group of plant hormones or their derivatives thereof, shown to play a role in plant development. Evolutionary forces have driven the development of mechanisms in plants that allow adaptive adjustments to a variety of different habitats by employing plasticity in shoot and root growth and development. The ability of SLs to regulate both shoot and root development suggests a role in the plant's response to its growth environment. To play this role, SL pathways need to be responsive to plant growth conditions, and affect plant growth toward increased adaptive adjustment. Here, the effects of SLs on shoot and root development are presented, and possible feedback loops between SLs and two environmental cues, light and nutrient status, are discussed; these might suggest a role for SLs in plants' adaptive adjustment to growth conditions.

  12. 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.

  13. The transcriptional regulator BZR1 mediates trade-off between plant innate immunity and growth

    PubMed Central

    Lozano-Durán, Rosa; Macho, Alberto P; Boutrot, Freddy; Segonzac, Cécile; Somssich, Imre E; Zipfel, Cyril

    2013-01-01

    The molecular mechanisms underlying the trade-off between plant innate immunity and steroid-mediated growth are controversial. Here, we report that activation of the transcription factor BZR1 is required and sufficient for suppression of immune signaling by brassinosteroids (BR). BZR1 induces the expression of several WRKY transcription factors that negatively control early immune responses. In addition, BZR1 associates with WRKY40 to mediate the antagonism between BR and immune signaling. We reveal that BZR1-mediated inhibition of immunity is particularly relevant when plant fast growth is required, such as during etiolation. Thus, BZR1 acts as an important regulator mediating the trade-off between growth and immunity upon integration of environmental cues. DOI: http://dx.doi.org/10.7554/eLife.00983.001 PMID:24381244

  14. 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.

  15. Retinoic acid receptor alpha mediates growth inhibition by retinoids in human colon carcinoma HT29 cells.

    PubMed

    Nicke, B; Kaiser, A; Wiedenmann, B; Riecken, E O; Rosewicz, S

    1999-08-11

    Although retinoids have been suggested to inhibit chemically induced colon carcinogenesis, the molecular mechanisms underlying retinoid-mediated growth regulation in colon carcinoma cells are unknown. Therefore, we investigated the biological effects of retinoids on growth in HT29 colon carcinoma cells. All-trans retinoic acid (ATRA) treatment of HT29 cells resulted in a profound inhibition of anchorage-independent growth without biochemical or morphological evidence for induction of differentiation. Treatment with the selective RARalpha agonist Ro 40-6055 completely mimicked the effects of ATRA on growth and transactivation of a betaRAREx2-luciferase reporter construct, while RARbeta- and gamma-specific analogues were ineffective. Furthermore, ATRA-regulated growth and transactivation could be completely blocked by a RARalpha-selective receptor antagonist. Thus, ATRA potently inhibits anchorage-independent growth in HT29 cells and this effect is mainly if not exclusively mediated by the retinoic acid receptor alpha.

  16. Genetic deletion of faim reveals its role in modulating c-FLIP expression during CD95-mediated apoptosis of lymphocytes and hepatocytes.

    PubMed

    Huo, J; Xu, S; Guo, K; Zeng, Q; Lam, K-P

    2009-07-01

    Fas-apoptosis inhibitory molecule (FAIM) is inducibly expressed in B lymphocytes and had been shown to antagonize Fas-mediated killing of B-cell lines in vitro. However, its mechanism and role in vivo are unknown. We have generated faim(-/-) mice and found these mutants to be viable. In contrast to fas(-/-) mice, faim(-/-) mice have normal B- and T-cell populations. However, faim(-/-) B cells and thymocytes show increased sensitivity to Fas-triggered apoptosis in vitro, and faim(-/-) mice suffer greater mortality and exhibit exacerbated liver damage in response to Fas (CD95) engagement in vivo. The lack of FAIM results in greater activation of caspase-8 and -3 in Fas-stimulated thymocytes. Detailed biochemical analyses further reveal the decreased expression of c-FLIP(L) and c-FLIP(R) in faim(-/-) thymocytes and increased association of caspase-8 with Fas in Fas-activated mutant cells. Decreased levels of c-FLIP(L) and c-FLIP(R) are also evident in faim(-/-) liver. Thus, FAIM plays a novel role in modulating Fas-mediated apoptosis and acts through influencing the expression of c-FLIP and regulating the physical binding of caspase-8 to Fas.

  17. 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.

  18. Hepatocyte nuclear factor 4 alpha ligand binding and F domains mediate interaction and transcriptional synergy with the pancreatic islet LIM HD transcription factor Isl1.

    PubMed

    Eeckhoute, J; Briche, I; Kurowska, M; Formstecher, P; Laine, B

    2006-12-01

    The orphan nuclear receptor HNF4alpha and the LIM homeodomain factor Isl1 are co-expressed in pancreatic beta-cells and are required for the differentiation and function of these endocrine cells. HNF4alpha activates numerous genes and mutations in its gene are associated with maturity onset diabetes of the young. Cofactors and transcription factors that interact with HNF4alpha are crucial to modulate its transcriptional activity, since the latter is not regulated by conventional ligands. These transcriptional partners interact mainly through the HNF4alpha AF-1 module and the ligand binding domain, which contains the AF-2 module. Here, we showed that Isl1 could enhance the HNF4alpha-mediated activation of transcription of the HNF1alpha, PPARalpha and insulin I promoters. Isl1 interacted with the HNF4alpha AF-2 but also required the HNF4alpha carboxy-terminal F domain for optimal interaction and transcriptional synergy. More specifically, we found that naturally occurring HNF4alpha isoforms, differing only in their F domain, exhibited different abilities to interact and synergize with Isl1, extending the crucial transcriptional modulatory role of the HNF4alpha F domain. HNF4alpha interacted with both the homeodomain and the first LIM domain of Isl1. We found that the transcriptional synergy between HNF4alpha and Isl1 involved an increase in HNF4alpha loading on promoter. The effect was more pronounced on the rat insulin I promoter containing binding sites for both HNF4alpha and Isl1 than on the human HNF1alpha promoter lacking an Isl1 binding site. Moreover, Isl1 could mediate the recruitment of the cofactor CLIM2 resulting in a further transcriptional enhancement of the HNF1alpha promoter activity.

  19. P-selectin-mediated platelet adhesion promotes tumor growth.

    PubMed

    Qi, Cuiling; Wei, Bo; Zhou, Weijie; Yang, Yang; Li, Bin; Guo, Simei; Li, Jialin; Ye, Jie; Li, Jiangchao; Zhang, Qianqian; Lan, Tian; He, Xiaodong; Cao, Liu; Zhou, Jia; Geng, Jianguo; Wang, Lijing

    2015-03-30

    Blood platelets foster carcinogenesis. We found that platelets are accumulated in human tumors. P-selectin deficiency and soluble P-selectin abolish platelet deposition within tumors, decreasing secretion of vascular endothelial growth factor and angiogenesis, thereby suppressing tumor growth. Binding of the P-selectin cytoplasmic tail to talin1 triggers the talin1 N-terminal head to interact with the β3 cytoplasmic tail. This activates αIIbβ3 and recruits platelets into tumors. Platelet infiltration into solid tumors occurs through a P-selectin-dependent mechanism.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Inhibition of heme oxygenase-1 partially reverses the arsenite-mediated decrease of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 catalytic activity in isolated rat hepatocytes.

    PubMed

    Anwar-Mohamed, Anwar; Klotz, Lars-Oliver; El-Kadi, Ayman O S

    2012-03-01

    Heme oxygenase (HO-1), the rate-limiting enzyme in the physiological breakdown of heme, is ubiquitous, and its expression can be increased by arsenite [As(III)], and similar other stimuli that induce cellular oxidative stress. Interestingly, it has been shown that the As(III)-induced HO-1 is inversely correlated with a decrease in cytochromes P450 (P450s) activity; however, the direct role for HO-1 in the inhibition of P450 enzymes remains unknown. Our results showed that As(III) at a concentration of 5 μM decreased the constitutive and inducible expression of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 at the mRNA, protein, and catalytic activity levels. Moreover, As(III) decreased the nuclear accumulation of aryl hydrocarbon receptor (AhR) and pregnane X receptor without increasing their degradation. As(III) also increased the binding of cytosolic AhR to heat shock protein 90 and hepatitis B virus X-associated protein 2. In the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin as an inducer for CYP1A and rifampin as an inducer for CYP3A, As(III) decreased the enzymatic activity of the four P450s more than it decreased their mRNA or protein expression levels. It is noteworthy that treatment with the competitive HO-1 inhibitor, tin-mesoporphyrin, or supplementing external heme partially reversed the As(III)-mediated decrease in activities of the four P450s. In conclusion, the current study provides the first evidence that As(III) decreases CYP1A1, CYP1A2, CYP3A23, and CYP3A2 expression in freshly isolated rat primary hepatocytes. Furthermore, inhibiting the As(III)-mediated induction of HO-1 partially restores the enzymatic activity of these P450s that was initially decreased by As(III), confirming the direct role of HO-1 in the inhibition of P450s.

  5. 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.

  6. [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

  7. Prolonged ER Stressed-Hepatocytes drives an Alternative Macrophage Polarization

    PubMed Central

    Xiu, Fangming; Catapano, Michael; Diao, Li; Stanojcic, Mile; Jeschke, Marc G.

    2015-01-01

    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 (TM) or Thapsigargin (TG), on the secretion of cytokines, expression of ER stress markers and polarization of PMA activated THP-1 cells (pTHP-1). We found that CM decreased the production of the pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and interleukin (IL)-1β as well as other cytokines and chemokines from pTHP-1 cells. These effects are mediated by the inhibition of TLR4 expression and NF-κB signaling pathway. In addition, hepatocytes CM increased the expression of binding immunoglobulin protein (BiP) and the transcription factor C/EBP homologous protein (CHOP) in pTHP-1 cells. Preconditioning with ER stress inhibitor, small molecular chaperone 4-phenylbutyrate (PBA) before addition of ER stressors, attenuated the ER stress in macrophages, the property of hepatocytes CM to alter TNF-α production and NF-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 (PPAR-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

  8. Mammalian mediator 19 mediates H1299 lung adenocarcinoma cell clone conformation, growth, and metastasis.

    PubMed

    Xu, Lu-Lu; Guo, Shu-Liang; Ma, Su-Ren; Luo, Yong-Ai

    2012-01-01

    Mammalian mediator (MED) is a multi-protein coactivator that has been identified by several research groups. The involvement of the MED complex subunit 19 (MED 19) in the metastasis of lung adenocarcinoma cell line (H1299), which expresses the MED 19 subunit, was here investigated. When MED 19 expression was decreased by RNA interference H1299 cells demonstrated reduced clone formation, arrest in the S phase of the cell cycle, and lowered metastatic capacity. Thus, MED 19 appears to play important roles in the biological behavior of non-small cell lung carcinoma cells. These findings may be important for the development of novel lung carcinoma treatments.

  9. Hepatocyte innervation in primates

    PubMed Central

    1977-01-01

    The efferent innervation and some characteristics of nerve fibers of the liver lobule in the tree shrew, a primate, are described. Nerve endings on hepatocytes were encountered regularly and were determined to be efferent adrenergic nerves. Transmission electron microscopy revealed nerve endings and varicosities in close apposition to the hepatocytes adjacent to the connective tissue of the triads as well as within the liver lobule in the space of Disse. Fluorescence microscopy indicated the existence of adrenergic nerves with a similar distribution. Autoradiography of the avid uptake of exogenous [3H]norepinephrine indicated that all intralobular nerves are potentially norepinephrinergic (adrenergic). Chemical sympathectomy with 6-OH-dopamine resulted in the degeneration of all intralobular liver nerve fibers as revealed by fluorescence microscopy and electron microscopy. Substantial regeneration occurred after 60-90 days but was not completed by that time. Some nerves were also observed in close association with von Kupffer cells and endothelial cells. The functional significance of the efferent liver innervation is discussed. PMID:406265

  10. Arsenite-mediated promotion of anchorage-independent growth of HaCaT cells through placental growth factor.

    PubMed

    Yajima, Ichiro; Kumasaka, Mayuko Y; Ohnuma, Shoko; Ohgami, Nobutaka; Naito, Hisao; Shekhar, Hossain U; Omata, Yasuhiro; Kato, Masashi

    2015-04-01

    Various cancers including skin cancer are increasing in 45 million people exposed to arsenic above the World Health Organization's guideline value of 10 μg l(-1). However, there is limited information on key molecules regulating arsenic-mediated carcinogenesis. Our fieldwork in Bangladesh demonstrated that levels of placental growth factor (PlGF) in urine samples from residents of cancer-prone areas with arsenic-polluted drinking water were higher than those in urine samples from residents of an area that was not polluted with arsenic. Our experimental study in human nontumorigenic HaCaT skin keratinocytes showed that arsenite promoted anchorage-independent growth with increased expression and secretion of PlGF, a ligand of vascular endothelial growth factor receptor1 (VEGFR1), and increased VEGFR1/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) activities. The arsenite-mediated promotion of anchorage-independent growth was strongly inhibited by PlGF depletion with decreased activities of the PlGF/VEGFR1/MEK/ERK pathway. Moreover, arsenite proteasome-dependently degrades metal-regulatory transcription factor-1 (MTF-1) protein, resulting in a decreased amount of MTF-1 protein binding to the PlGF promoter. MTF-1 negatively controlled PlGF transcription in HaCaT cells, resulting in increased PlGF transcription. These results suggest that arsenite-mediated MTF-1 degradation enhances the activity of PlGF/VEGFR1/MEK/ERK signaling, resulting in promotion of the malignant transformation of keratinocytes. Thus, this study proposed a molecular mechanism for arsenite-mediated development of skin cancer. PMID:25493652

  11. Arsenite-mediated promotion of anchorage-independent growth of HaCaT cells through placental growth factor.

    PubMed

    Yajima, Ichiro; Kumasaka, Mayuko Y; Ohnuma, Shoko; Ohgami, Nobutaka; Naito, Hisao; Shekhar, Hossain U; Omata, Yasuhiro; Kato, Masashi

    2015-04-01

    Various cancers including skin cancer are increasing in 45 million people exposed to arsenic above the World Health Organization's guideline value of 10 μg l(-1). However, there is limited information on key molecules regulating arsenic-mediated carcinogenesis. Our fieldwork in Bangladesh demonstrated that levels of placental growth factor (PlGF) in urine samples from residents of cancer-prone areas with arsenic-polluted drinking water were higher than those in urine samples from residents of an area that was not polluted with arsenic. Our experimental study in human nontumorigenic HaCaT skin keratinocytes showed that arsenite promoted anchorage-independent growth with increased expression and secretion of PlGF, a ligand of vascular endothelial growth factor receptor1 (VEGFR1), and increased VEGFR1/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) activities. The arsenite-mediated promotion of anchorage-independent growth was strongly inhibited by PlGF depletion with decreased activities of the PlGF/VEGFR1/MEK/ERK pathway. Moreover, arsenite proteasome-dependently degrades metal-regulatory transcription factor-1 (MTF-1) protein, resulting in a decreased amount of MTF-1 protein binding to the PlGF promoter. MTF-1 negatively controlled PlGF transcription in HaCaT cells, resulting in increased PlGF transcription. These results suggest that arsenite-mediated MTF-1 degradation enhances the activity of PlGF/VEGFR1/MEK/ERK signaling, resulting in promotion of the malignant transformation of keratinocytes. Thus, this study proposed a molecular mechanism for arsenite-mediated development of skin cancer.

