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Sample records for mediates hepatocyte growth

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

    PubMed Central

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

    2016-01-01

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

  2. 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.

  3. Hepatocyte growth factor mediates mesenchymal stem cell–induced recovery in multiple sclerosis models.

    PubMed

    Bai, Lianhua; Lennon, Donald P; Caplan, Arnold I; DeChant, Anne; Hecker, Jordan; Kranso, Janet; Zaremba, Anita; Miller, Robert H

    2012-06-01

    Mesenchymal stem cells (MSCs) have emerged as a potential therapy for a range of neural insults. In animal models of multiple sclerosis, an autoimmune disease that targets oligodendrocytes and myelin, treatment with human MSCs results in functional improvement that reflects both modulation of the immune response and myelin repair. Here we demonstrate that conditioned medium from human MSCs (MSC-CM) reduces functional deficits in mouse MOG35–55-induced experimental autoimmune encephalomyelitis (EAE) and promotes the development of oligodendrocytes and neurons. Functional assays identified hepatocyte growth factor (HGF) and its primary receptor cMet as critical in MSC-stimulated recovery in EAE, neural cell development and remyelination. Active MSC-CM contained HGF, and exogenously supplied HGF promoted recovery in EAE, whereas cMet and antibodies to HGF blocked the functional recovery mediated by HGF and MSC-CM. Systemic treatment with HGF markedly accelerated remyelination in lysolecithin-induced rat dorsal spinal cord lesions and in slice cultures. Together these data strongly implicate HGF in mediating MSC-stimulated functional recovery in animal models of multiple sclerosis.

  4. Ezrin Is an Effector of Hepatocyte Growth Factor–mediated Migration and Morphogenesis in Epithelial Cells

    PubMed Central

    Crepaldi, Tiziana; Gautreau, Alexis; Comoglio, Paolo M.; Louvard, Daniel; Arpin, Monique

    1997-01-01

    The dissociation, migration, and remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) entail modifications in cell adhesion and in the actin cytoskeleton through unknown mechanisms. Here we report that ezrin, a membrane–cytoskeleton linker, is crucial to HGF-mediated morphogenesis in a polarized kidney-derived epithelial cell line, LLC-PK1. Ezrin is a substrate for the tyrosine kinase HGF receptor both in vitro and in vivo. HGF stimulation causes enrichment of ezrin recovered in the detergent-insoluble cytoskeleton fraction. Overproduction of wild-type ezrin, by stable transfection in LLC-PK1 cells, enhances cell migration and tubulogenesis induced by HGF stimulation. Overproduction of a truncated variant of ezrin causes mislocalization of endogenous ezrin from microvilli into lateral surfaces. This is concomitant with altered cell shape, characterized by loss of microvilli and cell flattening. Moreover, the truncated variant of ezrin impairs the morphogenic and motogenic response to HGF, thus suggesting a dominant-negative mechanism of action. Site-directed mutagenesis of ezrin codons Y145 and Y353 to phenylalanine does not affect the localization of ezrin at microvilli, but perturbs the motogenic and morphogenic responses to HGF. These results provide evidence that ezrin displays activities that can control cell shape and signaling. PMID:9230083

  5. Hypoxia simultaneously alters satellite cell-mediated angiogenesis and hepatocyte growth factor expression.

    PubMed

    Flann, K L; Rathbone, C R; Cole, L C; Liu, X; Allen, R E; Rhoads, R P

    2014-05-01

    Skeletal muscle regeneration is a multifaceted process requiring the spatial and temporal coordination of myogenesis as well as angiogenesis. Hepatocyte growth factor (HGF) plays a pivotal role in myogenesis by activating satellite cells (SC) in regenerating muscle and likely plays a role as a contributor to revascularization. Moreover, repair of a functional blood supply is critical to ameliorate tissue ischemia and restore skeletal muscle function, however effects of hypoxia on satellite cell-mediated angiogenesis remain unclear. The objective of this study was to examine the role of HGF and effect of hypoxia on the capacity of satellite cells to promote angiogenesis. To characterize the role of HGF, a microvascular fragment (MVF) culture model coupled with satellite cell conditioned media (CM) was employed. The activity of HGF was specifically blocked in SC CM reducing sprout length compared to control CM. In contrast, MVF sprout number did not differ between control or HGF-deficient SC CM media. Next, we cultured MVF in the presence of CM from satellite cells exposed to normoxic (20% O2 ) or hypoxic (1% O2 ) conditions. Hypoxic CM recapitulated a MVF angiogenic response identical to HGF deficient satellite cell CM. Hypoxic conditions increased satellite cell HIF-1α protein abundance and VEGF mRNA abundance but decreased HGF mRNA abundance compared to normoxic satellite cells. Consistent with reduced HGF gene expression, HGF promoter activity decreased during hypoxia. Taken together, this data indicates that hypoxic modulation of satellite cell-mediated angiogenesis involves a reduction in satellite cell HGF expression.

  6. 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.

  7. 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

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

    PubMed

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

    2016-06-01

    Impaired vascular endothelial growth factor (VEGF) signaling contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). We hypothesized that the effects of VEGF on lung structure during development may be mediated through its downstream effects on both endothelial nitric oxide synthase (eNOS) and hepatocyte growth factor (HGF) activity, and that, in the absence of eNOS, trophic effects of VEGF would be mediated through HGF signaling. To test this hypothesis, we performed an integrative series of in vitro (fetal rat lung explants and isolated fetal alveolar and endothelial cells) and in vivo studies with normal rat pups and eNOS(-/-) mice. Compared with controls, fetal lung explants from eNOS(-/-) mice had decreased terminal lung bud formation, which was restored with recombinant human VEGF (rhVEGF) treatment. Neonatal eNOS(-/-) mice were more susceptible to hyperoxia-induced inhibition of lung growth than controls, which was prevented with rhVEGF treatment. Fetal alveolar type II (AT2) cell proliferation was increased with rhVEGF treatment only with mesenchymal cell (MC) coculture, and these effects were attenuated with anti-HGF antibody treatment. Unlike VEGF, HGF directly stimulated isolated AT2 cells even without MC coculture. HGF directly stimulates fetal pulmonary artery endothelial cell growth and tube formation, which is attenuated by treatment with JNJ-38877605, a c-Met inhibitor. rHGF treatment preserves alveolar and vascular growth after postnatal exposure to SU-5416, a VEGF receptor inhibitor. We conclude that the effects of VEGF on AT2 and endothelial cells during lung development are partly mediated through HGF-c-Met signaling and speculate that reciprocal VEGF-HGF signaling between epithelia and endothelia is disrupted in infants who develop BPD. Copyright © 2016 the American Physiological Society.

  9. 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.

  10. Ganglioside GM3 exerts opposite effects on motility via epidermal growth factor receptor and hepatocyte growth factor receptor-mediated migration signaling.

    PubMed

    Li, Ying; Huang, Xiaohua; Wang, Congcong; Li, Yuzhong; Luan, Mingchun; Ma, Keli

    2015-04-01

    The ganglioside GM3 exerts its different effects via various growth factor receptors. The present study investigated and comparatively analyzed the opposing effects exerted by GM3 on the migration of mouse hepatocellular carcinoma Hepa1‑6 cells via epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR/cMet). The results demonstrated that GM3 inhibited EGF‑stimulated motility, but promoted HGF‑stimulated motility of the Hepa1‑6 cells via phosphatidylinositol 3‑kinase/Akt‑mediated migration signaling. It is well established that the main cytokines modulating cell proliferation, invasion and metastasis are different in different types of tumor. This difference may, at least in part, explain why GM3 exerted its actions in a tumor‑type specific manner.

  11. Hepatocyte Growth Factor Mediates Enhanced Wound Healing Responses and Resistance to Transforming Growth Factor-β₁-Driven Myofibroblast Differentiation in Oral Mucosal Fibroblasts.

    PubMed

    Dally, Jordanna; Khan, Jabur S; Voisey, Alex; Charalambous, Chrisandrea; John, Hannah L; Woods, Emma L; Steadman, Robert; Moseley, Ryan; Midgley, Adam C

    2017-08-24

    Oral mucosal wounds are characterized by rapid healing with minimal scarring, partly attributable to the "enhanced" wound healing properties of oral mucosal fibroblasts (OMFs). Hepatocyte growth factor (HGF) is a pleiotropic growth factor, with potential key roles in accelerating healing and preventing fibrosis. HGF can exist as full-length or truncated (HGF-NK), NK1 and NK2 isoforms. As OMFs display elevated HGF expression compared to dermal fibroblasts (DFs), this study investigated the extent to which HGF mediates the preferential cellular functions of OMFs, and the influence of pro-fibrotic, transforming growth factor-β₁ (TGF-β₁) on these responses. Knockdown of HGF expression in OMFs by short-interfering RNA (siHGF) significantly inhibited OMF proliferative and migratory responses. Supplementation with exogenous TGF-β₁ also significantly inhibited proliferation and migration, concomitant with significantly down-regulated HGF expression. In addition, knockdown abrogated OMF resistance to TGF-β₁-driven myofibroblast differentiation, as evidenced by increased α-smooth muscle actin (α-SMA) expression, F-actin reorganisation, and stress fibre formation. Responses were unaffected in siHGF-transfected DFs. OMFs expressed significantly higher full-length HGF and NK1 levels compared to patient-matched DFs, whilst NK2 expression was similar in both OMFs and DFs. Furthermore, NK2 was preferentially expressed over NK1 in DFs. TGF-β₁ supplementation significantly down-regulated full-length HGF and NK1 expression by OMFs, while NK2 was less affected. This study demonstrates the importance of HGF in mediating "enhanced" OMF cellular function. We also propose that full-length HGF and HGF-NK1 convey desirable wound healing properties, whilst fibroblasts preferentially expressing more HGF-NK2 readily undergo TGF-β₁-driven differentiation into myofibroblasts.

  12. 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

  13. 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

    2015-07-23

    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.

  14. Enhancement of Gastric Ulcer Healing and Angiogenesis by Hepatocyte Growth Factor Gene Mediated by Attenuated Salmonella in Rats.

    PubMed

    Ha, Xiaoqin; Peng, Junhua; Zhao, Hongbin; Deng, Zhiyun; Dong, Juzi; Fan, Hongyan; Zhao, Yong; Li, Bing; Feng, Qiangsheng; Yang, Zhihua

    2017-02-01

    The present study developed an oral hepatocyte growth factor (HGF) gene therapy strategy for gastric ulcers treatment. An attenuated Salmonella typhimurium that stably expressed high HGF (named as TPH) was constructed, and the antiulcerogenic effect of TPH was evaluated in a rat model of gastric ulcers that created by acetic acid subserosal injection. From day 5 after injection, TPH (1 × 10⁹ cfu), vehicle (TP, 1 × 10⁹ cfu), or sodium bicarbonate (model control) was administered orally every alternate day for three times. Then ulcer size was measured at day 21 after ulcer induction. The ulcer area in TPH-treated group was 10.56 ± 3.30 mm², which was smaller when compared with those in the TP-treated and model control groups (43.47 ± 4.18 and 56.25 ± 6.38 mm², respectively). A higher level of reepithelialization was found in TPH-treated group and the crawling length of gastric epithelial cells was significantly longer than in the other two groups (P < 0.05). The microvessel density in the ulcer granulation tissues of the TPH-treated rats was 39.9 vessels/mm², which was greater than in the TP-treated and model control rats, with a significant statistical difference. These results suggest that TPH treatment significantly accelerates the healing of gastric ulcers via stimulating proliferation of gastric epithelial cells and enhancing angiogenesis on gastric ulcer site.

  15. Enhancement of Gastric Ulcer Healing and Angiogenesis by Hepatocyte Growth Factor Gene Mediated by Attenuated Salmonella in Rats

    PubMed Central

    2017-01-01

    The present study developed an oral hepatocyte growth factor (HGF) gene therapy strategy for gastric ulcers treatment. An attenuated Salmonella typhimurium that stably expressed high HGF (named as TPH) was constructed, and the antiulcerogenic effect of TPH was evaluated in a rat model of gastric ulcers that created by acetic acid subserosal injection. From day 5 after injection, TPH (1 × 109 cfu), vehicle (TP, 1 × 109 cfu), or sodium bicarbonate (model control) was administered orally every alternate day for three times. Then ulcer size was measured at day 21 after ulcer induction. The ulcer area in TPH-treated group was 10.56 ± 3.30 mm2, which was smaller when compared with those in the TP-treated and model control groups (43.47 ± 4.18 and 56.25 ± 6.38 mm2, respectively). A higher level of reepithelialization was found in TPH-treated group and the crawling length of gastric epithelial cells was significantly longer than in the other two groups (P < 0.05). The microvessel density in the ulcer granulation tissues of the TPH-treated rats was 39.9 vessels/mm2, which was greater than in the TP-treated and model control rats, with a significant statistical difference. These results suggest that TPH treatment significantly accelerates the healing of gastric ulcers via stimulating proliferation of gastric epithelial cells and enhancing angiogenesis on gastric ulcer site. PMID:28049228

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

    SciTech Connect

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

    2010-07-30

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

  17. Phosphorylation of the type II transmembrane serine protease, TMPRSS13, in hepatocyte growth factor activator inhibitor-1 and -2-mediated cell-surface localization.

    PubMed

    Murray, Andrew S; Varela, Fausto A; Hyland, Thomas E; Schoenbeck, Andrew J; White, Jordan M; Tanabe, Lauren M; Todi, Sokol V; List, Karin

    2017-09-08

    TMPRSS13 is a member of the type II transmembrane serine protease (TTSP) family. Although various TTSPs have been characterized in detail biochemically and functionally, the basic properties of TMPRSS13 remain unclear. Here, we investigate the activation, inhibition, post-translational modification, and localization of TMPRSS13. We show that TMPRSS13 is a glycosylated, active protease and that its own proteolytic activity mediates zymogen cleavage. Full-length, active TMPRSS13 exhibits impaired cell-surface expression in the absence of the cognate Kunitz-type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 or HAI-2. Concomitant presence of TMPRSS13 with either HAI-1 or -2 mediates phosphorylation of residues in the intracellular domain of the protease, and it coincides with efficient transport of the protease to the cell surface and its subsequent shedding. Cell-surface labeling experiments indicate that the dominant form of TMPRSS13 on the cell surface is phosphorylated, whereas intracellular TMPRSS13 is predominantly non-phosphorylated. These data provide novel insight into the cellular properties of TMPRSS13 and highlight phosphorylation of TMPRSS13 as a novel post-translational modification of this TTSP family member and potentially other members of this family of proteases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development

    PubMed Central

    1993-01-01

    Scatter factor/hepatocyte growth factor (SF/HGF) has potent motogenic, mitogenic, and morphogenetic activities on epithelial cells in vitro. The cell surface receptor for this factor was recently identified: it is the product of the c-met protooncogene, a receptor-type tyrosine kinase. We report here the novel and distinct expression patterns of SF/HGF and its receptor during mouse development, which was determined by a combination of in situ hybridization and RNase protection experiments. Predominantly, we detect transcripts of c-met in epithelial cells of various developing organs, whereas the ligand is expressed in distinct mesenchymal cells in close vicinity. In addition, transient SF/HGF and c-met expression is found at certain sites of muscle formation; transient expression of the c-met gene is also detected in developing motoneurons. SF/HGF and the c-met receptor might thus play multiple developmental roles, most notably, mediate a signal given by mesenchyme and received by epithelial. Mesenchymal signals are known to govern differentiation and morphogenesis of many epithelia, but the molecular nature of the signals has remained poorly understood. Therefore, the known biological activities of SF/HGF in vitro and the embryonal expression pattern reported here indicate that this mesenchymal factor can transmit morphogenetic signals in epithelial development and suggest a molecular mechanism for mesenchymal epithelial interactions. PMID:8408200

  19. 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

  20. 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

    2016-05-03

    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.

  1. Epidermal growth factor-stimulated protein phosphorylation in rat hepatocytes

    SciTech Connect

    Connelly, P.A.; Sisk, R.B.; Johnson, R.M.; Garrison, J.C.

    1987-05-01

    Epidermal growth factor (EGF) causes a 6-fold increase in the phosphorylation state of a cytosolic protein (pp36, M/sub r/ = 36,000, pI = 5.5) in hepatocytes isolated from fasted, male, Wistar rats. Stimulation of /sup 32/P incorporation is observed as early as 1 min following treatment of hepatocytes with EGF and is still present at 30 min after exposure to the growth factor. The phosphate incorporated into pp36 in response to EGF is located predominantly in serine but not tyrosine residues. Phosphorylation of pp36 does not occur in response to insulin or to agents which specifically activate the cAMP-dependent protein kinase (S/sub p/ -cAMPS), protein kinase C (PMA) or Ca/sup 2 +//calmodulin-dependent protein kinases (A23187) in these cells. Prior treatment of hepatocytes with the cAMP analog, S/sub p/-cAMPS, or ADP-ribosylation of N/sub i/, the inhibitory GTP-binding protein of the adenylate cyclase complex, does not prevent EGF-stimulated phosphorylation of pp36. However, as seen in other cell types, pretreatment of hepatocytes with PMA abolishes all EGF-mediated responses including phosphorylation of pp36. These results suggest that EGP specifically activates an uncharacterized, serine protein kinase in hepatocytes that is distal to the intrinsic EGF receptor tyrosine protein kinase. The rapid activation of this kinase suggests that it may play an important role in the early response of the cell to EGF.

  2. Repopulation of human origin hepatocyte progenitor-like cell line, THLE-5b, in the SCID mouse liver under p21-mediated cell growth-arresting conditions.

    PubMed

    Enosawa, Shin; Yamazaki, Taisuke; Kohsaka, Hitoshi; Tokiwa, Takayoshi

    2012-01-01

    The in vivo repopulation of hepatocytes depends on donor cell growth potential and recipient conditioning. We herein demonstrate the successful cell transplantation of a human hepatocyte cell line, THLE-5b, into the SCID mouse liver by means of a rather mild conditioning using a 55% hepatectomy and p21 transfection. Adult human liver-derived cells, THLE-5b, are SV40 T antigen-immortalized epithelial cells. A phenotypic examination of THLE-5b showed they expressed hepatic stem cell markers such as EpCAM, OCT3/4, and Thy-1, thus indicating the immature nature of the cells. A three-dimensional aggregate culture of THLE-5b showed a higher expression level of liver-specific genes such as albumin, α1-antitrypsin, and CYP3A4, thus suggesting that THLE-5b possess the capability to differentiate into hepatocytes. In a cell transplantation experiment, the cell cycle regulator p21 was transfected with adenoviral vector into the SCID mouse liver. On the next day, 8 × 10(5) cells of GFP-transfected THLE-5b were injected intrasplenically, together with the intraperitoneal administration of anti-asialo GM1 antibodies. The following day, a partial hepatectomy was performed. The GFP-THLE-5b cells were observed to have migrated and become integrated into the liver parenchyma 14 days after transplantation. The present protocol is thus considered to be a novel experimental model to elucidate the mechanism of hepatocyte repopulation and to develop efficient stem cell therapy in the liver.

  3. 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.

  4. Caspase-mediated proteolysis of the sorting nexin 2 disrupts retromer assembly and potentiates Met/hepatocyte growth factor receptor signaling

    PubMed Central

    Duclos, Catherine M; Champagne, Audrey; Carrier, Julie C; Saucier, Caroline; Lavoie, Christine L; Denault, Jean-Bernard

    2017-01-01

    The unfolding of apoptosis involves the cleavage of hundreds of proteins by the caspase family of cysteinyl peptidases. Among those substrates are proteins involved in intracellular vesicle trafficking with a net outcome of shutting down the crucial processes governing protein transport to organelles and to the plasma membrane. However, because of the intertwining of receptor trafficking and signaling, cleavage of specific proteins may lead to unintended consequences. Here we show that in apoptosis, sorting nexin 1 and 2 (SNX1 and SNX2), two proteins involved in endosomal sorting, are cleaved by initiator caspases and also by executioner caspase-6 in the case of SNX2. Moreover, SNX1 is cleaved at multiple sites, including following glutamate residues. Cleavage of SNX2 results in a loss of association with the endosome-to-trans-Golgi network transport protein Vps35 and in a delocalization from endosomes of its associated partner Vps26. We also demonstrate that SNX2 depletion causes an increase in hepatocyte growth factor receptor tyrosine phosphorylation and Erk1/2 signaling in cells. Finally, we show that SNX2 mRNA and protein levels are decreased in colorectal carcinoma and that lower SNX2 gene expression correlates with an increase in cancer patient mortality. Our study reveals the importance to characterize the cleavage fragments produced by caspases of specific death substrates given their potential implication in the mechanism of regulation of physiological (signaling/trafficking) pathways or in the dysfunction leading to pathogenesis. PMID:28179995

  5. 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.

  6. Autocrine expression of hepatocyte growth factor and its cytoprotective effect on hepatocyte poisoning

    PubMed Central

    He, Yong; Zhou, Jun; Dou, Ke-Feng; Chen, Yong; Yan, Qing-Guo; Li, Hai-Min

    2004-01-01

    AIM: To construct pEGFP-hepatocyte growth factor (HGF) expression vector, the to detect its expression in transfected human hepatocytes, and to investigate the influence of autocrine HGF expression on the proliferative potential and cytoprotective effects in human hepatocytes. METHODS: Human HGF cDNA was ligated to the pEGFP vector. Recombinant plasmid was transfected into human hepatocyte line QZG with liposome. Expression of HGF protein was observed by fluorescence microscopy and immunohistochemistry. Hepatic cells were collected 24, 48, and 72 h after transfection to detect the number of [3H]-TdR uptake in DNA. DNA synthesis was observed by using PCNA stain immunohistochemistry. Acute liver cell damage was induced by carbon tetrachloride. Cytoprotective effect was observed by examining the survival rate of hepatocytes and leakage of intracellular alanine transaminase (ALT) and potassium ions. RESULTS: HGF identification of pEGFP-HGF by enzyme digestion showed that HGF fragment was cloned into BamH I and Sal I sites of pEGFP-N3. Expression of GFP in transfected hepatocytes was observed with fluorescence microscopy. The [3H]-TdR uptake became 7 times as many as in the control group 96 h after transfection. After HGF transfection, the survival rate of hepatocytes poisoned by CCl4 significantly increased (83% vs 61%, P < 0.05), and the leakage of intracellular alanine transaminase and potassium ions decreased (586 nkat/L vs 1089 nkat/L, P < 0.01; and 5.59 mmol/L vs 6.02 mmol/L, P < 0.01 respectively). Culture of transfected hepatic cells promoted the proliferation of other non-transfected cells. CONCLUSION: Transfected HGF is expressed in hepatic cells and has the activity of promoting cell division and protecting hepatic cells against poisoning. PMID:15334679

  7. 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. PMID:27635169

  8. Hepatocyte growth factor-modulated rat Leydig cell functions.

    PubMed

    Del Bravo, Jessica; Catizone, Angela; Ricci, Giulia; Galdieri, Michela

    2007-01-01

    Hepatocyte growth factor (HGF) regulates many cellular functions acting through c-Met, its specific tyrosine kinase receptor. We previously reported that in prepuberal rats HGF is secreted by the peritubular myoid cells during the entire postnatal testicular development and by the Sertoli cells only at puberty. We have also demonstrated that germ cells at different stages of development express c-Met and that HGF modulates germ cell proliferation and apoptosis. In the present article, we extend our study to the interstitial compartment of the testis and demonstrate that the c-Met protein is present on Leydig cells. The receptor is functionally active as demonstrated by the detected effects of HGF. We report in this article that HGF significantly increases the amount of testosterone secreted by the Leydig cells and decreases the number of Leydig cells undergoing apoptosis. The antiapoptotic effect of HGF is mediated by caspase-3 activity because the amount of the active fragment of the enzyme is decreased in Leydig cells cultured in the presence of HGF. However, treatment with the growth factor does not modify the expression levels of caspase-3 mRNA. These data indicate that HGF regulates the functional activities of Leydig cells. Interestingly, the steroidogenetic activity of the cells is increased by HGF in cultured explants of testicular tissues as well as the antiapoptotic effect of HGF. Therefore, our data indicate that HGF has a crucial role in the regulation of male fertility.