  12. The Caenorhabditis elegans HNF4alpha Homolog, NHR-31, mediates excretory tube growth and function through coordinate regulation of the vacuolar ATPase.

    PubMed

    Hahn-Windgassen, Annett; Van Gilst, Marc R

    2009-07-01

    Nuclear receptors of the Hepatocyte Nuclear Factor-4 (HNF4) subtype have been linked to a host of developmental and metabolic functions in animals ranging from worms to humans; however, the full spectrum of physiological activities carried out by this nuclear receptor subfamily is far from established. We have found that the Caenorhabditis elegans nuclear receptor NHR-31, a homolog of mammalian HNF4 receptors, is required for controlling the growth and function of the nematode excretory cell, a multi-branched tubular cell that acts as the C. elegans renal system. Larval specific RNAi knockdown of nhr-31 led to significant structural abnormalities along the length of the excretory cell canal, including numerous regions of uncontrolled growth at sites near to and distant from the cell nucleus. nhr-31 RNAi animals were sensitive to acute challenge with ionic stress, implying that the osmoregulatory function of the excretory cell was also compromised. Gene expression profiling revealed a surprisingly specific role for nhr-31 in the control of multiple genes that encode subunits of the vacuolar ATPase (vATPase). RNAi of these vATPase genes resulted in excretory cell defects similar to those observed in nhr-31 RNAi animals, demonstrating that the influence of nhr-31 on excretory cell growth is mediated, at least in part, through coordinate regulation of the vATPase. Sequence analysis revealed a stunning enrichment of HNF4alpha type binding sites in the promoters of both C. elegans and mouse vATPase genes, arguing that coordinate regulation of the vATPase by HNF4 receptors is likely to be conserved in mammals. Our study establishes a new pathway for regulation of excretory cell growth and reveals a novel role for HNF4-type nuclear receptors in the development and function of a renal system.

  13. 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

  14. 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)

  15. 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

  16. 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

  17. 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

  18. 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

  19. MicroRNA Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1.

    PubMed

    Jung, Kwang Hwa; McCarthy, Ryan L; Zhou, Chong; Uprety, Nadima; Barton, Michelle Craig; Beretta, Laura

    2016-05-01

    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 overexpression 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. Stem Cells 2016;34:1284-1296.

  20. 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.

  1. Effect of carbohydrates attached to polystyrene on hepatocyte morphology on sugar-derivatized polystyrene matrices.

    PubMed

    Kim, Sang-Heon; Hoshiba, Takashi; Akaike, Toshihiro

    2003-12-15

    Sugar-carrying polymers have been utilized as artificial matrices for cell adhesion in tissue engineering. We have developed sugar-derivatized polystyrenes (PV-sugars) as artificial matrices, which control hepatocyte adhesion and hepatic function. Hepatocytes adhere to PV-sugar matrices in a receptor-mediated manner. In this study, we designed a new galactose-derivatized PV-sugar, poly-(6-O-p-vinylbenzyl-alpha-D-galactose) (PV6Gal) and evaluated the role of carbohydrate attached to polystyrene (PS) backbone in the morphological difference of hepatocyte cultured on PV-sugar matrices. Hepatocytes spread on monosaccharide-derivatized PV-sugars but not on disaccharide-derivatized PV-sugars. The actin filament remained aggregated in the central area of the cell body on disaccharide-derivatized PV-sugars. Hepatocyte cell bodies fully were spread on collagen, and the actin filament was almost completely reorganized. Hepatocyte spreading on monosaccharide-derivatized PV-sugars, however, was caused by protrusive cell-matrix contact like lamellipodia and the actin filament was not completely reorganized. This indicated that hepatocyte spreading on PV-sugar matrices was restricted compared with ECM-mediated cell spreading. In addition, typical spheroid formation of hepatocytes was promoted on disaccharide-derivatized PV-sugars compared with monosaccharide-derivatized PV-sugars. Although hepatocytes adhered with different affinities to PV-sugar matrices, hepatocyte morphology was not affected by the adhesion affinity. We suggest that the type of carbohydrate attached to the PS backbone governs the morphology of hepatocyte cultured on PV-sugar matrices.

  2. Pharmacological Inhibition of Microsomal Prostaglandin E Synthase-1 Suppresses Epidermal Growth Factor Receptor-Mediated Tumor Growth and Angiogenesis

    PubMed Central

    Bocci, Elena; Coletta, Isabella; Polenzani, Lorenzo; Mangano, Giorgina; Alisi, Maria Alessandra; Cazzolla, Nicola; Giachetti, Antonio; Ziche, Marina; Donnini, Sandra

    2012-01-01

    Background Blockade of Prostaglandin (PG) E2 production via deletion of microsomal Prostaglandin E synthase-1 (mPGES-1) gene reduces tumor cell proliferation in vitro and in vivo on xenograft tumors. So far the therapeutic potential of the pharmacological inhibition of mPGES-1 has not been elucidated. PGE2 promotes epithelial tumor progression via multiple signaling pathways including the epidermal growth factor receptor (EGFR) signaling pathway. Methodology/Principal Findings Here we evaluated the antitumor activity of AF3485, a compound of a novel family of human mPGES-1 inhibitors, in vitro and in vivo, in mice bearing human A431 xenografts overexpressing EGFR. Treatment of the human cell line A431 with interleukin-1beta (IL-1β) increased mPGES-1 expression, PGE2 production and induced EGFR phosphorylation, and vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) expression. AF3485 reduced PGE2 production, both in quiescent and in cells stimulated by IL-1β. AF3485 abolished IL-1β-induced activation of the EGFR, decreasing VEGF and FGF-2 expression, and tumor-mediated endothelial tube formation. In vivo, in A431 xenograft, AF3485, administered sub-chronically, decreased tumor growth, an effect related to inhibition of EGFR signalling, and to tumor microvessel rarefaction. In fact, we observed a decrease of EGFR phosphorylation, and VEGF and FGF-2 expression in tumours explanted from treated mice. Conclusion Our work demonstrates that the pharmacological inhibition of mPGES-1 reduces squamous carcinoma growth by suppressing PGE2 mediated-EGFR signalling and by impairing tumor associated angiogenesis. These results underscore the potential of mPGES-1 inhibitors as agents capable of controlling tumor growth. PMID:22815767

  3. Intercellular transfer of GPRC5B via exosomes drives HGF-mediated outward growth

    PubMed Central

    Kwon, Sang-Ho; Liu, Kathleen D.; Mostov, Keith E.

    2014-01-01

    How cells communicate during development and regeneration is a critical question. One mechanism of intercellular communication is via exosomes, extracellular vesicles that originate by the fusion of multivesicular endosomes with the plasma membrane [1-8]. To model exosome-based intercellular communication, we used MDCK cell cysts grown in 3D gels of extracellular matrix, which form tubules in response to Hepatocyte Growth Factor (HGF). We report that GPRC5B, an orphan G protein coupled receptor, is in exosomes produced by HGF-treated cysts and released into the cyst lumen. Exosomal GPRC5B is taken up by nearby cells, and together with HGF, promotes ERK1/2 activation and tubulogenesis, even under conditions where tubulogenesis would otherwise not occur. Recovery from injury, such as acute kidney injury (AKI), often recapitulates developmental processes. Here, we show that GPRC5B is elevated in urinary exosomes from patients with AKI. Our results elucidate how GPRC5B is carried by exosomes and augments HGF-induced morphogenesis. The unexpected role of exosomes in transporting GPRC5B between cells during morphogenesis and the ability of GPRC5B to predict the disease state of AKI elucidate a novel mechanism for intercellular communication during development and repair. PMID:24412205

  4. 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.

  5. 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.

  6. Caveolin-1-dependent activation of the metalloprotease TACE/ADAM17 by TGF-β in hepatocytes requires activation of Src and the NADPH oxidase NOX1.

    PubMed

    Moreno-Càceres, Joaquim; Mainez, Jèssica; Mayoral, Rafael; Martín-Sanz, Paloma; Egea, Gustavo; Fabregat, Isabel

    2016-04-01

    Transforming growth factor-β (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, the balance between which decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β. We have previously shown that caveolin-1 (CAV1) is required for activation of the metalloprotease tumour necrosis factor (TNF)-α-converting enzyme/a disintegrin and metalloproteinase 17 (TACE/ADAM17), and hence transactivation of the EGFR pathway. The specific mechanism by which TACE/ADAM17 is activated has not yet been determined. Here we show that TGF-β induces phosphorylation of sarcoma kinase (Src) in hepatocytes, a process that is impaired in Cav1(-/-) hepatocytes, coincident with a decrease in phosphorylated Src in detergent-resistant membrane fractions. TGF-β-induced activation of TACE/ADAM17 and EGFR phosphorylation were blocked using the Src inhibitor PP2. Cav1(+/+) hepatocytes showed early production of reactive oxygen species (ROS) induced by TGF-β, which was not seen in Cav1(-/-) cells. Production of ROS was inhibited by both the NADPH oxidase 1 (NOX1) inhibitor STK301831 and NOX1 knock-down, which also impaired TACE/ADAM17 activation and thus EGFR phosphorylation. Finally, neither STK301831 nor NOX1 silencing impaired Src phosphorylation, but PP2 blocked early ROS production, showing that Src is involved in NOX1 activation. As expected, inhibition of Src or NOX1 increased TGF-β-induced cell death in Cav1(+/+) cells. In conclusion, CAV1 is required for TGF-β-mediated activation of TACE/ADAM17 through a mechanism that involves phosphorylation of Src and NOX1-mediated ROS production.

  7. 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.

  8. PTEN modulates vascular endothelial growth factor-mediated signaling and angiogenic effects.

    PubMed

    Huang, Jianhua; Kontos, Christopher D

    2002-03-29

    Phosphatidylinositol 3-kinase is activated by vascular endothelial growth factor (VEGF), and many of the angiogenic cellular responses of VEGF are regulated by the lipid products of phosphatidylinositol 3-kinase. The tumor suppressor PTEN has been shown to down-regulate phosphatidylinositol 3-kinase signaling, yet the effects of PTEN on VEGF-mediated signaling and angiogenesis are unknown. Inhibition of endogenous PTEN in cultured endothelial cells by adenovirus-mediated overexpression of a dominant negative PTEN mutant (PTEN-C/S) enhanced VEGF-mediated Akt phosphorylation, and this effect correlated with decreases in caspase-3 cleavage, caspase-3 activity, and DNA degradation after induction of apoptosis with tumor necrosis factor-alpha. Overexpression of PTEN-C/S also enhanced VEGF-mediated endothelial cell proliferation and migration. In contrast, overexpression of wild-type PTEN inhibited the anti-apoptotic, proliferative, and chemotactic effects of VEGF. Moreover, PTEN-C/S increased the length of vascular sprouts in the rat aortic ring assay and modulated VEGF-mediated tube formation in an in vitro angiogenesis assay, whereas PTEN-wild type inhibited these effects. Taken together, these findings demonstrate that PTEN potently modulates VEGF-mediated signaling and function and that PTEN is a viable target in therapeutic approaches to promote or inhibit angiogenesis.

  9. A Comprehensive Analysis of Plasmodium Circumsporozoite Protein Binding to Hepatocytes.

    PubMed

    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

  10. 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

  11. Hypertonic stress regulates amino acid transport and cell cycle proteins in chick embryo hepatocytes.

    PubMed

    Bruscalupi, Giovannella; Massimi, Mara; Spagnuolo, Silvana; Fiore, Anna Maria; Leoni, Silvia

    2012-02-01

    Hyperosmotic stress affects cell growth, decreasing cell volume and increasing the uptake of organic osmolytes. However, the sensitivity of embryonic cells to osmotic treatment remains to be established. We have analysed some aspects of cell-cycle control and amino-acid transport in hypertonic conditions during prenatal life. The effects of hyperosmotic stress on amino-acid uptake mediated by system A, (3)H-thymidine incorporation, and regulation of cell-cycle proteins were analysed in chick embryo hepatocytes. Hypertonic stress increased system A activity and caused cell-cycle delay. Effects on amino-acid transport involved p38 kinase activation and new carrier synthesis. Cyclin D1, cdk4 (cyclin-dependent kinase 4) and PCNA (proliferating-cell nuclear antigen) levels decreased, whereas cyclin E, p21 and p53 levels were unchanged. Incorporation of (3)H-leucine indicated decreased synthesis of cyclin D1. In contrast, analysis of mRNA by qRT-PCR (quantitative real-time PCR) showed a net increase of cyclin D1 transcripts, suggesting post-transcriptional regulation. The data show that chick embryo hepatocytes respond to hyperosmotic conditions by arresting cell growth to prevent DNA damage and increasing osmolyte uptake to regulate cell volume, indicating that the adaptive response to environmental stress exists during prenatal life.