  9. Necrotic cell-derived high mobility group box 1 attracts antigen-presenting cells but inhibits hepatocyte growth factor-mediated tropism of mesenchymal stem cells for apoptotic cell death.

    PubMed

    Vogel, S; Börger, V; Peters, C; Förster, M; Liebfried, P; Metzger, K; Meisel, R; Däubener, W; Trapp, T; Fischer, J C; Gawaz, M; Sorg, R V

    2015-07-01

    Tissue damage due to apoptotic or necrotic cell death typically initiates distinct cellular responses, leading either directly to tissue repair and regeneration or to immunological processes first, to clear the site, for example, of potentially damage-inducing agents. Mesenchymal stem cells (MSC) as well as immature dendritic cells (iDC) and monocytes migrate to injured tissues. MSC have regenerative capacity, whereas monocytes and iDC have a critical role in inflammation and induction of immune responses, including autoimmunity after tissue damage. Here, we investigated the influence of apoptotic and necrotic cell death on recruitment of MSC, monocytes and iDC, and identified hepatocyte growth factor (HGF) and the alarmin high mobility group box 1 (HMGB1) as key factors differentially regulating these migratory responses. MSC, but not monocytes or iDC, were attracted by apoptotic cardiomyocytic and neuronal cells, whereas necrosis induced migration of monocytes and iDC, but not of MSC. Only apoptotic cell death resulted in HGF production and HGF-mediated migration of MSC towards the apoptotic targets. In contrast, HMGB1 was predominantly released by the necrotic cells and mediated recruitment of monocytes and iDC via the receptor of advanced glycation end products. Moreover, necrotic cardiomyocytic and neuronal cells caused an HMGB1/toll-like receptor-4-dependent inhibition of MSC migration towards apoptosis or HGF, while recruitment of monocytes and iDC by necrosis or HMGB1 was not affected by apoptotic cells or HGF. Thus, the type of cell death differentially regulates recruitment of either MSC or monocytes and iDC through HGF and HMGB1, respectively, with a dominant, HMGB1-mediated role of necrosis in determining tropism after tissue injury.

  10. Necrotic cell-derived high mobility group box 1 attracts antigen-presenting cells but inhibits hepatocyte growth factor-mediated tropism of mesenchymal stem cells for apoptotic cell death

    PubMed Central

    Vogel, S; Börger, V; Peters, C; Förster, M; Liebfried, P; Metzger, K; Meisel, R; Däubener, W; Trapp, T; Fischer, J C; Gawaz, M; Sorg, R V

    2015-01-01

    Tissue damage due to apoptotic or necrotic cell death typically initiates distinct cellular responses, leading either directly to tissue repair and regeneration or to immunological processes first, to clear the site, for example, of potentially damage-inducing agents. Mesenchymal stem cells (MSC) as well as immature dendritic cells (iDC) and monocytes migrate to injured tissues. MSC have regenerative capacity, whereas monocytes and iDC have a critical role in inflammation and induction of immune responses, including autoimmunity after tissue damage. Here, we investigated the influence of apoptotic and necrotic cell death on recruitment of MSC, monocytes and iDC, and identified hepatocyte growth factor (HGF) and the alarmin high mobility group box 1 (HMGB1) as key factors differentially regulating these migratory responses. MSC, but not monocytes or iDC, were attracted by apoptotic cardiomyocytic and neuronal cells, whereas necrosis induced migration of monocytes and iDC, but not of MSC. Only apoptotic cell death resulted in HGF production and HGF-mediated migration of MSC towards the apoptotic targets. In contrast, HMGB1 was predominantly released by the necrotic cells and mediated recruitment of monocytes and iDC via the receptor of advanced glycation end products. Moreover, necrotic cardiomyocytic and neuronal cells caused an HMGB1/toll-like receptor-4-dependent inhibition of MSC migration towards apoptosis or HGF, while recruitment of monocytes and iDC by necrosis or HMGB1 was not affected by apoptotic cells or HGF. Thus, the type of cell death differentially regulates recruitment of either MSC or monocytes and iDC through HGF and HMGB1, respectively, with a dominant, HMGB1-mediated role of necrosis in determining tropism after tissue injury. PMID:25571972

  11. Alternative Proteolytic Processing of Hepatocyte Growth Factor during Wound Repair

    PubMed Central

    Buchstein, Nils; Hoffmann, Daniel; Smola, Hans; Lang, Sabina; Paulsson, Mats; Niemann, Catherin; Krieg, Thomas; Eming, Sabine A.

    2009-01-01

    Wound healing is a crucial regenerative process in all organisms. We examined expression, integrity, and function of the proteins in the hepatocyte growth factor (HGF)/c-Met signaling pathway in normally healing and non-healing human skin wounds. Whereas in normally healing wounds phosphorylation of c-Met was most prominent in keratinocytes and dermal cells, in non-healing wounds phosphorylation of c-Met was barely detectable, suggesting reduced c-Met activation. In wound exudates obtained from non-healing, but not from healing wounds, HGF protein was a target of substantial proteolytic processing that was different from the classical activation by known serine proteases. Western blot analysis and protease inhibitor studies revealed that HGF is a target of neutrophil elastase and plasma kallikrein during skin repair. Proteolytic processing of HGF by each of these proteases significantly attenuated keratinocyte proliferation, wound closure capacity in vitro, and c-Met signal transduction. Our findings reveal a novel pathway of HGF processing during skin repair. Conditions in which proteases are imbalanced and tend toward increased proteolytic activity, as in chronic non-healing wounds, might therefore compromise HGF activity due to the inactivation of the HGF protein and/or the generation of HGF fragments that ultimately mediate a dominant negative effect and limit c-Met activation. PMID:19389925

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

    SciTech Connect

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

    2011-01-14

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

  13. 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.

  14. Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells.

    PubMed

    Devhare, Pradip B; Sasaki, Reina; Shrivastava, Shubham; Di Bisceglie, Adrian M; Ray, Ranjit; Ray, Ratna B

    2017-03-15

    Fibrogenic pathways in the liver are principally regulated by activation of hepatic stellate cells (HSC). Fibrosis is associated with chronic hepatitis C virus (HCV) infection, although the mechanism is poorly understood. HSC comprise the major population of nonparenchymal cells in the liver. Since HCV does not replicate in HSC, we hypothesized that exosomes secreted from HCV-infected hepatocytes activate HSC. Primary or immortalized human hepatic stellate (LX2) cells were exposed to exosomes derived from HCV-infected hepatocytes (HCV-exo), and the expression of fibrosis-related genes was examined. Our results demonstrated that HCV-exo internalized to HSC and increased the expression of profibrotic markers. Further analysis suggested that HCV-exo carry miR-19a and target SOCS3 in HSC, which in turn activates the STAT3-mediated transforming growth factor β (TGF-β) signaling pathway and enhances fibrosis marker genes. The higher expression of miR-19a in exosomes was also observed from HCV-infected hepatocytes and in sera of chronic HCV patients with fibrosis compared to healthy volunteers and non-HCV-related liver disease patients with fibrosis. Together, our results demonstrated that miR-19a carried through the exosomes from HCV-infected hepatocytes activates HSC by modulating the SOCS-STAT3 axis. Our results implicated a novel mechanism of exosome-mediated intercellular communication in the activation of HSC for liver fibrosis in HCV infection.IMPORTANCE HCV-associated liver fibrosis is a critical step for end-stage liver disease progression. However, the molecular mechanisms for hepatic stellate-cell activation by HCV-infected hepatocytes are underexplored. Here, we provide a role for miR-19a carried through the exosomes in intercellular communication between HCV-infected hepatocytes and HSC in fibrogenic activation. Furthermore, we demonstrate the role of exosomal miR-19a in activation of the STAT3-TGF-β pathway in HSC. This study contributes to the

  15. Sulodexide induces hepatocyte growth factor release in humans.

    PubMed

    Borawski, Jacek; Dubowski, Miroslaw; Pawlak, Krystyna; Mysliwiec, Michal

    2007-03-08

    Heparin influences numerous pleiotropic growth factors, including hepatocyte growth factor (HGF), partially by their release from endothelial and extracellular matrix stores. The effects of sulodexide, a heparin-like glycosaminoglycan medication of growing importance in medicine, on HGF liberation are not known. We performed a 2-week open-label sulodexide trial in healthy male volunteers. The drug was initially administered intravenously (i.v.) in a single dose of 1200 Lipoprotein Lipase Releasing Units (LRU), then -- orally for 12 days (500 LRU twice a day), and -- again by i.v. route (1200 LRU) on day 14. Intravenous sulodexide injections were repeatedly found to induce marked and reproducible increases in immunoreactive plasma HGF levels (more than 3500% vs baseline after 10 min, and more than 1200% after 120 min), and remained unchanged when measured 120 min following oral sulodexide administration. The percentage increments in plasma HGF evoked by i.v. sulodexide at both time points and on both days inversely correlated with baseline levels of the growth factor. On day 14, the HGF levels after 120 min and their percentage increase vs baseline were strongly and directly dependent on i.v. sulodexide dose per kg of body weight. This study shows that sulodexide has a novel, remarkable and plausibly biologically important stimulating effect on the release of pleiotropic hepatocyte growth factor in humans.

  16. Hepatocyte Growth Factor and Interleukin-6 in Prostate Cancer Bone Metastasis

    DTIC Science & Technology

    2005-03-01

    AD Award Number: DAMD17-02-1-0159 TITLE: Hepatocyte Growth Factor and Interleukin - 6 in Prostate Cancer Bone Metastasis PRINCIPAL INVESTIGATOR...TITLE AND SUBTITLE 5. FUNDING NUMBERS Hepatocyte Growth Factor and Interleukin - 6 in Prostate DAMD17-02-1-0159 Cancer Bone Metastasis 6 . AUTHOR(S...DAMD17-02-1-0159 "Hepatocyte Growth Factor and Interleukin - 6 in Prostate Cancer Bone Metastasis" INTRODUCTION: The hypothesis of this grant proposal

  17. 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.

  18. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product

    SciTech Connect

    Bottaro, D.P.; Rubin, J.S.; Chan, A.M.L.; Aaronson, S.A. ); Faletto, D.L.; Kmiecik, T.E.; Vande Woude, G.F. )

    1991-02-15

    Hepatocyte growth factor (HGF) is a plasminogen-like protein thought to be a humoral mediator of liver regeneration. A 145-kilodalton tyrosyl phosphoprotein observed in rapid response to HGF treatment of intact target cells was identified by immunoblot analysis as the {beta} subunit of the c-met proto-oncogene product, a membrane-spanning tyrosine kinase. Covalent cross-linking of {sup 125}I-labeled ligand to cellular proteins of appropriate size that were recognized by antibodies to c-met directly established the c-met product as the cell-surface receptor for HGF.

  19. Hepatocyte growth factor: a regenerative drug for acute hepatitis and liver cirrhosis.

    PubMed

    Mizuno, Shinya; Nakamura, Toshikazu

    2007-03-01

    Liver cirrhosis is a major cause of morbidity worldwide and is characterized by the loss of hepatocytes with interstitial fibrosis. In this review, we discuss the potential uses of hepatocyte growth factor for treating hepatic diseases, focusing on the molecular mechanisms whereby hepatocyte growth factor reverses liver cirrhosis. Hepatic myofibroblasts play a central role in the development of liver cirrhosis, while myofibroblasts acquire c-Met. Using a rat model of liver cirrhosis, we recently delineated the direct effect of hepatocyte growth factor toward myofibroblasts: the induction of apoptotic cell death associated with matrix degradation, the inhibition of overproliferation and the suppression of transforming growth factor-beta1 production in myofibroblasts. Hepatocyte growth factor elicits mitogenic, anti-apoptotic and anti-inflammatory functions in hepatocytes, therefore contributing to reversing liver dysfunction. Considering the insufficient production of hepatocyte growth factor is responsible for the manifestation of chronic hepatitis, supplementation with or reinduction of hepatocyte growth factor represents a new strategy for attenuating intractable liver diseases.

  20. Hepatocyte growth factor (HGF) and hemodialysis: physiopathology and clinical implications.

    PubMed

    Libetta, Carmelo; Esposito, Pasquale; Martinelli, Claudia; Grosjean, Fabrizio; Gregorini, Marilena; Rampino, Teresa; Dal Canton, Antonio

    2016-06-01

    Hepatocyte growth factor (HGF) is a pleiotropic cytokine which exerts a variety of effects on several cells, being involved in the regulation of many biological processes, such as inflammation, tissue repair, morphogenesis, angiogenesis, tumour propagation, immunomodulation of viral infections and cardio-metabolic activities. Patients undergoing regular hemodialysis (HD) present elevated levels of HGF, mainly due to the leukocyte activation associated with HD treatment. High HGF levels might account for specific clinical features of HD patients, i.e. mild liver damage in course of HCV-infection and high cardiovascular risk profile. Moreover, in patients with acute kidney injury, the induction of HGF may represent a crucial step to promote renal recovery, which can have important prognostic consequences in the short and long-term. In this review we discuss the mechanisms underlying HGF production in HD patients, the role of HGF in this particular patient population and the potential clinical implications derived from the study of HGF in HD patients.

  1. Lipid-mediated transfection of normal adult human hepatocytes in primary culture.

    PubMed

    Ourlin, J C; Vilarem, M J; Daujat, M; Harricane, M C; Domergue, J; Joyeux, H; Baulieux, J; Maurel, P

    1997-04-05

    The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.

  2. Hepatocyte growth factor in renal failure: promise and reality.

    PubMed

    Vargas, G A; Hoeflich, A; Jehle, P M

    2000-04-01

    Can science discover some secrets of Greek mythology? In the case of Prometheus, we can now suppose that his amazing hepatic regeneration was caused by a peptide growth factor called hepatocyte growth factor (HGF). Increasing evidence indicates that HGF acts as a multifunctional cytokine on different cell types. This review addresses the molecular mechanisms that are responsible for the pleiotropic effects of HGF. HGF binds with high affinity to its specific tyrosine kinase receptor c-met, thereby stimulating not only cell proliferation and differentiation, but also cell migration and tumorigenesis. The three fundamental principles of medicine-prevention, diagnosis, and therapy-may be benefited by the rational use of HGF. In renal tubular cells, HGF induces mitogenic and morphogenetic responses. In animal models of toxic or ischemic acute renal failure, HGF acts in a renotropic and nephroprotective manner. HGF expression is rapidly up-regulated in the remnant kidney of nephrectomized rats, inducing compensatory growth. In a mouse model of chronic renal disease, HGF inhibits the progression of tubulointerstitial fibrosis and kidney dysfunction. Increased HGF mRNA transcripts were detected in mesenchymal and tubular epithelial cells of rejecting kidney. In transplanted patients, elevated HGF levels may indicate renal rejection. When HGF is considered as a therapeutic agent in human medicine, for example, to stimulate kidney regeneration after acute injury, strategies need to be developed to stimulate cell regeneration and differentiation without an induction of tumorigenesis.

  3. 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.

  4. Nonviral gene transfer of hepatocyte growth factor attenuates neurologic injury after spinal cord ischemia in rabbits.

    PubMed

    Shi, Enyi; Jiang, Xiaojing; Kazui, Teruhisa; Washiyama, Naoki; Yamashita, Katsushi; Terada, Hitoshi; Bashar, Abul Hasan Muhammad

    2006-10-01

    Paraplegia caused by spinal cord ischemia remains a serious complication after surgical repair of thoracoabdminal aortic aneurysms. Hepatocyte growth factor is a potent angiogenic and neurotrophic factor. We sought to investigate the neuroprotective effect of gene transfer of hepatocyte growth factor on spinal cord ischemia in rabbits. Human hepatocyte growth factor expression plasmid was combined with hemagglutinating virus of Japan envelope vector. Hemagglutinating virus of Japan envelope vector containing the hepatocyte growth factor gene was injected intrathecally into the experimental rabbits, whereas control vector or saline was given to the control animals. Five days later, spinal cord ischemia was induced by means of infrarenal aortic occlusion for 30 minutes. Hind-limb motor function was assessed during a 14-day recovery period with Tarlov criteria. Human hepatocyte growth factor was detected in the cerebrospinal fluid 3 days after gene transfer, and the level peaked on day 5. Compared with the control animals, hepatocyte growth factor gene transfer significantly increased the capillary density in the gray matter and decreased the spinal cord edema. All rabbits pretreated with saline or control vector had hind-limb paraplegia (Tarlov score = 0) 14 days after spinal cord ischemia. However, previous transfection of the hepatocyte growth factor gene remarkably enhanced the Tarlov scores, and 8 of the 9 rabbits showed normal motor function (Tarlov score = 5) after a 14-day recovery period. Histologic examination showed that the intact motor neurons were preserved to a much greater extent in the rabbits transfected with the hepatocyte growth factor gene. Gene transfer of hepatocyte growth factor attenuates neurologic injury after spinal cord ischemia.

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

    SciTech Connect

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

    2010-11-01

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

  6. Estradiol-17beta and dihydrotestosterone differentially regulate vitellogenin and insulin-like growth factor-I production in primary hepatocytes of the tilapia Oreochromis mossambicus.

    PubMed

    Riley, Larry G; Hirano, Tetsuya; Grau, E Gordon

    2004-06-01

    Effects of estradiol-17beta (E2) and 5alpha-dihydrotestosterone (DHT) on the production of vitellogenin (Vg), insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) were examined in vitro using primary hepatocyte culture of the tilapia. Estradiol produced a significant and concentration-related stimulation of Vg release and concomitant, concentration-related reduction in IGF-I mRNA expression in both male and female hepatocytes. In male hepatocytes, DHT significantly increased IGF-I expression, whereas DHT inhibited IGF-I expression and stimulated Vg release in female hepatocytes. Estradiol treatment significantly reduced the release of 25 kDa IGFBP, while stimulating the release of 30 kDa IGFBP from male hepatocytes. In female hepatocytes, E2 significantly increased both 25 and 30 kDa IGFBPs. In male hepatocytes, DHT significantly reduced 25 kDa IGFBP without affecting 30 kDa IGFBP. Conversely, DHT treatment of hepatocytes from female fish significantly increased both the 25 and 30 kDa IGFBPs. The different growth rates observed between male and female tilapia may be a result of gonadal steroid hormones eliciting direct and antagonistic effects on production of IGF-I (growth) and Vg (reproduction) in the liver. Indeed, the different growth patterns likely result from a difference in the sensitivity of male and female hepatocytes to gonadal steroid hormones. These results also indicate direct effects of gonadal steroid hormones on production of IGFBPs, which may play a role in regulating IGF-I mediated growth.

  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. Engineering hepatocyte functional fate through growth factor dynamics: the role of cell morphologic priming.

    PubMed

    Semler, E J; Moghe, P V

    2001-12-05

    We have reported previously that cellular stimulation induced by variable mechanochemical properties of the extracellular microenvironment can significantly alter liver-specific function in cultured hepatocytes (Semler et al., Biotech Bioeng 69:359-369, 2000). Cell activation via time-invariant presentation of biochemical growth factors was found to either enhance or repress cellular differentiation of cultured hepatocytes depending on the mechanical properties of the underlying substrate. In this work, we investigated the effects of dynamic growth factor stimulation on the cell growth and differentiation behavior of hepatocytes cultured on either compliant or rigid substrates. Specifically, hepatotrophic growth factors (epidermal and hepatocyte) were either temporally added or withdrawn from hepatocyte cultures on Matrigel that was crosslinked to yield differential degrees of mechanical compliance. We determined that the functional responsiveness of hepatocytes to fluctuations in GF stimulation is substrate specific but only in conditions in which the initial mechanochemical environment induced significant cell morphogenesis. Our studies indicate that in conditions under which hepatocytes adopted a "rounded" phenotype, they exhibited increased levels of differentiated function upon soluble stimulation and markedly decreased function upon the depletion of GF stimulation. In contrast, hepatocytes that assumed a "spread" phenotype exhibited slightly increased function upon the depletion of GF stimulation. By examining the functional responsiveness of hepatocytes of differential morphology to varied fluctuations in GF activation, insights into the ability of cell shape to "prime" hepatocyte behavior in dynamic microenvironments were elucidated. We report on the possibility of uncoupling and, thus, selectively manipulating, the concerted contributions of GF-induced cellular activation and substrate- and GF-induced cell morphogenesis toward induction of cell function

  9. Hepatocyte growth factor, a biomarker of macroangiopathy in diabetes mellitus

    PubMed Central

    Konya, Hiroyuki; Miuchi, Masayuki; Satani, Kahori; Matsutani, Satoshi; Tsunoda, Taku; Yano, Yuzo; Katsuno, Tomoyuki; Hamaguchi, Tomoya; Miyagawa, Jun-Ichiro; Namba, Mitsuyoshi

    2014-01-01

    Atherosclerotic involvements are an essential causal element of prospect in diabetes mellitus (DM), with carotid atherosclerosis (CA) being a common risk-factor for prospective crisis of coronary artery diseases (CAD) and/or cerebral infarction (CI) in DM subjects. From another point of view, several reports have supplied augmenting proof that hepatocyte growth factor (HGF) has a physiopathological part in DM involvements. HGF has been a mesenchymal-derived polyphenic factor which modulates development, motion, and morphosis of diverse cells, and has been regarded as a humor intermediator of epithelial-mesenchymal interplays. The serum concentrations of HGF have been elevated in subjects with CAD and CI, especially during the acute phase of both disturbances. In our study with 89 type 2 DM patients, the association between serum concentrations of HGF and risk-factors for macrovascular complications inclusive of CA were examined. The average of serum HGF levels in the subjects was more elevated than the reference interval. The serum HGF concentrations associated positively with both intimal-media thickness (IMT) (r = 0.24, P = 0.0248) and plaque score (r = 0.27, P = 0.0126), indicating a relationship between the elevated HGF concentrations and advancement of CA involvements. Multivariate statistical analysis accentuated that serum concentrations of HGF would be associated independently with IMT (standardized = 0.28, P = 0.0499). The review indicates what is presently known regarding serum HGF might be a new and meaningful biomarker of macroangiopathy in DM subjects. PMID:25317245

  10. 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

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

    SciTech Connect

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

    2008-01-15

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

  12. Sensitive hepatocyte-mediated assay for the metabolism of nitrosamines to mutagens for mammalian cells

    SciTech Connect

    Jones, C.A.; Huberman, E.