  12. Beta-naphthoflavone causes an AhR-independent inhibition of invasion and intracellular multiplication of Listeria monocytogenes in murine hepatocytes

    PubMed Central

    Shi, Lewis Zhichang; Czuprynski, Charles J.

    2009-01-01

    We recently reported a heretofore unknown role for the aryl hydrocarbon receptor in host resistance to listeriosis in mice. Hepatocytes are an important site for Listeria monocytogenes multiplication in vivo. In this study, we investigated whether activation of AhR in TIB73 murine embryonic hepatocytes affects the ingestion and intracellular multiplication of L. monocytogenes. Treatment of TIB73 cells with the AhR agonist β-naphthoflavone (BNF) significantly inhibited the ingestion and intracellular growth of L. monocytogenes. The inhibitory effects of BNF were dose-dependent and correlated with up-regulation of CYP1A1. Surprisingly, pretreatment with AhR antagonists (3′-MNF or α-naphthoflavone) or knocking-down of AhR with siRNA did not abolish the inhibitory effects of BNF. Moreover, the inhibitory effects of BNF on invasion and intracellular growth of L. monocytogenes by BNF were observed in AhR-deficient (CRL-2710), or ARNT-dysfunctional (CRL-2717) Hepa cells. We also observed similar inhibitory effects of BNF treatment using primary hepatocytes recovered from AhR+/- or AhR-/- mice. Moreover, the prototypic AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) did not inhibit the invasion and intracellular growth of L. monocytogenes in TIB73 cells. Mechanistic studies demonstrated that ROS, but not TNF-α or iNOS, plays an important role in mediating BNF-induced inhibition. In conclusion, BNF caused an AhR-independent inhibition of ingestion and intracellular multiplication of L. monocytogenes in murine hepatocytes, mediated in part by production of ROS. PMID:19715752

  13. Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture

    SciTech Connect

    Sawada, N.; Tomomura, A.; Sattler, C.A.; Sattler, G.L.; Kleinman, H.K.; Pitot, H.C.

    1986-12-01

    The effects of several extracellular matrix components (EMCs) - fibronectin (Fn), laminin (Ln), type I (C-I) and type IV (C-IV) collagen - on DNA synthesis in rat hepatocytes in primary culture were examined by both quantitative scintillation spectrometry and autoradiography of (/sup 3/H)thymidine incorporation. Hepatocytes cultured on Fn showed the most active DNA synthesis initiated by epidermal growth factor (EGF) with decreasing levels of (/sup 3/H)thymidine uptake exhibited in the cell cultured on C-IV, C-I, and Ln, respectively. The decreasing level of DNA synthesis in hepatocytes cultured on Fn, C-IV, C-I, and Ln respectively was not influenced by cell density. The number of EGF receptors of hepatocytes was also not influenced by EMCs. These data suggest that EMCs modify hepatocyte DNA synthesis by means of post-EGF-receptor mechanisms which are regulated by both growth factors and cell density.

  14. Current status of hepatocyte xenotransplantation.

    PubMed

    Meier, Raphael P H; Navarro-Alvarez, Nalu; Morel, Philippe; Schuurman, Henk-Jan; Strom, Stephen; Bühler, Leo H

    2015-11-01

    The treatment of acute liver failure, a condition with high mortality, comprises optimal clinical care, and in severe cases liver transplantation. However, there are limitations in availability of organ donors. Hepatocyte transplantation is a promising alternative that could fill the medical need, in particular as the bridge to liver transplantation. Encapsulated porcine hepatocytes represent an unlimited source that could function as a bioreactor requiring minimal immunosuppression. Besides patients with acute liver failure, patients with alcoholic hepatitis who are unresponsive to a short course of corticosteroids are a target for hepatocyte transplantation. In this review we present an overview of the innate immune barriers in hepatocyte xenotransplantation, including the role of complement and natural antibodies; the role of phagocytic cells and ligands like CD47 in the regulation of phagocytic cells; and the role of Natural Killer cells. We present also some illustrations of physiological species incompatibilities in hepatocyte xenotransplantation, such as incompatibilities in the coagulation system. An overview of the methodology for cell microencapsulation is presented, followed by proof-of-concept studies in rodent and nonhuman primate models of fulminant liver failure: these studies document the efficacy of microencapsulated porcine hepatocytes which warrants progress towards clinical application. Lastly, we present an outline of a provisional clinical trial, that upon completion of preclinical work could start within the upcoming 2-3 years.

  15. Collapsin response mediator protein 4 regulates growth cone dynamics through the actin and microtubule cytoskeleton.

    PubMed

    Khazaei, Mohamad R; Girouard, Marie-Pier; Alchini, Ricardo; Ong Tone, Stephan; Shimada, Tadayuki; Bechstedt, Susanne; Cowan, Mitra; Guillet, Dominique; Wiseman, Paul W; Brouhard, Gary; Cloutier, Jean Francois; Fournier, Alyson E

    2014-10-24

    Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4-/- mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4-/- mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton. PMID:25225289

  16. Surface observation for seed-mediated growth attachment of gold nanoparticles on a glassy carbon substrate.

    PubMed

    Oyama, Munetaka; Yamaguchi, Shin-Ya; Zhang, Jingdong

    2009-02-01

    A seed-mediated growth method for surface modification was applied to the attachment of gold nanoparticles (AuNPs) to glassy carbon (GC) surfaces. By simply immersing a GC plate at first into a seed solution containing 4 nm Au nano-seed particles and then into a growth solution containing HAuCl(4), ascorbic acid and cetyltrimethyammonium bromide, AuNPs could be successfully attached to the GC surface via the growth of nanoparticles. A possible control of the size and density of AuNPs on GC was examined by observing surface images with a field-emission scanning electron microscope (FE-SEM) after several preparations with different immersion times. Compared with previous results on the growth of AuNPs on indium tin oxide (ITO) surfaces, it was characteristic that the AuNPs attached to GC surfaces exhibited smaller size and higher density as well as a flatter and non-crystal-like morphology. In addition, for performing the dense attachment of regular nano-sized AuNPs on GC surfaces, immersion for 2 h into the growth solution was sufficient. Longer immersion for 24 h caused an irregular growth of bold Au micro-crystals, while 24 h was necessary in the case of AuNPs on ITO surfaces. Shorter seeding and growth times were found to be effective for a sparse attachment of smaller Au nanoparticles whose size was ca. 20 nm. It was clarified that the seed-mediated growth method for surface modification was valid for fabricating a nanointerface composed of AuNPs on GC surfaces.

  17. 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.

  18. IL4 receptor α mediates enhanced glucose and glutamine metabolism to support breast cancer growth.

    PubMed

    Venmar, Katherine T; Kimmel, Danielle W; Cliffel, David E; Fingleton, Barbara

    2015-05-01

    The type II interleukin-4 receptor (IL4R) is expressed in human breast cancer, and in murine models thereof. It is activated by interleukin-4 (IL4), a cytokine produced predominantly by immune cells. Previously, we showed that expression of IL4Rα, a signaling component of IL4R, mediates enhanced metastatic growth through promotion of tumor cell survival and proliferation. In lymphocytes, these processes are supported by increased glucose and glutamine metabolism, and B lymphocyte survival is dependent upon IL4/IL4R-induced glucose metabolism. However, it is unknown whether IL4R-mediated metabolic reprogramming could support tumor growth. Here, we show that IL4Rα expression increases proliferation thus enhancing primary mammary tumor growth. In vitro, IL4-enhanced glucose consumption and lactate production in 4T1 cells was mediated by IL4Rα. Expression of the glucose transporter GLUT1 increased in response to IL4 in vitro, and enhanced GLUT1 expression was associated with the presence of IL4Rα in 4T1 mammary tumors in vivo. Although IL4 treatment did not induce changes in glucose metabolism in MDA-MB-231 human breast cancer cells, it increased expression of the main glutamine transporter, ASCT2, and enhanced glutamine consumption in both MDA-MB-231 and 4T1 cells. Pharmacologic inhibition of glutamine metabolism with compound 968 blocked IL4/IL4Rα-increased cell number in both cell lines. Our results demonstrate that IL4R mediates enhanced glucose and glutamine metabolism in 4T1 cancer cells, and that IL4-induced growth is supported by IL4/IL4R-enhanced glutamine metabolism in both human and murine mammary cancer cells. This highlights IL4Rα as a possible target for effective breast cancer therapy.

  19. 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

  20. 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

  1. Activation of PPARalpha inhibits IGF-I-mediated growth and survival responses in medulloblastoma cell lines.

    PubMed

    Urbanska, Katarzyna; Pannizzo, Paola; Grabacka, Maja; Croul, Sidney; Del Valle, Luis; Khalili, Kamel; Reiss, Krzysztof

    2008-09-01

    Recent studies suggest a potential role of lipid lowering drugs, fibrates and statins, in anticancer treatment. One candidate for tumor chemoprevention is fenofibrate, which is a potent agonist of peroxisome proliferator activated receptor alpha (PPARalpha). Our results demonstrate elevated expression of PPARalpha in the nuclei of neoplatic cells in 12 out of 13 cases of medulloblastoma, and of PPARgamma in six out of 13 cases. Further analysis demonstrated that aggressive mouse medulloblastoma cells, BsB8, express PPARalpha in the absence PPARgamma, and human medulloblastoma cells, D384 and Daoy, express both PPARalpha and PPARgamma. Mouse and human cells responded to fenofibrate by a significant increase of PPAR-mediated transcriptional activity, and by a gradual accumulation of cells in G1 and G2/M phase of the cell cycle, leading to the inhibition of cell proliferation and elevated apoptosis. Preincubation of BsB8 cells with fenofibrate attenuated IGF-I-induced IRS-1, Akt, ERKs and GSK3beta phosphorylation, and inhibited clonogenic growth. In Daoy and D384 cells, fenofibrate also inhibited IGF-I-mediated growth responses, and simultaneous delivery of fenofibrate with low dose of the IGF-IR inhibitor, NVP-AEW541, completely abolished their clonogenic growth and survival. These results indicate a strong supportive role of fenofibrate in chemoprevention against IGF-I-induced growth responses in medulloblastoma.

  2. GH3-mediated auxin homeostasis links growth regulation with stress adaptation response in Arabidopsis.

    PubMed

    Park, Jung-Eun; Park, Ju-Young; Kim, Youn-Sung; Staswick, Paul E; Jeon, Jin; Yun, Ju; Kim, Sun-Young; Kim, Jungmook; Lee, Yong-Hwan; Park, Chung-Mo

    2007-03-30

    Plants constantly monitor environmental fluctuations to optimize their growth and metabolism. One example is adaptive growth occurring in response to biotic and abiotic stresses. Here, we demonstrate that GH3-mediated auxin homeostasis is an essential constituent of the complex network of auxin actions that regulates stress adaptation responses in Arabidopsis. Endogenous auxin pool is regulated, at least in part, through negative feedback by a group of auxin-inducible GH3 genes encoding auxin-conjugating enzymes. An Arabidopsis mutant, wes1-D, in which a GH3 gene WES1 is activated by nearby insertion of the (35)S enhancer, exhibited auxin-deficient traits, including reduced growth and altered leaf shape. Interestingly, WES1 is also induced by various stress conditions as well as by salicylic acid and abscisic acid. Accordingly, wes1-D was resistant to both biotic and abiotic stresses, and stress-responsive genes, such as pathogenesis-related genes and CBF genes, were upregulated in this mutant. In contrast, a T-DNA insertional mutant showed reduced stress resistance. We therefore propose that GH3-mediated growth suppression directs reallocation of metabolic resources to resistance establishment and represents the fitness costs of induced resistance.

  3. Novel Regulation of Fibroblast Growth Factor 2 (FGF2)-mediated Cell Growth by Polysialic Acid*

    PubMed Central

    Ono, Sayaka; Hane, Masaya; Kitajima, Ken; Sato, Chihiro

    2012-01-01

    Polysialic acid (polySia) is a unique polysaccharide that modifies neural cell adhesion molecule (NCAM) spatiotemporally. Recently, we demonstrated that polySia functions as a reservoir for several neurotrophic factors and neurotransmitters. Here, we showed the direct interaction between polySia and fibroblast growth factor-2 (FGF2) by native-PAGE, gel filtration, and surface plasmon resonance. The minimum chain length of polySia required for the interaction with FGF2 was 17. Compared with heparan sulfate, a well known glycosaminoglycan capable of forming a complex with FGF2, polySia formed a larger complex with distinct properties in facilitating oligomerization of FGF2, as well as in binding to FGF receptors. In polySia-NCAM-expressing NIH-3T3 cells, which were established by transfecting cells with either of the plasmids for the expression of the polysialyltransferases ST8SiaII/STX and ST8SiaIV/PST that can polysialylate NCAM, FGF2-stimulated cell growth, but not cell survival, was inhibited. Taken together, these results suggest that polySia-NCAM might be involved in the regulation of FGF2-FGF receptor signaling through the direct binding of FGF2 in a manner distinct from heparan sulfate. PMID:22158871

  4. 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

  5. Modulation of HDL metabolism by the niacin receptor GPR109A in mouse hepatocytes.

    PubMed

    Li, Xiaoyu; Millar, John S; Brownell, Nicholas; Briand, François; Rader, Daniel J

    2010-11-01

    The niacin receptor GPR109A is a G(i)-protein-coupled receptor which mediates the effects of niacin on inhibiting intracellular triglyceride lipolysis in adipocytes. However, the role of GPR109A in mediating the effects of niacin on high density lipoprotein (HDL) metabolism is unclear. We found niacin has no effect on HDL-C in GPR109A knockout mice. Furthermore, niacin lowered intracellular cAMP in primary hepatocytes mediated by GPR109A. We used an adeno-associated viral (AAV) serotype 8 vector encoding GPR109A under the control of the hepatic-specific thyroxine-binding globulin promoter to specifically overexpress GPR109A in mouse liver. Plasma HDL-C, hepatic ABCA1 and the HDL cholesterol production rate were significantly reduced in mice overexpressing GPR109A. Overexpression of GPR109A reduced primary hepatocyte free cholesterol efflux to apoA-I; conversely, GPR109A deficient hepatocytes had increased ABCA1-mediated cholesterol efflux. These data support the concept that the HDL-C lowering effect of niacin in wild-type mice is mediated through stimulation of GPR109A in hepatocytes; such an effect then leads to reduced hepatocyte ABCA1 expression and activity, decreased cholesterol efflux to nascent apoA-I, and reduced HDL-C levels. These results indicate that niacin-mediated activation of GP109A in liver lowers ABCA1 expression leading to reduced hepatic cholesterol efflux to HDL.