    1980-02-01

    A sensitive cell-mediated assay has been developed for testing mutagenesis in Chinese hamster V79 cells by carcinogenic nitrosamines. Mutations were characterized by resistance to ouabian and 6-thioguanine. Since V79 cells do not metabolize nitrosamines, mutagenesis in the V79 cells was tested in the presence of primary hepatocytes capable of metabolizing nitrosamines. The hepatocytes were isolated after collagenase and hyaluronidase digestion of liver slices. All seven liver carcinogens of the nine tested nitrosamines exhibited a mutagenic response in this cell-mediated assay. The potent liver carcinogens nitrosodimethylamine, nitrosodiethylamine, nitrosoethylmethylamine, and nitrosodipropylamine could be detected with doses as low as 1 ..mu..m. The noncarcinogenic nitrosodiphenylamine was not mutagenic. Nitrosomethoxymethylamine was the only nitrosamine that exhibited mutagenic activity in the absence of hepatocytes, and this activity was diminished in the presence of hepatocytes. It is suggested that the use of hepatocytes prepared by the slicing method for carcinogen metabolism and mutable V79 cells offers a highly sensitive assay for determining the mutagenic potential of carcinogenic nitrosamines and probably of other classes of hazardous chemicals occurring in the environment.

  13. Autoradiographic analysis of hepatocytes in mirex-induced adaptive liver growth

    SciTech Connect

    Wilson, D.; Yarbrough, J.D. )

    1988-07-01

    The relationships between ({sup 3}H)thymidine incorporation into hepatocyte nuclei, cell enlargement, and mitotic index were studied in intact (INT) and adrenalectomized (ADX) mirex-dosed rats. In INT mirex-dosed rats the sequence of events included the following: a biphasic response in nuclear labeling of mononuclear hepatocyes with peaks at 48 and 66 h postmirex dose, a peak in mitotic activity 66 h postmirex dose, and a significant increase in binuclear hepatocyte size 48 h postmirex dose. In ADX mirex-dosed rats the sequence of events included the following: a biphasic response in nuclear labeling mononuclear hepatocytes with peaks at 24 and 48 h postmirex dose, a peak in mitotic activity 60 h postmirex dose, and a marginal increase in binuclear hepatocyte size 48 h postmirex dose. Corticosterone supplements to ADX mirex-dosed rats significantly reduced nuclear labeling of the mononuclear hepatocytes and increased the size of binuclear hepatocytes to that observed in INT mirex-dosed rats. This study demonstrates that adaptive liver growth consists of a hyperplastic response that involves mononuclear hepatocytes and a hypertrophic response that involves binuclear hepatocytes. Both responses appear to be modulated by corticosterone.

  14. [Sphingosine kinase regulates hepatocyte growth factor-induced migration of endothelial cells].

    PubMed

    Yi, Jun; Lu, Zhuao-Zhuang; Duan, Hai-Feng; Gai, Lu-Yue; Wang, Li-Sheng

    2006-05-01

    To elucidate the effect of sphingosine kinase (SPK) on the hepatocyte growth factor (HGF)-induced migration of endothelial cells. We constructed recombinant adenoviral vectors, which contain SPK gene and its mutant respectively. These adenoviral vectors were packaged and amplified in 293 cells. And intracellular SPK activity was assayed via measurement of [32]P radioisotope labeled S1P; the effect of SPK activation on HGF-induced migration of endothelial cell was observed by Transwell technique. Adenoviral mediated expression of SPK gene increased in ECV 304 cells intracellular SPK activity, which in turn enhanced the HGF-induced migration. Whereas these activities were blocked by the dominant negative SPK gene. These findings show that SPK activation plays important roles in the regulation of HGF-induced migration of endothelial cells.

  15. Hepatocyte growth factor stimulates root growth during the development of mouse molar teeth.

    PubMed

    Sakuraba, H; Fujiwara, N; Sasaki-Oikawa, A; Sakano, M; Tabata, Y; Otsu, K; Ishizeki, K; Harada, H

    2012-02-01

    It is well known that tooth root formation is initiated by the development of Hertwig's epithelial root sheath (HERS). However, relatively little is known about the regulatory mechanisms involved in root development. As hepatocyte growth factor (HGF) is one of the mediators of epithelial-mesenchymal interactions in rodent tooth, the objective of this study was to examine the effects of HGF on the root development of mouse molars. The HERS of mouse molars and HERS01a, a cell line originated from HERS, were used in this study. For detection of HGF receptors in vivo and in vitro, we used immunochemical procedures. Root development was assessed by implanting molar tooth germs along with HGF-soaked beads into kidney capsules, by counting cell numbers in HERS01a cell cultures and by performing a 5'-bromo-2'-deoxyuridine (BrdU) assay in an organ-culture system. HGF receptors were expressed in the enamel epithelium of molar germs as well as in HERS cells. HGF stimulated root development in the transplanted tooth germs, the proliferation of HERS01a cells in culture and HERS elongation in the organ-culture system. Examination using BrdU revealed that cell proliferation in HERS was increased by treatment with HGF, especially that in the outer layer of HERS. This effect was down-regulated when antibody against HGF receptor was present in the culture medium. Our results raise the possibility that HGF signaling controls root formation via the development of HERS. This study is the first to show that HGF is one of the stimulators of root development. © 2011 John Wiley & Sons A/S.

  16. Hepatocyte Growth Factor Regulates Angiotensin Converting Enzyme Expression*

    PubMed Central

    Day, Regina M.; Thiel, Gerald; Lum, Julie; Chévere, Rubén D.; Yang, Yongzhen; Stevens, Joanne; Sibert, Laura; Fanburg, Barry L.

    2008-01-01

    Hepatocyte growth factor (HGF) is a mitogen, morphogen, and motogen that functions in tissue healing and acts as an anti-fibrotic factor. The mechanism for this is not well understood. Recent studies implicate somatic angiotensin-converting enzyme (ACE) in fibrosis. We examined the effects of HGF on ACE expression in bovine pulmonary artery endothelial cells (BPAEC). Short term treatment of BPAEC with HGF transiently increased both ACE mRNA (3 h) and activity (24 h), as determined by ACE protease assays and reverse transcription-PCR. Incubation of BPAEC with HGF for longer periods suppressed ACE mRNA (6 h) and activity (72 h). In contrast, phorbol ester (PMA) treatment produced sustained increase in ACE mRNA and activity. We examined the short term molecular effects of HGF on ACE using PMA for comparison. HGF and PMA increased transcription from a luciferase reporter with the core ACE promoter, which contains a composite binding site for SP1/3 and Egr-1. Immunocytochemistry and electrophoretic mobility shift assay showed that both HGF and PMA increased Egr-1 binding. HGF also increased SP3 binding, as measured by EMSA. However, HGF and PMA increased the cellular activity of only Egr-1, not SP3, as measured by luciferase reporter assays. Deletion of the Egr-1 site in the reporter construct completely abrogated HGF-induced transcription but only ~50% of PMA-induced activity. Expression of dominant negative Egr-1 and SP3 blocked up-regulation of the ACE promoter by HGF but only reduced up-regulation by PMA. These results show that HGF transiently increases gene transcription of ACE via activation of Egr-1, whereas PMA regulation involves Egr-1 and additional factor(s). PMID:14679188

  17. 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.

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

    PubMed

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

    2015-10-01

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

  19. Functional role of hepatocyte growth factor receptor during sperm maturation.

    PubMed

    Catizone, A; Ricci, G; Galdieri, M

    2002-01-01

    Mammalian spermatozoa acquire motility and fertilizing capacity during their transit through the epididymis. Hepatocyte growth factor (HGF) is a pleiotropic cytokine with potent motogenic capacities that has been identified in different organs, including the mammalian male genital tract. In mice, HGF is present in the testis and, in large amounts, in the distal part of the epididymis. In prepuberal rats, we have demonstrated that HGF is synthesized by the peritubular myoid cells and in men, HGF is present in significant quantities in seminal plasma. It has been suggested that in mice, HGF has a role in initiating sperm motility, whereas in men, no significant correlations between HGF concentration and sperm motility have been found. In the present paper we report that in rats, HGF receptor, c-met, is expressed in testicular and epididymal spermatozoa. Through immunocytochemistry, we have found that c-met is exclusively localized on the head in testicular sperm. A different localization of c-met has been found in sperm isolated from caput and cauda epididymidis. Cells isolated from epididymal caput show a c-met localization exclusively restricted to the head in most cells. In a minority of caput epididymis spermatozoa the receptor is localized both in the cell head and along the flagellum. Spermatozoa isolated from the epididymal cauda were quite homogeneous, showing the receptor localized along the entire cell surface. We also report that HGF is synthesized and secreted by the rat epididymis as indicated by the scatter effect of epididymal cell homogenate and culture medium on MDCK cells. To clarify whether HGF is involved in the acquisition of sperm motility in the epididymis, its maintenance, or both, spermatozoa isolated from caput epididymidis have been cultured in medium alone or supplemented with HGF. The results obtained indicated that HGF has a positive effect on the maintenance of sperm motility which, in the absence of HGF, significantly decreases during

  20. [An experimental study on the protective effect of hepatocyte growth factor (HGF) for the primary cultured hepatocytes obtained from iron-loaded rats].

    PubMed

    Yoshida, J

    1995-01-01

    Pathological iron deposition in liver is often found in various liver diseases. The deposited iron is thought to be one of the most important factor of liver cell injury, not only in hemochromotosis but also in cirrhosis following hepatitis virus B or C infection. To investigate the influence of the deposited iron on damage and regeneration of hepatocyte, primary cultured hepatocytes obtained from carbonyl iron-loaded rats were treated with carbon tetrachloride (CCl4) in the presence or absence of hepatocyte growth factor (HGF). Although the section of liver from carbonyl iron-loaded rats showed no necrosis and fibrosis, iron-loaded hepatocytes contained about twofold more iron than control. The damage of iron-loaded hepatocytes induced by CCl4 was more serious than that of control, and HGF decreased this injury only in iron-loaded hepatocytes but not in control. There is no difference in DNA synthesis stimulated by HGF between iron-loaded hepatocytes and control. These findings suggest that the pathological iron deposition induces the fragility of hepatocyte and that cytoprotective effect of HGF is induced by this pathological iron.

  1. FGF19-induced hepatocyte proliferation is mediated through FGFR4 activation.

    PubMed

    Wu, Xinle; Ge, Hongfei; Lemon, Bryan; Vonderfecht, Steven; Weiszmann, Jennifer; Hecht, Randy; Gupte, Jamila; Hager, Todd; Wang, Zhulun; Lindberg, Richard; Li, Yang

    2010-02-19

    FGF19 and FGF21, unique members of the fibroblast growth factor (FGF) family, are hormones that regulate glucose, lipid, and energy homeostasis. Increased hepatocyte proliferation and liver tumor formation have also been observed in FGF19 transgenic mice. Here, we report that, in contrast to FGF19, FGF21 does not induce hepatocyte proliferation in vivo. To identify the mechanism for FGF19-induced hepatocyte proliferation, we explored similarities and differences in receptor specificity between FGF19 and FGF21. We find that although both are able to activate FGF receptors (FGFRs) 1c, 2c, and 3c, only FGF19 activates FGFR4, the predominant receptor in the liver. Using a C-terminal truncation mutant of FGF19 and a series of FGF19/FGF21 chimeric molecules, we determined that amino acids residues 38-42 of FGF19 are sufficient to confer both FGFR4 activation and increased hepatocyte proliferation in vivo to FGF21. These data suggest that activation of FGFR4 is the mechanism whereby FGF19 can increase hepatocyte proliferation and induce hepatocellular carcinoma formation.