  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 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

  8. Involvement of the nuclear high mobility group B1 peptides released from injured hepatocytes in murine hepatic fibrogenesis.

    PubMed

    Kao, Ying-Hsien; Lin, Yu-Chun; Tsai, Ming-Shian; Sun, Cheuk-Kwan; Yuan, Shyng-Shiou; Chang, Chih-Yang; Jawan, Bruno; Lee, Po-Huang

    2014-09-01

    This study investigated the pro-fibrogenic role of high mobility group box 1 (HMGB1) peptides in liver fibrogenesis. An animal model of carbon tetrachloride (CCl4)-induced liver fibrosis was used to examine the serum HMGB1 levels and its intrahepatic distribution. The increased serum HMGB1 levels were positively correlated with elevation of transforming growth factor-β1 (TGF-β1) and collagen deposition during fibrogenesis. The cytoplasmic distribution of HMGB1 was noted in the parenchymal hepatocytes of fibrotic livers. In vitro studies confirmed that exposure to hydrogen peroxide and CCl4 induced an intracellular mobilization and extracellular release of nuclear HMGB1 peptides in clone-9 and primary hepatocytes, respectively. An uptake of exogenous HMGB1 by hepatic stellate cells (HSCs) T6 cells indicated a possible paracrine action of hepatocytes on HSCs. Moreover, HMGB1 dose-dependently stimulated HSC proliferation, up-regulated de novo synthesis of collagen type I and α-smooth muscle actin (α-SMA), and triggered Smad2 phosphorylation and its nuclear translocation through a TGF-β1-independent mechanism. Blockade with neutralizing antibodies and gene silencing demonstrated the involvement of the receptor for advanced glycation end-products (RAGE), but not toll-like receptor 4, in cellular uptake of HMGB1 and the HMGB1-mediated Smad2 and ERK1/2 phosphorylation as well as α-SMA up-regulation in HSC-T6 cells. Furthermore, anti-RAGE treatment significantly ameliorated CCl4-induced liver fibrosis. In conclusion, the nuclear HMGB1 peptides released from parenchymal hepatocytes during liver injuries may directly activate HSCs through stimulating HSC proliferation and transformation, eventually leading to the fibrotic changes of livers. Blockade of HMGB1/RAGE signaling cascade may constitute a therapeutic strategy for treatment of liver fibrosis.

  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. 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

  11. Growth control in colon epithelial cells: gadolinium enhances calcium-mediated growth regulation.

    PubMed

    Attili, Durga; Jenkins, Brian; Aslam, Muhammad Nadeem; Dame, Michael K; Varani, James

    2012-12-01

    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.

  12. Epidermal growth factor-mediated effects on equine vascular smooth muscle cells

    SciTech Connect

    Grosenbaugh, D.A.; Amoss, M.S.; Hood, D.M.; Morgan, S.J.; Williams, J.D. )

    1988-10-01

    Epidermal growth factor (EGF) receptor binding kinetics and EGF-mediated stimulation of DNA synthesis and cellular proliferation were studied in cultured vascular smooth muscle cells (VSMC) from the equine thoracic aorta. Binding studies, using murine {sup 125}I-labeled EGF, indicate the presence of a single class of high-affinity binding sites, with an estimated maximal binding capacity of 5,800 sites/cells. EGF stimulated ({sup 3}H)thymidine uptake in confluent quiescent monolayers in a dose-dependent fashion, half-maximal stimulation occurring at 7.5 {times} 10{sup {minus}11} M. Likewise, EGF-mediated cellular proliferation was dose dependent under reduced serum concentrations. Equine VSMC contain specific receptors for EGF, and EGF can stimulate DNA synthesis and proliferation in these cultured cells, which suggests that EGF may participate in the proliferative changes observed in equine distal digital peripheral vascular disease.

  13. 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.

  14. A novel epigenetic CREB-miR-373 axis mediates ZIP4-induced pancreatic cancer growth.

    PubMed

    Zhang, Yuqing; Yang, Jingxuan; Cui, Xiaobo; Chen, Yong; Zhu, Vivian F; Hagan, John P; Wang, Huamin; Yu, Xianjun; Hodges, Sally E; Fang, Jing; Chiao, Paul J; Logsdon, Craig D; Fisher, William E; Brunicardi, F Charles; Chen, Changyi; Yao, Qizhi; Fernandez-Zapico, Martin E; Li, Min

    2013-09-01

    Changes in the intracellular levels of the essential micronutrient zinc have been implicated in multiple diseases including pancreatic cancer; however, the molecular mechanism is poorly understood. Here, we report a novel mechanism where increased zinc mediated by the zinc importer ZIP4 transcriptionally induces miR-373 in pancreatic cancer to promote tumour growth. Reporter, expression and chromatin immunoprecipitation assays demonstrate that ZIP4 activates the zinc-dependent transcription factor CREB and requires this transcription factor to increase miR-373 expression through the regulation of its promoter. miR-373 induction is necessary for efficient ZIP4-dependent enhancement of cell proliferation, invasion, and tumour growth. Further analysis of miR-373 in vivo oncogenic function reveals that it is mediated through its negative regulation of TP53INP1, LATS2 and CD44. These results define a novel ZIP4-CREB-miR-373 signalling axis promoting pancreatic cancer growth, providing mechanistic insights explaining in part how a zinc transporter functions in cancer cells and may have broader implications as inappropriate regulation of intracellular zinc levels plays an important role in many other diseases.

  15. rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling in KB cells.

    PubMed Central

    Nishiyama, T; Sasaki, T; Takaishi, K; Kato, M; Yaku, H; Araki, K; Matsuura, Y; Takai, Y

    1994-01-01

    Insulin and hepatocyte growth factor (HGF) induced morphologically different membrane rufflings in KB cells. Insulin-induced membrane ruffling was inhibited by microinjection of rho GDI, an inhibitory GDP/GTP exchange regulator for both rho p21 and rac p21 small GTP-binding proteins, but not inhibited by microinjection of botulinum exoenzyme C3, known to selectively ADP-ribosylate rho p21 and to impair its function. This rho GDI action was prevented by comicroinjection with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound rac1 p21. In contrast, HGF-induced membrane ruffling was inhibited by microinjection of rho GDI or C3. This rho GDI action was prevented by comicroinjection with GTP gamma S-bound rhoA p21, and this C3 action was prevented by comicroinjection with GTP gamma S-bound rhoAIle-41 p21, which is resistant to C3. Microinjection of either GTP gamma S-bound rac1 p21 or rhoA p21 alone induced membrane ruffling in the absence of the growth factors. The rac1 p21-induced membrane ruffling was morphologically similar to the insulin-induced kind, whereas rhoA p21-induced ruffling was apparently different from both the insulin- and HGF-induced kinds. Membrane ruffling was also induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, but not by Ca2+ ionophore or microinjection of a dominant active Ki-ras p21 mutant (Ki-rasVal-12 p21). The phorbol ester-induced membrane ruffling was morphologically similar to the rhoA p21-induced kind and inhibited by microinjection of rho GDI or C3. These results indicate that rac p21 and rho GDI are involved in insulin-induced membrane ruffling and that rho p21 and rho GDI are involved in HGF- and phorbol ester-induced membrane rufflings. Images PMID:8139548

  16. Early growth response-1 suppresses epidermal growth factor receptor-mediated airway hyperresponsiveness and lung remodeling in mice.

    PubMed

    Kramer, Elizabeth L; Mushaben, Elizabeth M; Pastura, Patricia A; Acciani, Thomas H; Deutsch, Gail H; Khurana Hershey, Gurjit K; Korfhagen, Thomas R; Hardie, William D; Whitsett, Jeffrey A; Le Cras, Timothy D

    2009-10-01

    Transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor receptor, are induced after lung injury and are associated with remodeling in chronic pulmonary diseases, such as pulmonary fibrosis and asthma. Expression of TGF-alpha in the lungs of adult mice causes fibrosis, pleural thickening, and pulmonary hypertension, in addition to increased expression of a transcription factor, early growth response-1 (Egr-1). Egr-1 was increased in airway smooth muscle (ASM) and the vascular adventitia in the lungs of mice conditionally expressing TGF-alpha in airway epithelium (Clara cell secretory protein-rtTA(+/-)/[tetO](7)-TGF-alpha(+/-)). The goal of this study was to determine the role of Egr-1 in TGF-alpha-induced lung disease. To accomplish this, TGF-alpha-transgenic mice were crossed to Egr-1 knockout (Egr-1(ko/ko)) mice. The lack of Egr-1 markedly increased the severity of TGF-alpha-induced pulmonary disease, dramatically enhancing airway muscularization, increasing pulmonary fibrosis, and causing greater airway hyperresponsiveness to methacholine. Smooth muscle hyperplasia, not hypertrophy, caused the ASM thickening in the absence of Egr-1. No detectable increases in pulmonary inflammation were found. In addition to the airway remodeling disease, vascular remodeling and pulmonary hypertension were also more severe in Egr-1(ko/ko) mice. Thus, Egr-1 acts to suppress epidermal growth factor receptor-mediated airway and vascular muscularization, fibrosis, and airway hyperresponsiveness in the absence of inflammation. This provides a unique model to study the processes causing pulmonary fibrosis and ASM thickening without the complicating effects of inflammation.

  17. 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.

  18. 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).

  19. Ethinyl estradiol-induced cell proliferation in rat liver. Involvement of specific populations of hepatocytes.

    PubMed

    Mayol, X; Neal, G E; Davies, R; Romero, A; Domingo, J

    1992-12-01

    Hepatocyte proliferation was analyzed in vivo during the time course of continuous administration to rats of the liver tumor promoter ethinyl estradiol (EE) at 10 p.p.m. in the diet. EE-induced acute liver hyperplasia was detected in male and female Sprague-Dawley rats as an increased mitotic index of hepatocytes after 2 days of treatment. 5'-Bromodeoxyuridine (BrdU) labeling showed that proliferating hepatocytes were randomly distributed throughout the hepatic lobule. Subsequently, and still during the first few days of continuous EE treatment, hepatocyte proliferation decreased to control levels, and a transient increase in the incidence of apoptosis in the liver was detected. Although consistent with the concept of liver growth regression after mitogen-induced hyperplasia, these results differ from others reported to date in that, in our experiments, the cessation of cell proliferation and the subsequent growth regression occurred without withdrawal of EE in our experiments. After returning to control levels, hepatocellular proliferation again increased between 3 and 6 months of chronic treatment and remained activated during the following months of continuous treatment, as seen by accumulative BrdU labeling. Proliferating hepatocytes were predominantly located in zone 2 of the hepatic lobule at this time, surrounding a periportal zone of vacuolated hepatocytes, which were also induced by the treatment. Moreover, hyperplasia of basophilic hepatocytes was also seen around some portal spaces. In another set of experiments, chronic EE-induced activation was characterized by flow cytometry on hepatocytes isolated from male Fischer rats. Ploidy analysis of hepatocyte cell suspensions showed that the normal polyploid pattern of hepatocytes was altered by EE, the proportion of diploid hepatocytes rising considerably. The results also showed that these diploid cells were the most susceptible hepatocyte population to EE-induced proliferation, as shown by a combination of

  20. Inhibition of tumor angiogenesis and growth by nanoparticle-mediated p53 gene therapy in mice.

    PubMed

    Prabha, S; Sharma, B; Labhasetwar, V

    2012-08-01

    Mutation of the p53 tumor suppressor gene, the most common genetic alteration in human cancers, results in more aggressive disease and increased resistance to conventional therapies. Aggressiveness may be related to the increased angiogenic activity of cancer cells containing mutant p53. To restore wild-type p53 function in cancer cells, we developed polymeric nanoparticles (NPs) for p53 gene delivery. Previous in vitro and in vivo studies demonstrated the ability of these NPs to provide sustained intracellular release of DNA, thus sustained gene transfection and decreased tumor cell proliferation. We investigated in vivo mechanisms involved in NP-mediated p53 tumor inhibition, with focus on angiogenesis. We hypothesize that sustained p53 gene delivery will help decrease tumor angiogenic activity and thus reduce tumor growth and improve animal survival. Xenografts of p53 mutant tumors were treated with a single intratumoral injection of p53 gene-loaded NPs (p53NPs). We observed intratumoral p53 gene expression corresponding to tumor growth inhibition, over 5 weeks. Treated tumors showed upregulation of thrombospondin-1, a potent antiangiogenic factor, and a decrease in microvessel density vs controls (saline, p53 DNA alone, and control NPs). Greater levels of apoptosis were also observed in p53NP-treated tumors. Overall, this led to significantly improved survival in p53NP-treated animals. NP-mediated p53 gene delivery slowed cancer progression and improved survival in an in vivo cancer model. One mechanism by which this was accomplished was disruption of tumor angiogenesis. We conclude that the NP-mediated sustained tumor p53 gene therapy can effectively be used for tumor growth inhibition.

  1. Testosterone mediates seasonal growth of the song control nuclei in a tropical bird

    PubMed Central

    Small, Thomas W.; Brenowitz, Eliot A.; Wojtenek, Winfried; Moore, Ignacio T.