  2. 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

  3. Effect of recombinant human growth hormone on age-related hepatocyte changes in old male and female Wistar rats.

    PubMed

    Castillo, Carmen; Salazar, Veronica; Ariznavarreta, Carmen; Vara, Elena; Tresguerres, Jesus A F

    2004-10-01

    Aging induces changes in several organs, such as the liver, and this process might be due to damage caused by free radicals and inflammatory mediators. The growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis shows a reduction with age, and this fact could be associated with some age-related changes. The aim of this study was to investigate the effect of GH administration on age-induced alterations in hepatocytes. Two and twenty two month-old male and female Wistar rats were used. Old rats were treated with human recombinant GH for 10 wk. At the end of the treatment, hepatocytes were isolated from the liver and cultured, and different parameters were measured in cells and medium. Plasma IGF-1 was also measured. Aging significantly decreased plasma IGF-1 in males. In females, plasma IGF-1 was also reduced, but not significantly. GH treatment restored plasma IGF-1 levels to values similar to young males. Aging was associated with a significant increase in lipid peroxidation (LPO), nitric oxide (NO), carbon monoxide (CO) and cyclic guanosyl-monophosphate (cGMP), as well as a reduction in adenosyl triphosphate (ATP) and phosphatidylcholine (PC) synthesis. GH administration partially prevented all these changes in males. In females, some of the parameters were significantly improved by GH (ATP, CO, cGMP), while others showed a tendency to improvement, although differences did not reach significance. In conclusion, GH administration could exert beneficial effects against age-related changes in hepatocytes, mainly in males.

  4. Hepatocyte Growth Factor Is Required for Mesenchymal Stromal Cell Protection Against Bleomycin-Induced Pulmonary Fibrosis

    PubMed Central

    Cahill, Emer F.; Kennelly, Helen; Carty, Fiona; Mahon, Bernard P.

    2016-01-01

    The incidence of idiopathic pulmonary fibrosis is on the rise and existing treatments have failed to halt or reverse disease progression. Mesenchymal stromal cells (MSCs) have potent cytoprotective effects, can promote tissue repair, and have demonstrated efficacy in a range of fibrotic lung diseases; however, the exact mechanisms of action remain to be elucidated. Chemical antagonists and short hairpin RNA knockdown were used to identify the mechanisms of action used by MSCs in promoting wound healing, proliferation, and inhibiting apoptosis. Using the bleomycin induced fibrosis model, the protective effects of early or late MSC administration were examined. The role for hepatocyte growth factor (HGF) in MSC protection against bleomycin lung injury was examined using HGF knockdown MSC. Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling assay was performed on ex vivo lung sections to examine the effects of MSC on apoptosis. MSC conditioned media (CM) enhanced wound closure and inhibited apoptosis of pulmonary cells in vitro. HGF was required for MSC CM enhancement of epithelial cell proliferation and inhibition of apoptosis. In contrast, MSC required COX-2 for CM to inhibit fibroblast proliferation. In a murine model, early administration of MSC protected against bleomycin induced lung fibrosis and correlated with reduced levels of the proinflammatory cytokine interleukin-1β, reduced levels of apoptosis, and significantly increased levels of HGF. These protective effects were in part mediated by MSC derived HGF as HGF knockdown MSC were unable to protect against fibrosis in vivo. These findings delineate the mechanisms of MSC protection in a preclinical model of fibrotic lung disease. Significance The mechanisms used by mesenchymal stromal cells (MSCs) in mediating protective effects in chronic models of lung disease are not understood and remain to be elucidated. These findings from in vitro studies highlight an important role for the MSC

  5. Cyclic adenosine monophosphate-mediated protection against bile acid-induced apoptosis in cultured rat hepatocytes.

    PubMed

    Webster, C R; Anwer, M S

    1998-05-01

    Cyclic adenosine monophosphate (cAMP) has been shown to modulate apoptosis. To evaluate the role of cAMP in bile acid-induced hepatocyte apoptosis, we studied the effect of agents that increase cAMP on the induction of apoptosis by glycochenodeoxycholate (GCDC) in cultured rat hepatocytes. GCDC induced apoptosis in 26.5%+/-1.1% of hepatocytes within 2 hours. Twenty-minute pretreatment of hepatocytes with 100 micromol/L 8-(4-chlorothiophenyl) cAMP (CP-cAMP) resulted in a reduction in the amount of apoptosis to 35.2%+/-3.8% of that seen in hepatocytes treated with GCDC alone. Other agents that increase intracellular cAMP, including dibutyryl cAMP (100 micromol/L), glucagon (200 nmol/L), and a combination of forskolin (20 micromol/L) and 3-isobutyl-1-methylxanthine (20 micromol/L), also inhibited GCDC-induced apoptosis to a similar extent. Pretreatment with the protein kinase A (PKA) inhibitor, KT5720, prevented the protective effect of CP-cAMP and inhibited CP-cAMP-induced activation of PKA activity. Inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin (50 nmol/L), or Ly 294002 (20 micromol/L) also prevented the cytoprotective effect of cAMP. PI3K assays confirmed that wortmannin (50 nmol/L) inhibited PI3K activity, while CP-cAMP had no effect on the activity of this lipid kinase. GCDC increased mitogen-activated protein kinase (MAPK) activity, but had no effect on stress-activated protein kinase (SAPK) activity in hepatocytes. cAMP decreased basal and GCDC-induced MAPK activity and increased SAPK activity. The MAPK kinase inhibitor, PD 98059, inhibited both GCDC-mediated MAPK activation and GCDC-induced apoptosis. 1) agents that increase intracellular cAMP protect against hepatocyte apoptosis induced by hydrophobic bile acids; 2) activation of MAPK by GCDC may be involved in bile acid-induced apoptosis; and 3) cAMP-mediated cytoprotection against bile acid-induced apoptosis appears to involve PKA, MAPK, and PI3K.

  6. 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

  7. 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.

  8. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.

    PubMed

    Storey, Stephen M; McIntosh, Avery L; Huang, Huan; Landrock, Kerstin K; Martin, Gregory G; Landrock, Danilo; Payne, H Ross; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

    2012-04-15

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2

  9. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes

    PubMed Central

    Storey, Stephen M.; McIntosh, Avery L.; Huang, Huan; Landrock, Kerstin K.; Martin, Gregory G.; Landrock, Danilo; Payne, H. Ross; Atshaves, Barbara P.; Kier, Ann B.

    2012-01-01

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2

  10. Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor

    SciTech Connect

    Matsumoto, K.; Hashimoto, K.; Yoshikawa, K.; Nakamura, T. )

    1991-09-01

    Effect of hepatocyte growth factor (HGF) on normal human epidermal keratinocytes cultured under conditions of low Ca2+ (0.1 mM, growth-promoting condition) and physiological Ca2+ (1.8 mM, differentiation-promoting condition) was investigated. In low Ca2+, HGF markedly enhanced the migration of keratinocytes while it suppressed cell growth and DNA synthesis in a dose-dependent manner. In contrast, HGF enhanced the migration, cell growth, and DNA synthesis of keratinocytes cultured under conditions of physiological Ca2+. The maximal stimulation of DNA synthesis (2.4-fold stimulation) in physiological Ca2+ was seen at 2.5-5 ng/ml HGF and the stimulatory effect of HGF was suppressed by transforming growth factor-beta 1. Analysis of the HGF receptor using 125I-HGF as a ligand showed that human keratinocytes expressed a single class of specific, saturable receptor for HGF in both low and physiological Ca2+ conditions, exhibiting a Kd = 17.3 pM and approximately 690 binding sites/cell under physiological Ca2+. Thus, HGF is a potent factor which enhances growth and migration of normal human keratinocytes under conditions of physiological Ca2+. HGF may play an important role in epidermal tissue repair as it enhances both the migration and growth of keratinocytes.

  11. 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.

  12. 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.

  13. Sphingosine kinase activation regulates hepatocyte growth factor induced migration of endothelial cells.

    PubMed

    Duan, Hai-Feng; Wu, Chu-Tse; Lu, Ying; Wang, Hua; Liu, Hong-Jun; Zhang, Qun-Wei; Jia, Xiang-Xu; Lu, Zhu-Zhuang; Wang, Li-Sheng

    2004-08-15

    Hepatocyte growth factor (HGF)-induced migration of endothelial cells is critical for angiogenesis. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms. The aim of this study was to investigate whether activation of SPK is involved in the migration of endothelial cells induced by HGF. The biological functions of HGF are mediated through the activation of its high-affinity tyrosine kinase receptor, c-met protooncogene. In the present study, Treatment of ECV304 endothelial cells with HGF resulted in tyrosine phosphorylation of c-Met and activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitor of the PI3K and ERK/MAPK pathways, respectively, blocked the HGF-induced activation of SPK. HGF stimulation significantly increased intracellular S1P level, but no detectable secretion of S1P into the cell culture medium was observed. Treatment of ECV304 cells with pertussis toxin (PTX) has no effect on the HGF-induced migration, indicating extracellular S1P is dispensable for this process. Overexpression of wild-type SPK gene in ECV 304 cells increased the intracellular S1P and enhanced the HGF-induced migration, whereas inhibition of cellular SPK activity by N,N-dimethylsphingosine (DMS), a potent inhibitor of SPK, or by expression of a dominant-negative SPK (DN-SK) blocked the HGF-induced migration of ECV 304 cells. It is suggested that PI3K and ERK/MAPK mediated the activation of SPK and would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which intracellularly generated S1P mediates signaling from HGF/c-Met to the endothelial cell migration.

  14. Integrated analysis of gene expression and DNA methylation changes induced by hepatocyte growth factor in human hepatocytes.

    PubMed

    Xie, Cheng-Rong; Sun, Hongguang; Wang, Fu-Qiang; Li, Zhao; Yin, Yi-Rui; Fang, Qin-Liang; Sun, Yu; Zhao, Wen-Xiu; Zhang, Sheng; Zhao, Wen-Xing; Wang, Xiao-Min; Yin, Zhen-Yu

    2015-09-01

    Hepatocellular carcinoma (HCC) is the one of most common malignant tumors. The tumor microenvironment has a role in not only supporting growth and survival of tumor cells, but also triggering tumor recurrence and metastasis. Hepatocyte growth factor (HGF), one of the important growth factors in the tumor microenvironment, has an important role in angiogenesis, tumorigenesis and regeneration. However, the exact mechanism by which HGF regulates HCC initiation and development via epigenetic reprogramming has remained elusive. The present study focused on the epigenetic modification and target tumor-suppressive genes of HGF treatment in HCC. Expression profiling and DNA methylation array were performed to investigate the function of HGF and examine global genomic DNA methylation changes, respectively. Integrated analysis of gene expression and DNA methylation revealed potential tumor suppressor genes (TSGs) in HCC. The present study showed the multiple functions of HGF in tumorous and non‑tumorous pathways and global genomic DNA methylation changes. HGF treatment upregulated the expression of DNA methyltransferase 1 (DNMT1). Overexpression of DNMT1 in HCC patients correlated with the malignant potential and poor prognosis of HCC. Furthermore, integration analysis of gene expression and DNA methylation changes revealed novel potential tumor suppressor genes TSGs including MYOCD, PANX2 and LHX9. The present study has provided mechanistic insight into epigenetic repression of TSGs through HGF‑induced DNA hypermethylation.

  15. Direct transfer of hepatocyte growth factor gene into kidney suppresses cyclosporin A nephrotoxicity in rats.

    PubMed

    Yazawa, Koji; Isaka, Yoshitaka; Takahara, Shiro; Imai, Enyu; Ichimaru, Naotsugu; Shi, Yi; Namba, Yukiomi; Okuyama, Akihiko

    2004-04-01

    The clinical utility of cyclosporin A (CsA) has been limited by its nephrotoxicity, which is characterized by tubular atrophy, interstitial fibrosis and progressive renal impairment. Hepatocyte growth factor (HGF), which plays diverse roles in the regeneration of the kidney following acute renal failure, has been reported to protect against and salvage renal injury by acting as a renotropic and anti-fibrotic factor. Here, we investigated protective effects of HGF gene therapy on CsA-induced nephrotoxicity by using an electroporation-mediated gene transfer method. CsA was orally administered as a daily dose of 30 mg/kg in male Sprague-Dawley rats receiving a low sodium diet (0.03% sodium). Plasmid vector encoding HGF (200 micro g) was transferred into the kidney by electroporation. HGF gene transfer resulted in significant increases in plasma HGF levels. Morphological assessment revealed that HGF gene transfer reduced CsA-induced initial tubular injury and inhibited interstitial infiltration of ED-1-positive macrophages. In addition, northern blot analysis demonstrated that cortical mRNA levels of TGF-beta and type I collagen were suppressed in the HGF group. Finally, HGF gene transfer significantly reduced striped interstitial phenotypic alterations and fibrosis in CsA-treated rats, as assessed by alpha-smooth muscle actin expression and Masson's trichrome staining. These results suggest that HGF may prevent CsA-induced tubulointerstitial fibrosis, indicating that HGF gene transfer may provide a potential strategy for preventing renal fibrosis.