    2015-01-01

    In mid- to high-latitude songbirds seasonal reproduction is stimulated by increasing daylength accompanied by elevated plasma sex steroid levels, increased singing, and growth of the song control nuclei (SCN). Plasticity of the SCN and song behavior are primarily mediated by testosterone (T) and its metabolites in most species studied thus far. However, the majority of bird species are tropical and have less pronounced seasonal reproductive cycles. We have previously documented that equatorial rufous-collared sparrows (Zonotrichia capensis) exhibit seasonal neuroplasticity in the SCN. Manipulating T in these birds, however, did not alter singing behavior. In the current study we investigated whether T mediates plasticity of the SCN in a similar manner to temperate songbirds. In the first experiment we treated captive male birds with T or blank implants during the non-breeding season. In a second experiment we treated captive males with either blank implants, T-filled implants, T with Flutamide (FLU; an androgen receptor antagonist) or T with FLU and 1,4,6-androstatriene-3,17-dione (ATD; an estrogen synthesis inhibitor) during the breeding season. In both experiments, the volumes of brain areas HVC, Area X, and RA were measured along with singing behavior. In summary, T stimulated growth of HVC and RA and the combined effect of FLU and ATD reversed this effect in HVC. Area X was not affected by testosterone treatment in either experiment. Neither T-treated birds nor controls sang in captivity during either experiment. Together these data indicate that T mediates seasonal changes in the HVC and RA of both tropical and higher latitude bird species even if the environmental signals differ. However, unlike most higher latitude songbirds, we found no evidence that motivation to sing or growth of Area X are stimulated by T under captive conditions. PMID:26346733

  2. Insulin and IGFs enhance hepatocyte differentiation from human embryonic stem cells via the PI3K/AKT pathway.

    PubMed

    Magner, Nataly L; Jung, Yunjoon; Wu, Jian; Nolta, Jan A; Zern, Mark A; Zhou, Ping

    2013-10-01

    Human embryonic stem cells (hESCs) can be progressively differentiated into definitive endoderm (DE), hepatic progenitors, and hepatocytes, and thus provide an excellent model system for the mechanistic study of hepatocyte differentiation, which is currently poorly understood. Here, we found that insulin enhanced hepatocyte differentiation from hESC-derived DE. Insulin activated the PI3K/AKT pathway, but not the mitogen-activated protein kinase pathway in the DE cells, and inhibition of the PI3K/AKT pathways by inhibitors markedly inhibited hepatocyte differentiation. In addition, insulin-like growth factor 1 (IGF1) and IGF2 also activated the PI3K/AKT pathway in DE cells and their expression was robustly upregulated during hepatocyte differentiation from DE. Furthermore, inhibition of IGF receptor 1 (IGF1R) by a small molecule inhibitor PPP or knockdown of the IGF1R by shRNA attenuated hepatocyte differentiation. Moreover, simultaneous knockdown of the IGF1R and the insulin receptor with shRNAs markedly reduced the activation of AKT and substantially impaired hepatocyte differentiation. The PI3K pathway specifically enhanced the expression of HNF1 and HNF4 to regulate hepatocyte differentiation from DE. Although inhibition of the PI3K pathway was previously shown to be required for the induction of DE from hESCs, our study revealed a positive role of the PI3K pathway in hepatocyte differentiation after the DE stage, and has advanced our understanding of hepatocyte cell fate determination.

  3. Involvement of hexokinase1 in plant growth promotion as mediated by Burkholderia phytofirmans.

    PubMed

    Park, Jae Min; Lazarovits, George

    2014-06-01

    Potato plantlets inoculated with strain PsJN of the bacterium Burkholderia phytofirmans exhibit consistent and significant increases in plant growth under in vitro conditions, when compared with uninoculated plants. The greatest influence on the degree and type of growth enhancement that develops has been shown to be mediated by the sugar concentration in the agar media. Bacterial growth promotion has been suggested in other studies to be regulated by the sugar sensor enzyme hexokinase1, the role of which is activation of glucose phosphorylation. In this present study, we examined the co-relationship between root and stem development in potato plants treated with PsJN and the activity of hexokinase1. Plants grown in the presence of 1.5% and 3% sucrose showed increased levels of hexokinase1 activity only in the roots of inoculated plants, suggesting that the increased enzyme levels may be associated with root growth. Analysis for mRNA using reverse transcriptase did not reveal any significant differences in transcription levels of the gene between inoculated and uninoculated plants. When PsJN-inoculated plants were grown in 1.5% and 3% concentrations of glucose and fructose, stem height and mass, leaf number, root mass, and overall biomass increased. No growth promotion occurred when PsJN-inoculated plants were grown in 3% maltose. Subsequently, a hexokinase1 activity assay showed that PsJN-induced growth of potato plants was found to only occur when plants were grown in the presence of sugars that are recognized by the plant hexokinase1. The results suggest that PsJN may enhance sugar uptake in plants by direct or indirect stimulation of hexokinase1 activity in roots and this results in enhanced overall plant growth.

  4. Octylphenol induces vitellogenin production and cell death in fish hepatocytes

    SciTech Connect

    Toomey, B.H.; Monteverdi, G.H.; Di Giulio, R.T.

    1999-04-01

    The effects of octylphenol (OP) on vitellogenin production and cell death in hepatocytes from brown bullhead catfish (Americurus nebulosus) were studied. Production of vitellogenin was induced in hepatocytes exposed to 10 to 50 {micro}M OP, whereas a higher concentration of OP (100 {micro}M) induced apoptotic cell death. By 3 h after the addition of 100 {micro}M OP, dying cells showed chromatin condensation and DNA fragmentation as determined by fluorescence microscopy and gel electrophoresis. Later stages of cell death (nuclear membrane breakdown and cell fragmentation into apoptotic bodies) were identified in cells exposed to OP for at least 6 h. Hepatocytes exposed to 100 {micro}M OP also produced less vitellogenin than cells exposed to 50 {micro}M OP. An estrogen receptor antagonist, tamoxifen, greatly decreased vitellogenin production in OP-exposed hepatocytes from male fish but did not decrease cell death in these cells. Thus, although the ability of OP to induce vitellogenin production is likely mediated through interactions with the estrogen receptor, the induction of apoptotic cell death by OP does not appear to be dependent on its estrogenic activity but may be a more general toxic effect.

  5. 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}.

  6. The complement cascade as a mediator of tissue growth and regeneration.

    PubMed

    Rutkowski, Martin J; Sughrue, Michael E; Kane, Ari J; Ahn, Brian J; Fang, Shanna; Parsa, Andrew T

    2010-11-01

    Recent evidence has demonstrated that the complement cascade is involved in a variety of physiologic and pathophysiologic processes in addition to its role as an immune effector. Research in a variety of organ systems has shown that complement proteins are direct participants in maintenance of cellular turnover, healing, proliferation and regeneration. As a physiologic housekeeper, complement proteins maintain tissue integrity in the absence of inflammation by disposing of cellular debris and waste, a process critical to the prevention of autoimmune disease. Developmentally, complement proteins influence pathways including hematopoietic stem cell engraftment, bone growth, and angiogenesis. They also provide a potent stimulus for cellular proliferation including regeneration of the limb and eye in animal models, and liver proliferation following injury. Here, we describe the complement cascade as a mediator of tissue growth and regeneration.

  7. Thyroid hormone-responsive genes mediate otolith growth and development during flatfish metamorphosis.

    PubMed

    Wang, X; Tan, Y; Sievers, Q; Sievers, B; Lee, M; Burrall, K; Schreiber, A M

    2011-01-01

    Flatfish begin life as up-right swimming, bilaterally symmetrical larvae that metamorphose into asymmetrically shaped juveniles that swim with a highly lateralized posture. We have previously shown that TH induces abrupt growth and mineralization of one component of the vestibular system, the otoliths, during early larval development and metamorphosis. Here we report that four of five vestibular-specific genes that we tested (alpha-tectorin, otogelin, otolith matrix protein, and otopetrins 1 and 2 that are known to be associated with otolith development in other vertebrates are up-regulated 1.5- to 7-fold in larval flatfish during spontaneous metamorphosis and/or following 72 h of TH treatment. These findings suggest that otolith growth and development are mediated by diverse TH-responsive genes during flatfish metamorphosis.

  8. 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

  9. Akt1-mediated fast/glycolytic skeletal muscle growth attenuates renal damage in experimental kidney disease.

    PubMed

    Hanatani, Shinsuke; Izumiya, Yasuhiro; Araki, Satoshi; Rokutanda, Taku; Kimura, Yuichi; Walsh, Kenneth; Ogawa, Hisao

    2014-12-01

    Muscle wasting is frequently observed in patients with kidney disease, and low muscle strength is associated with poor outcomes in these patients. However, little is known about the effects of skeletal muscle growth per se on kidney diseases. In this study, we utilized a skeletal muscle-specific, inducible Akt1 transgenic (Akt1 TG) mouse model that promotes the growth of functional skeletal muscle independent of exercise to investigate the effects of muscle growth on kidney diseases. Seven days after Akt1 activation in skeletal muscle, renal injury was induced by unilateral ureteral obstruction (UUO) in Akt1 TG and wild-type (WT) control mice. The expression of atrogin-1, an atrophy-inducing gene in skeletal muscle, was upregulated 7 days after UUO in WT mice but not in Akt1 TG mice. UUO-induced renal interstitial fibrosis, tubular injury, apoptosis, and increased expression of inflammatory, fibrosis-related, and adhesion molecule genes were significantly diminished in Akt1 TG mice compared with WT mice. An increase in the activating phosphorylation of eNOS in the kidney accompanied the attenuation of renal damage by myogenic Akt1 activation. Treatment with the NOS inhibitor L-NAME abolished the protective effect of skeletal muscle Akt activation on obstructive kidney disease. In conclusion, Akt1-mediated muscle growth reduces renal damage in a model of obstructive kidney disease. This improvement appears to be mediated by an increase in eNOS signaling in the kidney. Our data support the concept that loss of muscle mass during kidney disease can contribute to renal failure, and maintaining muscle mass may improve clinical outcome. PMID:25012168

  10. Akt1-mediated fast/glycolytic skeletal muscle growth attenuates renal damage in experimental kidney disease.

    PubMed

    Hanatani, Shinsuke; Izumiya, Yasuhiro; Araki, Satoshi; Rokutanda, Taku; Kimura, Yuichi; Walsh, Kenneth; Ogawa, Hisao

    2014-12-01

    Muscle wasting is frequently observed in patients with kidney disease, and low muscle strength is associated with poor outcomes in these patients. However, little is known about the effects of skeletal muscle growth per se on kidney diseases. In this study, we utilized a skeletal muscle-specific, inducible Akt1 transgenic (Akt1 TG) mouse model that promotes the growth of functional skeletal muscle independent of exercise to investigate the effects of muscle growth on kidney diseases. Seven days after Akt1 activation in skeletal muscle, renal injury was induced by unilateral ureteral obstruction (UUO) in Akt1 TG and wild-type (WT) control mice. The expression of atrogin-1, an atrophy-inducing gene in skeletal muscle, was upregulated 7 days after UUO in WT mice but not in Akt1 TG mice. UUO-induced renal interstitial fibrosis, tubular injury, apoptosis, and increased expression of inflammatory, fibrosis-related, and adhesion molecule genes were significantly diminished in Akt1 TG mice compared with WT mice. An increase in the activating phosphorylation of eNOS in the kidney accompanied the attenuation of renal damage by myogenic Akt1 activation. Treatment with the NOS inhibitor L-NAME abolished the protective effect of skeletal muscle Akt activation on obstructive kidney disease. In conclusion, Akt1-mediated muscle growth reduces renal damage in a model of obstructive kidney disease. This improvement appears to be mediated by an increase in eNOS signaling in the kidney. Our data support the concept that loss of muscle mass during kidney disease can contribute to renal failure, and maintaining muscle mass may improve clinical outcome.

  11. 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

  12. Nrf2 is involved in maintaining hepatocyte identity during liver regeneration.

    PubMed

    Zou, Yuhong; Lee, Joonyong; Nambiar, Shashank Manohar; Hu, Min; Rui, Wenjuan; Bao, Qi; Chan, Jefferson Y; Dai, Guoli

    2014-01-01

    Nrf2, a central regulator of the cellular defense against oxidative stress and inflammation, participates in modulating hepatocyte proliferation during liver regeneration. It is not clear, however, whether Nrf2 regulates hepatocyte growth, an important cellular mechanism to regain the lost liver mass after partial hepatectomy (PH). To determine this, various analyses were performed in wild-type and Nrf2-null mice following PH. We found that, at 60 h post-PH, the vast majority of hepatocytes lacking Nrf2 reduced their sizes, activated hepatic progenitor markers (CD133, TWEAK receptor, and trefoil factor family 3), depleted HNF4α protein, and downregulated the expression of a group of genes critical for their functions. Thus, the identity of hepatocytes deficient in Nrf2 was transiently but massively impaired in response to liver mass loss. This event was associated with the coupling of protein depletion of hepatic HNF4α, a master regulator of hepatocyte differentiation, and concomitant inactivation of hepatic Akt1 and p70S6K, critical hepatocyte growth signaling molecules. We conclude that Nrf2 participates in maintaining newly regenerated hepatocytes in a fully differentiated state by ensuring proper regulation of HNF4α, Akt1, and p70S6K during liver regeneration.

  13. Nrf2 Is Involved in Maintaining Hepatocyte Identity during Liver Regeneration

    PubMed Central

    Zou, Yuhong; Lee, Joonyong; Nambiar, Shashank Manohar; Hu, Min; Rui, Wenjuan; Bao, Qi; Chan, Jefferson Y.; Dai, Guoli

    2014-01-01

    Nrf2, a central regulator of the cellular defense against oxidative stress and inflammation, participates in modulating hepatocyte proliferation during liver regeneration. It is not clear, however, whether Nrf2 regulates hepatocyte growth, an important cellular mechanism to regain the lost liver mass after partial hepatectomy (PH). To determine this, various analyses were performed in wild-type and Nrf2-null mice following PH. We found that, at 60 h post-PH, the vast majority of hepatocytes lacking Nrf2 reduced their sizes, activated hepatic progenitor markers (CD133, TWEAK receptor, and trefoil factor family 3), depleted HNF4α protein, and downregulated the expression of a group of genes critical for their functions. Thus, the identity of hepatocytes deficient in Nrf2 was transiently but massively impaired in response to liver mass loss. This event was associated with the coupling of protein depletion of hepatic HNF4α, a master regulator of hepatocyte differentiation, and concomitant inactivation of hepatic Akt1 and p70S6K, critical hepatocyte growth signaling molecules. We conclude that Nrf2 participates in maintaining newly regenerated hepatocytes in a fully differentiated state by ensuring proper regulation of HNF4α, Akt1, and p70S6K during liver regeneration. PMID:25222179

  14. The wedelolactone derivative inhibits estrogen receptor-mediated breast, endometrial, and ovarian cancer cells growth.