  16. HIGH GLUCOSE POTENTIATES L-FABP MEDIATED FIBRATE INDUCTION OF PPARα IN MOUSE HEPATOCYTES

    PubMed Central

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

    2013-01-01

    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 (6 mM) glucose, fibrates (bezafibrate, fenofibrate) only weakly activated PPARα transcription of genes in LCFA β-oxidation; 2) High (11–20 mM) 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. PMID:23747828

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

    PubMed

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

    1994-11-01

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

  18. Hepatocyte growth factor and transforming growth factor beta regulate 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in rat hepatocyte primary cultures.

    PubMed Central

    Joaquin, M; Rosa, J L; Salvadó, C; López, S; Nakamura, T; Bartrons, R; Gil, J; Tauler, A

    1996-01-01

    Hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta) are believed to be of major importance for hepatic regeneration after liver damage. We have studied the effect of these growth factors on fructose 2,6-bisphosphate (Fru-2,6-P2) levels and the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-BPase) in rat hepatocyte primary cultures. Our results demonstrate that HGF activates the expression of the 6PF2K/Fru-2,6-BPase gene by increasing the levels of its mRNA. As a consequence of this activation, the amount of 6PF2K/Fru-2,6-BPase protein and 6-phosphofructo-2-kinase activity increased, which was reflected by a rise in Fru-2,6-P2 levels. In contrast, TGF-beta decreased the levels of 6PF2K/Fru-2,6-BPase mRNA, which led to a decrease in the amount of 6PF2K/Fru-2,6-BPase protein and Fru-2,6-P2. The different actions of HGF and TGF-beta on 6PF2K/Fru-2,6-BPase gene expression are concomitant with their effect on cell proliferation. Here we show that, in the absence of hormones, primary cultures of hepatocytes express the F-type isoenzyme. In addition, HGF increases the expression of this isoenzyme, and dexamethasone activates the L-type isoform. HGF and TGF-beta were able to inhibit this activation. PMID:8660288

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

    PubMed

    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

    2015-06-29

    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.

  20. Impairment of Transforming Growth Factor β Signaling in Caveolin-1-deficient Hepatocytes

    PubMed Central

    Mayoral, Rafael; Valverde, Ángela M.; Llorente Izquierdo, Cristina; González-Rodríguez, Águeda; Boscá, Lisardo; Martín-Sanz, Paloma

    2010-01-01

    Caveolin-1 (Cav-1) is the main structural protein of caveolae and plays an important role in various cellular processes such as vesicular transport, cholesterol homeostasis, and signal transduction pathways. The expression and functional role of Cav-1 have been reported in liver and in hepatocyte cell lines, in human cirrhotic liver, and in hepatocellular carcinomas. Previous studies demonstrated that Cav-1 was dispensable for liver regeneration, because Cav-1−/− animals survived and fully regenerated liver function and size after partial hepatectomy. In this study, we have investigated the mechanisms by which the lack of Cav-1 accelerates liver regeneration after partial hepatectomy. The data show that transforming growth factor β (TGF-β) signaling is impaired in regenerating liver of Cav-1−/− mice and in hepatocytes derived from these animals. TGF-β receptors I and II do not colocalize in the same membrane fraction in the hepatocytes derived from Cav-1−/− mice, as Smad2/3 signaling decreased in the absence of Cav-1 at the time that the transcriptional corepressor SnoN accumulates. Accordingly, the expression of TGF-β target genes, such as plasminogen activator inhibitor-1, is decreased due to the presence of the high levels of SnoN. Moreover, hepatocyte growth factor inhibited TGF-β signaling in the absence of Cav-1 by increasing SnoN expression. Taken together, these data might help to unravel why Cav-1-deficient mice exhibit an accelerated liver regeneration after partial hepatectomy and add new insights on the molecular mechanisms controlling the initial commitment to hepatocyte proliferation. PMID:19966340

  1. Effect of growth hormone and estrogen administration on hepatocyte alterations in old ovariectomized female wistar rats.

    PubMed

    Castillo, Carmen; Salazar, Veronica; Ariznavarreta, Carmen; Vara, Elena; Tresguerres, Jesus A F

    2005-02-01

    Aging could be due to the accumulation of oxidative damage. On the other hand, growth hormone (GH) and estrogen deficiency induce deleterious effects on different tissues, and hormonal replacement could counteract these effects. We have investigated whether GH and estrogen administration modify some parameters related to oxidative stress and inflammation in hepatocytes isolated from old ovariectomized female rats. Twenty-two month-old ovariectomized animals were divided into control rats, rats treated with GH, rats treated with estradiol, and rats treated with GH+estradiol. Two-month-old intact female rats were used as young reference group. Hepatocytes were isolated, cultured, and CO and NO release, ATP, cyclic-guanosyl monophosphate (cGMP), and lipid peroxide (LPO) content of cells, as well as phosphatidylcholine (PC)synthesis, were measured. Hepatocytes isolated from old ovariectomized rats showed a decrease in ATP content and PC synthesis compared to young rats. Age also induced an increase in LPO, NO, CO, and cGMP. Treating old rats with GH significantly increased ATP and reduced CO and cGMP levels. Estradiol administration improved all the parameters that were altered. Co-administration of GH and estrogens induced a more marked effect than estrogens alone only in cGMP content. In conclusion, administration of estrogens to old ovariectomized females seemed to prevent oxidative changes in hepatocytes, whereas the effect of GH is not so evident.

  2. Targeted gene transfer of human hepatocyte growth factor into rat skin by the use of laser-induced stress waves

    NASA Astrophysics Data System (ADS)

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

    2006-02-01

    We successfully delivered therapeutic gene, Hepatocyte Growth Factor (HGF), to rat skin in vivo. The level of hHGF protein increased by the application of LISWs when compared with that of control samples without LISW application.

  3. Gene transfer into hepatocytes mediated by herpes simplex virus-Epstein-Barr virus hybrid amplicons.

    PubMed

    Müller, Lars; Saydam, Okay; Saeki, Yoshinaga; Heid, Irma; Fraefel, Cornel

    2005-01-01

    Gene transfer into hepatocytes is highly desirable for the long-term goal of replacing deficient proteins and correcting metabolic disorders. Vectors based on herpes simplex virus type-1 (HSV-1) have been demonstrated to mediate efficient gene transfer into hepatocytes both in vitro and in vivo. Large transgene capacity and extrachromosomal persistence make HSV-1/EBV hybrid amplicon vectors an attractive candidate for hepatic gene replacement therapy. To assess liver-directed gene transfer, we constructed (i) a conventional HSV-1 amplicon vector encoding a secreted reporter protein (secreted alkaline phosphatase, SEAP) under the control of the HSV-1 immediate-early 4/5 promoter; (ii) a HSV-1 amplicon encoding SEAP under the control of the artificial CAG promoter (the chicken beta-actin promoter and cytomegalovirus (CMV) immediate-early enhancer); and (iii) a HSV-1/EBV hybrid amplicon, also encoding SEAP under the control of the CAG promoter. While all three vector constructs yielded high SEAP concentrations in vitro and in vivo, use of HSV-1/EBV hybrid amplicon vectors significantly prolonged the duration of gene expression. Using conventional amplicon vectors in cultured hepatocytes, SEAP was detected for two weeks, whereas SEAP was detected for at least six weeks when HSV-1/EBV amplicons were used. Intraparenchymal injection into the liver of SICD mice yielded high (up to 77 ng of SEAP per milliliter serum) and sustained (greater than three weeks) expression of SEAP. Serum transaminases (ALT/AST) were measured at different time points to monitor for hepatocellular damage. While initially elevated four times above baseline, the transaminase levels returned to normal after three to seven days. These results demonstrate the usefulness of HSV-1-based amplicons and SEAP for the evaluation of gene replacement strategies in the liver.

  4. Growth Hormone Mediates Its Protective Effect in Hepatic Apoptosis through Hnf6

    PubMed Central

    Wang, Kewei; Wang, Minhua; Gannon, Maureen

    2016-01-01

    Background and Aims Growth hormone (GH) not only supports hepatic metabolism but also protects against hepatocyte cell death. Hnf6 (or Oc1) belonging to the Onecut family of hepatocyte transcription factors known to regulate differentiated hepatic function, is a GH-responsive gene. We evaluate if GH mediates Hnf6 activity to attenuate hepatic apoptotic injury. Methods We used an animal model of hepatic apoptosis by bile duct ligation (BDL) with Hnf6 -/- (KO) mice in which hepatic Hnf6 was conditionally inactivated. GH was administered to adult wild type WT and KO mice for the 7 days of BDL to enhance Hnf6 expression. In vitro, primary hepatocytes derived from KO and WT liver were treated with LPS and hepatocyte apoptosis was assessed with and without GH treatment. Results In WT mice, GH treatment enhanced Hnf6 expression during BDL, inhibited Caspase -3, -8 and -9 responses and diminished hepatic apoptotic and fibrotic injury. GH-mediated upregulation of Hnf6 expression and parallel suppression of apoptosis and fibrosis in WT BDL liver were abrogated in KO mice. LPS activated apoptosis and suppressed Hnf6 expression in primary hepatocytes. GH/LPS co-treatment enhanced Hnf6 expression with corresponding attenuation of apoptosis in WT-derived hepatocytes, but not in KO hepatocytes. ChiP-on-ChiP and electromobility shift assays of KO and WT liver nuclear extracts identified Ciap1 (or Birc2) as an Hnf6-bound target gene. Ciap1 expression patterns closely follow Hnf6 expression in the liver and in hepatocytes. Conclusion GH broad protective actions on hepatocytes during liver injury are effected through Hnf6, with Hnf6 transcriptional activation of Ciap1 as an underlying molecular mediator. PMID:27936029

  5. The therapeutic potential of hepatocyte growth factor for myocardial infarction and heart failure.

    PubMed

    Jin, Hongkui; Wyss, J Michael; Yang, Renhui; Schwall, Ralph

    2004-01-01

    Hepatocyte growth factor (HGF) is a cytokine whose multipotent actions are mediated by c-Met receptor. This review focuses on effects of HGF on myocardial infarction (MI) and heart failure. Circulating concentrations of HGF and myocardial concentrations of HGF and c-Met mRNA and protein are substantially increased following acute MI. HGF has been shown to be cardioprotective towards acute cardiac ischemia-reperfusion injury. Gene transfection of HGF into rat hearts attenuates acute ischemia injury. Administration of HGF protein reduces infarct size and increases cardiac performance in a rat model of acute ischemia/reperfusion. In contrast, acute blockade of endogenous HGF increases infarct size and mortality. These acute effects of HGF appear to be related to angiogenic and anti-apoptotic mechanisms. Recent studies demonstrate that post-MI treatment with HGF gene or protein attenuates chronic cardiac remodeling and dysfunction. In rats, HGF gene transfer following large MI results in preserved cardiac function and geometry in association with angiogenesis and reduced apoptosis, and treatment with recombinant HGF also significantly improves cardiac performance measured 8 weeks after MI. In mice, post-MI HGF gene therapy improves cardiac remodeling and dysfunction through hypertrophy of cardiomyocytes, infarct wall thickening, preservation of vessels, and antifibrosis. In addition, gene transfer of HGF improves cardiac remodeling, angiogenesis and regional myocardial function in the chronic ischemic myocardium of dogs. Together, these preclinical data highlight the significant acute and chronic cardioprotective effects of HGF following ischemic heart failure. Clinical trials are needed to investigate the therapeutic potential of HGF for postinfarction heart failure in humans.