    PubMed

    Xu, Defeng; Lin, Tzu-Hua; Yeh, Chiuan-Ren; 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.

  15. 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.

  16. 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

  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.

  18. 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

  19. 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

  20. Yes is a central mediator of cell growth in malignant mesothelioma cells.

    PubMed

    Sato, Ayami; Sekine, Miki; Virgona, Nantiga; Ota, Masako; Yano, Tomohiro

    2012-11-01

    The constitutive activation of the Src family kinases (SFKs) has been established as a poor prognostic factor in malignant mesothelioma (MM), however, the family member(s) which contribute to the malignancy have not been defined. This study aimed to identify the SFK member(s) contributing to cell growth using RNA interference in various MM cell lines. Silencing of Yes but not of c-Src or Fyn in MM cells leads to cell growth suppression. This suppressive effect caused by Yes silencing mainly depends on G1 cell cycle arrest and partly the induction of apoptosis. Also, the knockout of Yes induces the inactivation of β-catenin signaling and subsequently decreases the levels of cyclin D necessary for G1-S transition in the cell cycle. In addition, Yes knockout has less effect on cell growth suppression in β-catenin-deficient H28 MM cells compared to other MM cells which express the catenin. Overall, we conclude that Yes is a central mediator for MM cell growth that is not shared with other SFKs such as c-Src.

  1. 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.

  2. Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

    PubMed Central

    Liu, Zhongle; Moran, Gary P.; Myers, Lawrence C.

    2016-01-01

    Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of ‘free,’ non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large ‘free’ pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the

  3. Interaction between Cartilage Oligomeric Matrix Protein and Extracellular Matrix Protein 1 Mediates Endochondral Bone Growth

    PubMed Central

    Kong, Li; Tian, Qingyun; Guo, Fengjin; Mucignat, Maria T.; Perris, Roberto; Sercu, Sandy; Merregaert, Joseph; Di Cesare, Paul E.; Liu, Chuan-ju

    2010-01-01

    In an effort to define the biological functions of COMP, a functional genetic screen was performed. This led to the identification of extracellular matrix protein 1 (ECM1) as a novel COMP-associated partner. COMP directly binds to ECM1 both in vitro and in vivo. The EGF domain of COMP and the C-terminus of ECM1 mediate the interaction between them. COMP and ECM1 Colocalize in the Growth Plates in Vivo. ECM1 inhibits chondrocyte hypertrophy, matrix mineralization, and endochondral bone formation, and COMP overcomes the inhibition by ECM1. In addition, COMP-mediated neutralization of ECM1 inhibition depends on their interaction, since COMP largely fails to overcome the ECM1 inhibition in the presence of the EGF domain of COMP, which disturbs the association of COMP and ECM1. These findings provide the first evidence linking the association of COMP and ECM1 and the biological significance underlying the interaction between them in regulating endochondral bone growth. PMID:20138147

  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. 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.

  6. 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

  7. Hormone-mediated growth dynamics of the barley pericarp as revealed by magnetic resonance imaging and transcript profiling.

    PubMed

    Pielot, Rainer; Kohl, Stefan; Manz, Bertram; Rutten, Twan; Weier, Diana; Tarkowská, Danuše; Rolčík, Jakub; Strnad, Miroslav; Volke, Frank; Weber, Hans; Weschke, Winfriede

    2015-11-01

    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

  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. Loss of α1,6-fucosyltransferase suppressed liver regeneration: implication of core fucose in the regulation of growth factor receptor-mediated cellular signaling.

    PubMed

    Wang, Yuqin; Fukuda, Tomohiko; Isaji, Tomoya; Lu, Jishun; Gu, Wei; Lee, Ho-Hsun; Ohkubo, Yasuhito; Kamada, Yoshihiro; Taniguchi, Naoyuki; Miyoshi, Eiji; Gu, Jianguo

    2015-02-05

    Core fucosylation is an important post-translational modification, which is catalyzed by α1,6-fucosyltransferase (Fut8). Increased expression of Fut8 has been shown in diverse carcinomas including hepatocarcinoma. In this study, we investigated the role of Fut8 expression in liver regeneration by using the 70% partial hepatectomy (PH) model, and found that Fut8 is also critical for the regeneration of liver. Interestingly, we show that the Fut8 activities were significantly increased in the beginning of PH (~4d), but returned to the basal level in the late stage of PH. Lacking Fut8 led to delayed liver recovery in mice. This retardation mainly resulted from suppressed hepatocyte proliferation, as supported not only by a decreased phosphorylation level of epidermal growth factor (EGF) receptor and hepatocyte growth factor (HGF) receptor in the liver of Fut8(-/-) mice in vivo, but by the reduced response to exogenous EGF and HGF of the primary hepatocytes isolated from the Fut8(-/-) mice. Furthermore, an administration of L-fucose, which can increase GDP-fucose synthesis through a salvage pathway, significantly rescued the delayed liver regeneration of Fut8(+/-) mice. Overall, our study provides the first direct evidence for the involvement of Fut8 in liver regeneration.

  10. Adhesion, invasion and intracellular replication of Salmonella typhimurium in a murine hepatocyte cell line. Effect of cytokines and LPS on antibacterial activity of hepatocytes.

    PubMed

    Lajarin, F; Rubio, G; Galvez, J; Garcia-Peñarrubia, P

    1996-11-01

    Elimination of pathogenic microorganisms in the liver may be an important effector mechanism in host defenses. In this paper we describe the adhesion, invasion and multiplication of Salmonella typhimurium in a murine embryonic hepatocyte cell line (ATCC TIB-73). Monolayers of hepatocytes treated with recombinant IFN gamma, IL1 beta, and LPS exhibit antibacterial activity against intracellular Salmonella. The dynamic of the infection process in stimulated vs unstimulated hepatocytes was determined by counting the number of survival bacteria in the cell monolayers at 4 and 28h after gentamicin was added to the infected cells. Salmonella typhimurium is able to adhere, invade and replicate inside the hepatocytes. The maximum number of cell-associated bacteria is approximately 15 bacteria per cell, whereas the invasive capacity of Salmonella is 0.003 bacteria per hepatocyte. Stimulated cultures display antibacterial activity compared to unstimulated controls. The antibacterial activity does not seem to be mediated by nitric oxide (NO) since inhibition of NO production by using NG-Monomethyl-L-Arginine did not revert the antibacterial activity. Also, high amounts of NO induced by adding L-Arginine to the cell cultures did not enhance hepatocyte antibacterial activity.

  11. The matricellular protein Cyr61 is a key mediator of platelet-derived growth factor-induced cell migration.

    PubMed

    Zhang, Fuqiang; Hao, Feng; An, Dong; Zeng, Linlin; Wang, Yi; Xu, Xuemin; Cui, Mei-Zhen

    2015-03-27

    Platelet-derived growth factor (PDGF), a potent chemoattractant, induces cell migration via the MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified the matricellular protein Cyr61, which is de novo synthesized in response to PDGF stimulation, as the key downstream mediator of the ERK and JNK pathways, independent of the p38 MAPK and AKT pathways, and, thereby, it mediates PDGF-induced smooth muscle cell migration but not proliferation. Our results revealed that, when Cyr61 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with the plasma membrane integrins α6β1 and αvβ3. We further demonstrate that Cyr61 and integrins are integral components of the PDGF signaling pathway via an "outside-in" signaling route to activate intracellular focal adhesion kinase (FAK), leading to cell migration. Therefore, this study provides the first evidence that the PDGF-induced endogenous extracellular matrix component Cyr61 is a key mediator in modulating cell migration by connecting intracellular PDGF-ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular Cyr61 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor-induced cell migration. The discovered signaling pathway may represent an important therapeutic target in growth factor-mediated cell migration/invasion-related vascular diseases and tumorigenesis.

  12. 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.

  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. 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

  15. 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

  16. 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

  17. 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.

  18. Activation of Epidermal Growth Factor Receptor in Macrophages Mediates Feedback Inhibition of M2 Polarization and Gastrointestinal Tumor Cell Growth.

    PubMed

    Zhao, Gang; Liu, Liping; Peek, Richard M; Hao, Xishan; Polk, D Brent; Li, Hui; Yan, Fang

    2016-09-23

    EGF receptor (EGFR) in tumor cells serves as a tumor promoter. However, information about EGFR activation in macrophages in regulating M2 polarization and tumor development is limited. This study aimed to investigate the effects of EGFR activation in macrophages on M2 polarization and development of gastrointestinal tumors. IL-4, a cytokine to elicit M2 polarization, stimulated release of an EGFR ligand, HB-EGF, and transactivation and down-regulation of EGFR in Raw 264.7 cells and peritoneal macrophages from WT mice. Knockdown of HB-EGF in macrophages inhibited EGFR transactivation by IL-4. IL-4-stimulated STAT6 activation, Arg1 and YM1 gene expression, and HB-EGF production were further enhanced by inhibition of EGFR activity in Raw 264.7 cells using an EGFR kinase inhibitor and in peritoneal macrophages from Egfr(wa5) mice with kinase inactive EGFR and by knockdown of EGFR in peritoneal macrophages from Egfr(fl/fl) LysM-Cre mice with myeloid cell-specific EGFR deletion. Chitin induced a higher level of M2 polarization in peritoneal macrophages in Egfr(fl/fl) LysM-Cre mice than that in Egfr(fl/fl) mice. Accordingly, IL-4-conditioned medium stimulated growth and epithelial-to-mesenchymal transition in gastric epithelial and colonic tumor cells, which were suppressed by that from Raw 264.7 cells with HB-EGF knockdown but promoted by that from Egfr(wa5) and Egfr(fl/fl) LysM-Cre peritoneal macrophages. Clinical assessment revealed that the number of macrophages with EGFR expression became less, indicating decreased inhibitory effects on M2 polarization, in late stage of human gastric cancers. Thus, IL-4-stimulated HB-EGF-dependent transactivation of EGFR in macrophages may mediate inhibitory feedback for M2 polarization and HB-EGF production, thereby inhibiting gastrointestinal tumor growth.

  19. Growth Hormone-Releasing Hormone and Its Analogues: Significance for MSCs-Mediated Angiogenesis

    PubMed Central

    Tao, Quanwei; Ma, Qunchao; Chen, Huiqiang; Wang, Jian'an

    2016-01-01

    Mesenchymal stromal cells (MSCs) are promising candidates for regenerative medicine because of their multipotency, immune-privilege, and paracrine properties including the potential to promote angiogenesis. Accumulating evidence suggests that the inherent properties of cytoprotection and tissue repair by native MSCs can be enhanced by various preconditioning stimuli implemented prior to cell transplantation. Growth hormone-releasing hormone (GHRH), a stimulator in extrahypothalamus systems including tumors, has attracted great attentions in recent years because GHRH and its agonists could promote angiogenesis in various tissues. GHRH and its agonists are proangiogenic in responsive tissues including tumors, and GHRH antagonists have been tested as antitumor agents through their ability to suppress angiogenesis and cell growth. GHRH-R is expressed by MSCs and evolving work from our laboratory indicates that treatment of MSCs with GHRH agonists prior to cell transplantation markedly enhanced the angiogenic potential and tissue reparative properties of MSCs through a STAT3 signaling pathway. In this review we summarized the possible effects of GHRH analogues on cell growth and development, as well as on the proangiogenic properties of MSCs. We also discussed the relationship between GHRH analogues and MSC-mediated angiogenesis. The analyses provide new insights into molecular pathways of MSCs-based therapies and their augmentation by GHRH analogues. PMID:27774107

  20. FCA mediates thermal adaptation of stem growth by attenuating auxin action in Arabidopsis.

    PubMed

    Lee, Hyo-Jun; Jung, Jae-Hoon; Cortés Llorca, Lucas; Kim, Sang-Gyu; Lee, Sangmin; Baldwin, Ian T; Park, Chung-Mo

    2014-11-17

    Global warming is predicted to profoundly affect plant distribution and crop yield in the near future. Higher ambient temperature can influence diverse aspects of plant growth and development. In Arabidopsis, the basic helix-loop-helix transcription factor Phytochrome-Interacting Factor 4 (PIF4) regulates temperature-induced adaptive responses by modulating auxin biosynthesis. At high temperature, PIF4 directly activates expression of YUCCA8 (YUC8), a gene encoding an auxin biosynthetic enzyme, resulting in auxin accumulation. Here we demonstrate that the RNA-binding protein FCA attenuates PIF4 activity by inducing its dissociation from the YUC8 promoter at high temperature. At 28 °C, auxin content is elevated in FCA-deficient mutants that exhibit elongated stems but reduced in FCA-overexpressing plants that exhibit reduced stem growth. We propose that the FCA-mediated regulation of YUC8 expression tunes down PIF4-induced architectural changes to achieve thermal adaptation of stem growth at high ambient temperature.

  1. 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

  2. Promotion of mitochondrial energy metabolism during hepatocyte apoptosis in a rat model of acute liver failure

    PubMed Central

    CHEN, LI-YAN; YANG, BAOSHAN; ZHOU, LI; REN, FENG; DUAN, ZHONG-PING; MA, YING-JI

    2015-01-01

    Hepatocyte apoptosis and energy metabolism in mitochondria have an important role in the mechanism of acute liver failure (ALF). However, data on the association between apoptosis and the energy metabolism of hepatocytes are lacking. The current study assessed the activity of several key enzymes in mitochondria during ALF, including citrate synthase (CS), carnitine palmitoyltransferase-1 (CPT-1) and cytochrome c oxidase (COX), which are involved in hepatocyte energy metabolism. A total of 40 male Sprague-Dawley rats were divided into five groups and administered D-galactosamine and lipopolysaccharide to induce ALF. Hepatic pathology and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling examinations indicated that hepatocyte apoptosis was observed at 4 h and increased 8 h after ALF. Hepatocyte necrosis appeared at 12 h and was significantly higher at 24 h with inflammatory cell invasion. The results measured by electron microscopy indicated that ultrastructural changes in mitochondria began at 4 h and the mitochondrial outer membrane was completely disrupted at 24 h resulting in mitochondrial collapse. The expression of CS, CPT-1 and COX was measured and analyzed using assay kits. The activity and protein expression of CS, CPT-1 and COX began to increase at 4 h, reached a peak at 8 h and decreased at 12 h during ALF. The activities of CS, CPT-1 and COX were enhanced during hepatocyte apoptosis suggesting that these enzymes are involved in the initiation and development of ALF. Therefore, these results demonstrated that energy metabolism is important in hepatocyte apoptosis during ALF and hepatocyte apoptosis is an active and energy-consuming procedure. The current study on how hepatocyte energy metabolism affects the transmission of death signals may provide a basis for the early diagnosis and development of an improved therapeutic strategy for ALF. PMID:26135512

  3. Effect of albumin on net copper accumulation by fibroblasts and hepatocytes

    SciTech Connect

    Waldrop, G.L.; Palida, F.A.; Hadi, M.; Lonergan, P.A.; Ettinger, M.J. )

    1990-08-01

    The liver accumulates copper rapidly and preferentially from plasma. The effects of albumin on net copper accumulation by fibroblasts and hepatocytes were compared to determine whether preferential uptake involves hepatocyte-specific sequestering of copper. Although albumin inhibits the initial rates (30 s) of copper transport by fibroblasts and hepatocytes similarly, the effects of albumin on net copper accumulation (4 h) by these cell types were strikingly different. Fibroblasts accumulate only approximately 15% as much copper when equimolar albumin is present as from albumin-free media; hepatocytes accumulate about the same amount of copper with or without extracellular albumin present. Copper efflux data show that the special capacity of hepatocytes to accumulate copper in the presence of extracellular albumin is due to greater copper retention by hepatocytes than fibroblasts. The ability of hepatocytes to accumulate copper does not seem to be due to albumin-receptor-mediated uptake, since albumin was not co-transported with copper. The data are consistent with an equilibrium model of copper accumulation in which intracellular and extracellular copper are in equilibrium with intracellular and extracellular ligands. A high-affinity, copper-binding fraction that was previously identified in cytosols from hepatocytes was low or absent in fibroblasts. This may contain a liver-specific protein(s) that helps hepatocytes sequester and retain copper from albumin or serum-containing media. Irrespective of the exact species involved, the data are consistent with rapid, preferential copper uptake by the liver being due in part to a liver-specific, intracellular copper-binding protein(s) with a high binding affinity for copper.

  4. Functional expression and regulation of drug transporters in monolayer- and sandwich-cultured mouse hepatocytes.

    PubMed

    Noel, Gregory; Le Vee, Marc; Moreau, Amélie; Stieger, Bruno; Parmentier, Yannick; Fardel, Olivier

    2013-04-11

    Primary hepatocyte cultures are now considered as convenient models for in vitro analyzing liver drug transport. However, if primary human and rat hepatocytes have been well-characterized with respect to drug transporter expression and regulation, much less is known for primary mouse hepatocytes. The present study was therefore designed to gain insights about this point. The profile of sinusoidal and canalicular drug transporter mRNA expression in short time (4h)-cultured mouse hepatocytes was found to be highly correlated with that of freshly isolated hepatocytes; by contrast, those of counterparts cultured for a longer time (until 4 days) either in monolayer configurations on plastic or collagen or in sandwich configuration with matrigel were profoundly altered: uptake drug transporters such as Oct1, Oatps and Oat2 were thus down-regulated, whereas most of efflux transporters such as Mdr1a/b, Mrp3, Mrp4 and Bcrp were induced. Moreover, short time-cultured hepatocytes exhibited the highest levels of sinusoidal influx transporter activities. Transporter-mediated drug secretion into canalicular networks was however only observed in sandwich-cultured hepatocytes. Mouse hepatocytes cultured either in monolayer or sandwich configurations were finally shown to exhibit up-regulation of referent transporters in response to exposure to prototypical activators of the drug sensing receptors pregnane X receptor, aryl hydrocarbon receptor or constitutive androstane receptor. Taken together, these data demonstrate the feasibility of using primary mouse hepatocytes for investigating potential interactions of xenobiotics with hepatic transporter activity or regulation, provided that adequate culture conditions are retained. PMID:23396053

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. P2y receptor-mediated angiogenesis via vascular endothelial growth factor receptor 2 signaling.

    PubMed

    Rumjahn, Sharif M; Baldwin, Karla A; Buxton, Iain L O

    2007-01-01

    Pathological as well as physiological angiogenesis is known to be regulated by such factors as nucleotides and Vascular Endothelial Growth Factor (VEGF). Activated P2Y nucleotide receptors have been observed to associate and transactivate VEGF Receptor 2 (VEGFR2), suggesting a cooperation between nucleotide and VEGF signaling in angiogenesis. P2YR mediated VEGFR2 signaling therefore may be important in describing the angiogenic signaling of nucleotides such as ATP. Here, we provide evidence that supports the notion of P2YR-VEGFR2 signaling. The significant angiogenic effect of P2Y1/2 receptor agonists (100 microM ATP and 10 microM 2MS-ATP) on endothelial cell tubulogenesis was suppressed back to near control levels upon addition of 1 microM SU1498 (specific VEGFR2 tyrosine kinase inhibitor). We believe that this P2YR-VEFGR2 signaling is an important component of pathological, as well as physiological angiogenesis.

  10. 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

  11. 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

  12. 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

  13. Epidermal growth factor mediated healing in stem cell-derived vocal fold mucosa

    PubMed Central

    Palencia, Liliana; Das, Amritava; Palecek, Sean P; Thibeault, Susan; Leydon, Ciara

    2015-01-01

    Background The goal of vocal fold wound healing is the reconstitution of functional tissue, including a structurally and functionally intact epithelium. Mechanisms underlying reepithelialization in vocal folds are not known, although it is suspected that healing involves the interplay between several growth factors. We used a three-dimensional human embryonic stem cell-derived model of vocal fold mucosa to examine the effects of one growth factor, exogenous epidermal growth factor (EGF), on wound healing. Materials and methods A scratch wound was created in the in vitro model. Rate of wound healing, epidermal growth factor receptor (EGFR) activation, and cell proliferation post-injury were analyzed with and without application of both exogenous EGF and an EGFR inhibitor, Gefitinib. Results Wound repair after injury was significantly hastened by application of exogenous EGF (13.3 µm/hour, ±2.63) compared to absence of exogenous EGF (7.1 µm/hour ±2.84), but inhibited with concurrent addition of Gefitinib (5.2 µm/hour, ±2.23), indicating that EGF mediates wound healing in an EGFR-dependent manner. Immunohistochemistry revealed that EGFR activation occurred only in the presence of exogenous EGF. While not statistically significant, increased density of Ki67 staining in epithelium adjacent to the scratch wound was observed following treatment with EGF, suggesting a tendency for exogenous EGF to increase epithelial cell proliferation. Conclusions Exogenous EGF increases the rate of wound healing in an EGFR-dependent manner in a three-dimensional stem cell-derived model of vocal fold mucosa. This model of wound healing can be used to gain insight into the mechanisms that regulate vocal fold epithelial repair following injury. PMID:25818979

  14. EHD1 mediates vesicle trafficking required for normal muscle growth and transverse tubule development.

    PubMed

    Posey, Avery D; Swanson, Kaitlin E; Alvarez, Manuel G; Krishnan, Swathi; Earley, Judy U; Band, Hamid; Pytel, Peter; McNally, Elizabeth M; Demonbreun, Alexis R

    2014-03-15

    EHD proteins have been implicated in intracellular trafficking, especially endocytic recycling, where they mediate receptor and lipid recycling back to the plasma membrane. Additionally, EHDs help regulate cytoskeletal reorganization and induce tubule formation. It was previously shown that EHD proteins bind directly to the C2 domains in myoferlin, a protein that regulates myoblast fusion. Loss of myoferlin impairs normal myoblast fusion leading to smaller muscles in vivo but the intracellular pathways perturbed by loss of myoferlin function are not well known. We now characterized muscle development in EHD1-null mice. EHD1-null myoblasts display defective receptor recycling and mislocalization of key muscle proteins, including caveolin-3 and Fer1L5, a related ferlin protein homologous to myoferlin. Additionally, EHD1-null myoblast fusion is reduced. We found that loss of EHD1 leads to smaller muscles and myofibers in vivo. In wildtype skeletal muscle EHD1 localizes to the transverse tubule (T-tubule), and loss of EHD1 results in overgrowth of T-tubules with excess vesicle accumulation in skeletal muscle. We provide evidence that tubule formation in myoblasts relies on a functional EHD1 ATPase domain. Moreover, we extended our studies to show EHD1 regulates BIN1 induced tubule formation. These data, taken together and with the known interaction between EHD and ferlin proteins, suggests that the EHD proteins coordinate growth and development likely through mediating vesicle recycling and the ability to reorganize the cytoskeleton.

  15. EHD1 mediates vesicle trafficking required for normal muscle growth and tubule development

    PubMed Central

    Posey, Avery D.; Swanson, Kaitlin E.; Alvarez, Manuel G.; Krishnan, Swathi; Earley, Judy E.; Band, Hamid; Pytel, Peter; McNally, Elizabeth M.; Demonbreun, Alexis R.

    2014-01-01

    EHD proteins have been implicated in intracellular trafficking, especially endocytic recycling, where they mediate receptor and lipid recycling back to the plasma membrane. Additionally, EHDs help regulate cytoskeletal reorganization and induce tubule formation. It was previously shown that EHD proteins bind directly to the C2 domains in myoferlin, a protein that regulates myoblast fusion. Loss of myoferlin impairs normal myoblast fusion leading to smaller muscles in vivo but the intracellular pathways perturbed by loss of myoferlin function are not well known. We now characterized muscle development in EHD1-null mice. EHD1-null myoblasts display defective receptor recycling and mislocalization of key muscle proteins, including caveolin-3 and Fer1L5, a related ferlin protein homologous to myoferlin. Additionally, EHD1-null myoblast fusion is reduced. We found that loss of EHD1 leads to smaller muscles and myofibers in vivo. In wildtype skeletal muscle EHD1 localizes to the transverse tubule (T-tubule), and loss of EHD1 results in overgrowth of T-tubules with excess vesicle accumulation in skeletal muscle. We provide evidence that tubule formation in myoblasts relies on a functional EHD1 ATPase domain. Moreover, we extended our studies to show EHD1 regulates BIN1 induced tubule formation. These data, taken together and with the known interaction between EHD and ferlin proteins, suggests that the EHD proteins coordinate growth and development likely through mediating vesicle recycling and the ability to reorganize the cytoskeleton. PMID:24440153

  16. Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

    PubMed

    Roth, Lise; Prahst, Claudia; Ruckdeschel, Tina; Savant, Soniya; Weström, Simone; Fantin, Alessandro; Riedel, Maria; Héroult, Mélanie; Ruhrberg, Christiana; Augustin, Hellmut G

    2016-04-26

    Neuropilin-1 (NRP1) regulates developmental and pathological angiogenesis, arteriogenesis, and vascular permeability, acting as a coreceptor for semaphorin 3A (Sema3A) and the 165-amino acid isoform of vascular endothelial growth factor A (VEGF-A165). NRP1 is also the receptor for the CendR peptides, a class of cell- and tissue-penetrating peptides with a specific R-x-x-R carboxyl-terminal motif. Because the cytoplasmic domain of NRP1 lacks catalytic activity, NRP1 is mainly thought to act through the recruitment and binding to other receptors. We report here that the NRP1 intracellular domain mediates vascular permeability. Stimulation with VEGF-A165, a ligand-blocking antibody, and a CendR peptide led to NRP1 accumulation at cell-cell contacts in endothelial cell monolayers, increased cellular permeability in vitro and vascular leakage in vivo. Biochemical analyses, VEGF receptor-2 (VEGFR-2) silencing, and the use of a specific VEGFR blocker established that the effects induced by the CendR peptide and the antibody were independent of VEGFR-2. Moreover, leakage assays in mice expressing a mutant NRP1 lacking the cytoplasmic domain revealed that this domain was required for NRP1-induced vascular permeability in vivo. Hence, these data define a vascular permeability pathway mediated by NRP1 but independent of VEGFR-2 activation.

  17. 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.

  18. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation.

    PubMed

    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.

  19. The growth and reproduction performance of TALEN-mediated β-lactoglobulin-knockout bucks.

    PubMed

    Ge, Hengtao; Cui, Chenchen; Liu, Jun; Luo, Yan; Quan, Fusheng; Jin, Yaping; Zhang, Yong

    2016-10-01

    With the technological development of several engineered endonucleases (EENs), such as zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9, gene targeting by homologous recombination has been efficiently improved to generate site-specifically genetically modified livestock. However, few studies have been done to investigate the health and fertility of these animals. The purpose of the present study is to investigate if gene targeting events and a recloning procedure would affect the production traits of EEN-mediated gene targeted bucks. TALEN-mediated β-lactoglobulin (BLG) gene mono-allelic knockout (BLG (+/-)) goats and bi-allelic knockout (BLG (-/-)) buck produced by using sequential gene targeting combined with recloning in fibroblasts from BLG (+/-) buck were used to evaluate their health and fertility. Birth weight and postnatal growth of BLG (+/-) bucks were similar to the wild-type goats. None of the parameters for both fresh and frozen-thawed semen quality were significantly different in BLG (+/-) or BLG (-/-) bucks compared to their corresponding comparators. In vitro fertilization (IVF) test revealed that the proportion of IVF oocytes developing to the blastocyst stage was identical among BLG (+/-), BLG (-/-) and wild-type bucks. Conception rates of artificial insemination were respectively 42.3, 38.0 and 42.6 % for frozen-thawed semen from the BLG (+/-), BLG (-/-) and wild-type bucks. In addition, germline transmission of the targeted BLG modification was in accordance with Mendelian rules. These results demonstrated that the analyzed growth and reproductive traits were not impacted by targeting BLG gene and recloning, implicating the potential for dairy goat breeding of BLG (+/-) and BLG (-/-) bucks.

  20. The growth and reproduction performance of TALEN-mediated β-lactoglobulin-knockout bucks.

    PubMed

    Ge, Hengtao; Cui, Chenchen; Liu, Jun; Luo, Yan; Quan, Fusheng; Jin, Yaping; Zhang, Yong

    2016-10-01

    With the technological development of several engineered endonucleases (EENs), such as zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9, gene targeting by homologous recombination has been efficiently improved to generate site-specifically genetically modified livestock. However, few studies have been done to investigate the health and fertility of these animals. The purpose of the present study is to investigate if gene targeting events and a recloning procedure would affect the production traits of EEN-mediated gene targeted bucks. TALEN-mediated β-lactoglobulin (BLG) gene mono-allelic knockout (BLG (+/-)) goats and bi-allelic knockout (BLG (-/-)) buck produced by using sequential gene targeting combined with recloning in fibroblasts from BLG (+/-) buck were used to evaluate their health and fertility. Birth weight and postnatal growth of BLG (+/-) bucks were similar to the wild-type goats. None of the parameters for both fresh and frozen-thawed semen quality were significantly different in BLG (+/-) or BLG (-/-) bucks compared to their corresponding comparators. In vitro fertilization (IVF) test revealed that the proportion of IVF oocytes developing to the blastocyst stage was identical among BLG (+/-), BLG (-/-) and wild-type bucks. Conception rates of artificial insemination were respectively 42.3, 38.0 and 42.6 % for frozen-thawed semen from the BLG (+/-), BLG (-/-) and wild-type bucks. In addition, germline transmission of the targeted BLG modification was in accordance with Mendelian rules. These results demonstrated that the analyzed growth and reproductive traits were not impacted by targeting BLG gene and recloning, implicating the potential for dairy goat breeding of BLG (+/-) and BLG (-/-) bucks. PMID:27272006

  1. Effect of coenzyme Q10 on proteomic profile of blood plasma and cytosolic and microsomal fractions of rat hepatocytes during ontogeny.

    PubMed

    Sharanova, N E; Toropygin, I Yu; Khriapova, E V; Vasilyev, A V; Gapparov, M M G

    2012-11-01

    The proteomic features of blood plasma and subcellular fractions of rat hepatocytes were studied during long-term dietary consumption of coenzyme Q10 as the endogenous mediator of antioxidant and energy homeostasis in the cell. Long-term coenzyme Q10 consumption was followed by the formation of specific nutriproteomes of the microsomal and cytosolic fractions of rat hepatocytes.

  2. 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

  3. 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

  4. 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

  5. The effect of a brief sprint interval exercise on growth factors and inflammatory mediators.

    PubMed

    Meckel, Yoav; Eliakim, Alon; Seraev, Mariana; Zaldivar, Frank; Cooper, Dan M; Sagiv, Michael; Nemet, Dan

    2009-01-01

    Exercise training efficiency depends on the intensity, volume, duration, and frequency of training, as well as on the athlete's ability to tolerate it. Recent efforts to quantify the effects of aerobic exercise training on hormonal response have suggested that exercise leads to simultaneous changes of antagonistic mediators. The effects of anaerobic exercise on these mediators are not known. Therefore, the aim of the present study was to evaluate the effects of a brief sprint interval session on the balance between anabolic (growth hormone [GH]--> insulin-like growth factor [IGF]-I axis) and catabolic hormones (cortisol), and circulating inflammatory cytokines such as interleukin (IL)-6. Twelve healthy elite junior handball players (17-20 years) participated in the study. Exercise consisted of a 4 x 250-m run on a treadmill, at a constant intensity of 80% of the personal maximal speed. Each run was separated by 3 minutes of rest. Blood samples were collected before, immediately after each 250-m run, and 1 hour after the last run. Exercise led to significant increases in GH (0.3 +/- 0.2 to 5.1 +/- 2.2 ngxml, p < 0.05), IGF binding protein (IGFBP)-3 (4191 +/- 2.48 to 4875 +/- 301 ngxml, p < 0.05), IL-6 (1.3 +/- 0.2 to 2.1 +/- 0.3 pgxml, p < 0.002), testosterone, and testosterone/cortisol ratio, and to a significant decrease in IGFBP-1 levels. Levels of IL-6 remained elevated 1 hour after the end of exercise. Exercise had no significant effects on IGF-I and cortisol levels. Changes in the GH-IGF-I axis and testosterone/cortisol ratio after the brief sprint interval exercise suggested exercise-related anabolic adaptations. The increase in IL-6 may indicate its important role in muscle tissue repair after anaerobic exercise. Changes in the anabolic-catabolic hormonal balance and in inflammatory mediators can be used as an objective tool to gauge the training intensity of different types of anaerobic exercises and training periods.

  6. 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}.

  7. Mitochondrial dysfunction mediated by cytoplasmic acidification results in pollen tube growth cessation in Pyrus pyrifolia.

    PubMed

    Gao, Yongbin; Zhou, Hongsheng; Chen, Jianqing; Jiang, Xueting; Tao, Shutian; Wu, Juyou; Zhang, Shaoling

    2015-04-01

    The length of pollen tubes grown in synthetic media is normally shorter than those grown in vivo. However, the mechanism(s) underlying the cessation of pollen tube growth under culture conditions remain(s) largely unknown. Here, we report a previously unknown correlation between vacuolar function and the cell's ability to sustain mitochondrial functions in pear pollen tubes. The pear pollen tubes in vitro grew slowly after 15 hours post-cultured (HPC) and nearly ceased growth at 18 HPC. There was increased malondialdehyde content and membrane ion leakage at 15 HPC compared with 12 HPC. Furthermore, cytoplasmic acidification mainly mediated by decreased vacuolar H(+)-ATPase [V-ATPase, Enzyme Commission (EC) 3.6.1.3] activity was observed in pollen tubes after 15 HPC, and this further resulted in mitochondrial dysfunction, including mitochondrial structure disruption, mitochondrial membrane potential collapse and decreases in both oxygen consumption and ATP production. Our findings suggest that vacuoles and mitochondria intimately linked in regulating pollen tube elongation.

  8. Maturational conversion of dendritic early endosomes and their roles in L1-mediated axon growth.

    PubMed

    Lasiecka, Zofia M; Yap, Chan Choo; Katz, Joshua; Winckler, Bettina

    2014-10-29

    The function of endosomes is intricately linked to cellular function in all cell types, including neurons. Intriguingly, neurons express cell type-specific proteins that localize to endosomes, but little is known about how these neuronal proteins interface with canonical endosomes and ubiquitously expressed endosomal components, such as EEA1 (Early Endosomal Antigen 1). NEEP21 (Neuronal Early Endosomal Protein 21 kDa) localizes to somatodendritic endosomes, and downregulation of NEEP21 perturbs the correct trafficking of multiple receptors, including glutamate receptors (GluA2) during LTP and amyloidogenic processing of βAPP. Our own work implicated NEEP21 in correct trafficking of the axonal cell adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM). NEEP21 dynamically localizes with EEA1-positive early endosomes but is also found in EEA1-negative endosomes. Live imaging reveals that NEEP21-positive, EEA1-negative endosomes arise as a consequence of maturational conversion of EEA1/NEEP21 double-positive endosomes. Interfering with EEA1 function causes missorting of L1/NgCAM, axon outgrowth defects on the L1 substrate, and disturbance of NEEP21 localization. Last, we uncover evidence that functional interference with NEEP21 reduces axon and dendrite growth of primary rat hippocampal neurons on L1 substrate but not on N-cadherin substrate, thus implicating endosomal trafficking through somatodendritic early endosomes in L1-mediated axon growth. PMID:25355216

  9. 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

  10. Medial gastrocnemius muscle growth during adolescence is mediated by increased fascicle diameter rather than by longitudinal fascicle growth.

    PubMed

    Weide, Guido; Huijing, Peter A; Maas, Josina C; Becher, Jules G; Harlaar, Jaap; Jaspers, Richard T

    2015-06-01

    Using a cross-sectional design, the purpose of this study was to determine how pennate gastrocnemius medialis (GM) muscle geometry changes as a function of adolescent age. Sixteen healthy adolescent males (aged 10-19 years) participated in this study. GM muscle geometry was measured within the mid-longitudinal plane obtained from a 3D voxel-array composed of transverse ultrasound images. Images were taken at footplate angles corresponding to standardised externally applied footplate moments (between 4 Nm plantar flexion and 6 Nm dorsal flexion). Muscle activity was recorded using surface electromyography (EMG), expressed as a percentage of maximal voluntary contraction (%MVC). To minimise the effects of muscle excitation, EMG inclusion criteria were set at <10% of MVC. In practice, however, normalised EMG levels were much lower. For adolescent subjects with increasing ages, GM muscle (belly) length increased due to an increase in the length component of the physiological cross-sectional area measured within the mid-longitudinal plane. No difference was found between fascicles at different ages, but the aponeurosis length and pennation angle increased by 0.5 cm year(-1) and 0.5° per year, respectively. Footplate angles corresponding to externally applied 0 and 4 Nm plantarflexion moments were not associated with different adolescent ages. In contrast, footplate angles corresponding to externally applied 4 and 6 Nm dorsal flexion moments decreased by 10° between 10 and 19 years. In conclusion, we found that in adolescents' pennate GM muscles, longitudinal muscle growth is mediated predominantly by increased muscle fascicle diameter.

  11. Active ERK2 is sufficient to mediate growth arrest and differentiation signaling

    PubMed Central

    Wu, Pui-Kei; Hong, Seung-Keun; Yoon, Seung-Hee; Park, Jong-In

    2015-01-01

    Although extracellular signal-regulated kinase (ERK) ½ has been shown for its necessity for a variety of the Raf/MEK/ERK pathway signaling, its sufficiency in mediating the pathway signaling has not been firmly established. In an effort to address this, we evaluated previously reported ERK2 mutants that exhibit enhanced activity of autophosphorylation of TEY sites in the activation loop for their ability to induce growth arrest and differentiation in LNCaP and PC12 cells. Here, we demonstrate that expression of ERK2-L73P/S151D, containing Lys73Pro and Ser151Asp replacements that synergistically promote ERK autophosphorylation, is sufficient to induce growth arrest and differentiation whereas ERK2-I84A and ERK2-R65S/D319N are not as effective. When compared to the constitutively active MEK1-ΔN3/S218E/S222D, expression of ERK2-L73P/S151D could only mildly increase ERK kinase activity in cells, as determined by the ERK substrates, p90RSK and ELK1. Nevertheless, ERK2-L73P/S151D expression effectively induced downregulation of androgen receptor, Rb and E2F1, and upregulation of p16INK4A and p21CIP1, which were accompanied by cell cycle arrest and morphological differentiation, in LNCaP cells and neurite-like processing in PC12 cells. These effects and TEY site phosphorylation of ERK2-L73P/S151D were abrogated upon introducing the active site-disabling Lys52Arg mutation, confirming its sufficiency in this signaling. Moreover, introduction of the mutations (producing Asp316/319Ala or Asp319Asn) that impair the common docking site/D-domain-based physical interaction of ERK did not significantly affect the ERK2-L73P/S151D signaling, suggesting that ERK2 can mediate growth arrest and differentiation independently of the conventional ERK-target interaction mechanism. Our study presents a convincing example of ERK sufficiency for Raf/MEK/ERK signaling. PMID:25639353

  12. Hypoxia-mediated carbohydrate metabolism and transport promote early-stage murine follicle growth and survival.

    PubMed

    Makanji, Yogeshwar; Tagler, David; Pahnke, Jennifer; Shea, Lonnie D; Woodruff, Teresa K

    2014-04-15

    Oxygen tension is critical for follicle growth and metabolism, especially for early-stage follicles, where vascularity is limited. Its role and underlying mechanism in the in vitro activation and maturation of immature to ovulatory follicles is largely unknown. In this study, early secondary (110 μm) murine follicles were isolated and encapsulated in alginate hydrogels to replicate the in vivo environment of the growing/maturing follicle. Encapsulated follicles were cultured for 8 days at either 2.5 or 20% O2. Survival (2.6-fold) and growth (1.2-fold) were significantly higher for follicles cultured at 2.5% compared with 20% O2. Using a mouse hypoxia-signaling pathway qRT-PCR array and GeneGo Metacore analysis, we found that direct target genes of the hypoxia-activated HIF1-complex were significantly upregulated in follicles cultured for 8 days at 2.5% compared with 20% O2, including the carbohydrate transport and metabolism genes Slc2a3, Vegfa, Slc2a1, Edn1, Pgk1, Ldha, and Hmox1. Other upregulated genes included carbohydrate transporters (Slc2a1, Slc2a3, and Slc16a3) and enzymes essential for glycolysis (Pgk1, Hmox1, Hk2, Gpi1, Pfkl, Pfkp, Aldoa, Gapdh, Pgam1, Eno1, Pkm2, and Ldha). For follicles cultured at 2.5% O2, a 7.2-fold upregulation of Vegfa correlated to an 18-fold increase in VEGFA levels, and a 3.2-fold upregulation of Ldha correlated to a 4.8-fold increase in lactate levels. Both VEGFA and lactate levels were significantly higher in follicles cultured at 2.5% compared with 20% O2. Therefore, enhanced hypoxia-mediated glycolysis is essential for growth and survival of early secondary follicles and provides vital insights into improving in vitro culture conditions.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. YAP inhibition restores hepatocyte differentiation in advanced HCC leading to tumor regression

    PubMed Central

    Fitamant, Julien; Kottakis, Filippos; Benhamouche, Samira; Tian, Helen S.; Chuvin, Nicolas; Parachoniak, Christine A.; Nagle, Julia M.; Perera, Rushika M.; Lapouge, Marjorie; Deshpande, Vikram; Zhu, Andrew X.; Lai, Albert; Min, Bosun; Hoshida, Yujin; Avruch, Joseph; Sia, Daniela; Campreciós, Genís; McClatchey, Andrea I.; Llovet, Josep M.; Morrissey, David; Raj, Lakshmi; Bardeesy, Nabeel

    2015-01-01

    SUMMARY Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC). Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat maintaining metabolic specialization, differentiation and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs controlling hepatocyte quiescence and differentiation. Importantly, treatment with siRNA-lipid nanoparticles (siRNA-LNPs) targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of positional identity of hepatocytes, supports targeting YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype potentially responsive to this approach. PMID:25772357

  18. 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

  19. 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 in