  6. Natural pyrethrins induces apoptosis in human hepatocyte cells via Bax- and Bcl-2-mediated mitochondrial pathway.

    PubMed

    Yang, Yun; Zong, Mimi; Xu, Wenping; Zhang, Yang; Wang, Bo; Yang, Mingjun; Tao, Liming

    2017-01-25

    Natural pyrethrins have been widely used for pest control in organic farming and for residential indoor pest managements. Although the specific mechanisms underlying their activity are incompletely understood, natural pesticides are considered the safest based on their target specificity and rapid degradation in the environment. Here, we used in vitro bioassays to characterize the cytotoxic effects of natural pyrethrins and attempted to delineate the cellular and molecular mechanisms of their cytotoxicity against human hepatocytes. The results demonstrate that natural pyrethrins reduce cell viability and enhance apoptosis in HepG2 cells. In addition, the current data indicate that natural pyrethrins cause a reduction in the mitochondrial membrane potential (Δψm), increase reactive oxygen species production, and up-regulate the Bax/Bcl-2 expression, leading to the release of cytochrome-c into the cytosol, activation of caspase-9 and caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). Taken together, the results indicate that natural pyrethrins has potentially exert adverse effects on human health by inducing caspase-dependent apoptosis in hepatocytes through Bax- and Bcl-2-mediated mitochondrial pathway.

  7. 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

  8. Development of a chemically defined medium and discovery of new mitogenic growth factors for mouse hepatocytes: mitogenic effects of FGF1/2 and PDGF.

    PubMed

    Bowen, William C; Michalopoulos, Amantha W; Orr, Anne; Ding, Michael Q; Stolz, Donna B; Michalopoulos, George K

    2014-01-01

    Chemically defined serum-free media for rat hepatocytes have been useful in identifying EGFR ligands and HGF/MET signaling as direct mitogenic factors for rat hepatocytes. The absence of such media for mouse hepatocytes has prevented screening for discovery of such mitogens for mouse hepatocytes. We present results obtained by designing such a chemically defined medium for mouse hepatocytes and demonstrate that in addition to EGFR ligands and HGF, the growth factors FGF1 and FGF2 are also important mitogenic factors for mouse hepatocytes. Smaller mitogenic response was also noticed for PDGF AB. Mouse hepatocytes are more likely to enter into spontaneous proliferation in primary culture due to activation of cell cycle pathways resulting from collagenase perfusion. These results demonstrate unanticipated fundamental differences in growth biology of hepatocytes between the two rodent species.

  9. 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.

  10. Targeted gene transfer of hepatocyte growth factor to alveolar type II epithelial cells reduces lung fibrosis in rats.

    PubMed

    Gazdhar, Amiq; Temuri, Almas; Knudsen, Lars; Gugger, Mathias; Schmid, Ralph A; Ochs, Matthias; Geiser, Thomas

    2013-01-01

    Inefficient alveolar wound repair contributes to the development of pulmonary fibrosis. Hepatocyte growth factor (HGF) is a potent growth factor for alveolar type II epithelial cells (AECII) and may improve repair and reduce fibrosis. We studied whether targeted gene transfer of HGF specifically to AECII improves lung fibrosis in bleomycin-induced lung fibrosis. A plasmid encoding human HGF expressed from the human surfactant protein C promoter (pSpC-hHGF) was designed, and extracorporeal electroporation-mediated gene transfer of HGF specifically to AECII was performed 7 days after bleomycin-induced lung injury in the rat. Animals were killed 7 days after hHGF gene transfer. Electroporation-mediated HGF gene transfer resulted in HGF expression specifically in AECII at biologically relevant levels. HGF gene transfer reduced pulmonary fibrosis as assessed by histology, hydroxyproline determination, and design-based stereology compared with controls. Our results indicate that the antifibrotic effect of HGF is due in part to a reduction of transforming growth factor-β(1), modulation of the epithelial-mesenchymal transition, and reduction of extravascular fibrin deposition. We conclude that targeted HGF gene transfer specifically to AECII decreases bleomycin-induced lung fibrosis and may therefore represent a novel cell-specific gene transfer technology to treat pulmonary fibrosis.

  11. Loss of wild-type carrier-mediated L-carnitine transport activity in hepatocytes of juvenile visceral steatosis mice.

    PubMed

    Yokogawa, K; Yonekawa, M; Tamai, I; Ohashi, R; Tatsumi, Y; Higashi, Y; Nomura, M; Hashimoto, N; Nikaido, H; Hayakawa, J; Nezu, J; Oku, A; Shimane, M; Miyamoto, K; Tsuji, A

    1999-10-01

    Juvenile visceral steatosis (JVS) mice, which show systemic L-carnitine deficiency, may be an animal model of Reye's syndrome because of its phenotype of fat deposition and mitochondrial abnormalities in the liver. In this study, we compared the characteristics of the L-carnitine transport in isolated hepatocytes from wild-type and JVS mice. The uptake of L-carnitine by wild-type hepatocytes was saturable and the Eadie-Hofstee plot showed 2 distinct components. The apparent Michaelis constant (K(m)) and the maximum transport rate (V(max)) were 4.6 micromol/L and 59.5 pmol/15 min/10(6) cells, respectively, for the high-affinity component, and 404 micromol/L and 713 pmol/15 min/10(6) cells, respectively, for the low-affinity component. The high-affinity L-carnitine uptake occurred via an active carrier-mediated transport mechanism, which is characterized by Na(+)-, energy-, and pH-dependency. On the other hand, the high-affinity uptake was absent in JVS hepatocytes, and the values of K(m) and V(max) for the low-affinity uptake were 475 micromol/L and 557 pmol/15 min/10(6) cells, respectively. The hepatic carnitine transport properties in wild-type hepatocytes were similar to those of high-affinity mouse Octn2-transfected HEK293 cells. This study suggests that Octn2-type carnitine transporter is dysfunctional in hepatocytes of JVS mice.

  12. Inhibition of Drp1 protects against senecionine-induced mitochondria-mediated apoptosis in primary hepatocytes and in mice.

    PubMed

    Yang, Xiao; Wang, Hua; Ni, Hong-Min; Xiong, Aizhen; Wang, Zhengtao; Sesaki, Hiromi; Ding, Wen-Xing; Yang, Li

    2017-03-02

    Pyrrolizidine alkaloids (PAs) are a group of compounds found in various plants and some of them are widely consumed in the world as herbal medicines and food supplements. PAs are potent hepatotoxins that cause irreversible liver injury in animals and humans. However, the mechanisms by which PAs induce liver injury are not clear. In the present study, we determined the hepatotoxicity and molecular mechanisms of senecionine, one of the most common toxic PAs, in primary cultured mouse and human hepatocytes as well as in mice. We found that senecionine administration increased serum alanine aminotransferase levels in mice. H&E and TUNEL staining of liver tissues revealed increased hemorrhage and hepatocyte apoptosis in liver zone 2 areas. Mechanistically, senecionine induced loss of mitochondrial membrane potential, release of mitochondrial cytochrome c as well as mitochondrial JNK translocation and activation prior to the increased DNA fragmentation and caspase-3 activation in primary cultured mouse and human hepatocytes. SP600125, a specific JNK inhibitor, and ZVAD-fmk, a general caspase inhibitor, alleviated senecionine-induced apoptosis in primary hepatocytes. Interestingly, senecionine also caused marked mitochondria fragmentation in hepatocytes. Pharmacological inhibition of dynamin-related protein1 (Drp1), a protein that is critical to regulate mitochondrial fission, blocked senecionine-induced mitochondrial fragmentation and mitochondrial release of cytochrome c and apoptosis. More importantly, hepatocyte-specific Drp1 knockout mice were resistant to senecionine-induced liver injury due to decreased mitochondrial damage and apoptosis. In conclusion, our results uncovered a novel mechanism of Drp1-mediated mitochondrial fragmentation in senecionine-induced liver injury. Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by PAs.

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

    PubMed Central

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

    2012-01-01

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

  14. 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

  15. Differential effects of hepatocyte growth factor and keratinocyte growth factor on corneal epithelial cell cycle protein expression, cell survival, and growth

    PubMed Central

    Chandrasekher, Gudiseva; Pothula, Swetha; Bazan, Haydee.E.P.

    2014-01-01

    Purpose Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are secreted in the cornea in response to injury. In this study, we investigated the HGF- and KGF-mediated effect on the expression of cell cycle and apoptosis controlling proteins, cell survival, and growth in the corneal epithelium to better understand the possible role of their signaling mechanisms in repairing epithelial injuries. Methods The cell survival capability of HGF and KGF in epithelial primary cultures was evaluated by using a staurosporine-induced apoptosis model. Apoptosis was quantified with image analysis following nuclear staining with Hoechst fluorescent dye and DNA laddering. Western immunoblotting was used to study the effect of growth factors on the expression of cell cycle- and apoptosis-regulating proteins. Results HGF and KGF protected cells from apoptosis for a short duration (10 h), but only KGF exhibited cell survival capability and maintained cell growth for a longer period (24 h). The onset of apoptosis was accompanied by a significant increase in cell cycle inhibitor p27kip. HGF and KGF suppressed p27kip levels in the apoptosis environment; however, KGF- but not HGF-dependent downregulation in p27kip expression was sustained for a longer duration. Inhibition of phosphatidylinositol 3-kinase/Akt activation blocked HGF- and KGF-mediated control of p27kip expression. Further, when compared to HGF, the presence of KGF produced significant downregulation of p53 and poly(adenosine diphosphate-ribose) polymerase, the key proteins involved in apoptosis and blocked the degradation of G1/S cell cycle progression checkpoint protein retinoblastoma. HGF and KGF upregulated the levels of p21cip, cyclins A, D, and E and cyclin-dependent kinases (CDK2 and CDK4) as well, but the KGF-mediated effect on the expression of these molecules lasted longer. Conclusions Sustained effect of KGF on cell survival and proliferation could be attributed to its ability to inhibit p53

  16. Differential effects of hepatocyte growth factor and keratinocyte growth factor on corneal epithelial cell cycle protein expression, cell survival, and growth.

    PubMed

    Chandrasekher, Gudiseva; Pothula, Swetha; Maharaj, Glenn; Bazan, Haydee E P

    2014-01-01

    Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are secreted in the cornea in response to injury. In this study, we investigated the HGF- and KGF-mediated effect on the expression of cell cycle and apoptosis controlling proteins, cell survival, and growth in the corneal epithelium to better understand the possible role of their signaling mechanisms in repairing epithelial injuries. The cell survival capability of HGF and KGF in epithelial primary cultures was evaluated by using a staurosporine-induced apoptosis model. Apoptosis was quantified with image analysis following nuclear staining with Hoechst fluorescent dye and DNA laddering. Western immunoblotting was used to study the effect of growth factors on the expression of cell cycle- and apoptosis-regulating proteins. HGF and KGF protected cells from apoptosis for a short duration (10 h), but only KGF exhibited cell survival capability and maintained cell growth for a longer period (24 h). The onset of apoptosis was accompanied by a significant increase in cell cycle inhibitor p27(kip). HGF and KGF suppressed p27(kip) levels in the apoptosis environment; however, KGF- but not HGF-dependent downregulation in p27(kip) expression was sustained for a longer duration. Inhibition of phosphatidylinositol 3-kinase/Akt activation blocked HGF- and KGF-mediated control of p27(kip) expression. Further, when compared to HGF, the presence of KGF produced significant downregulation of p53 and poly(adenosine diphosphate-ribose) polymerase, the key proteins involved in apoptosis and blocked the degradation of G1/S cell cycle progression checkpoint protein retinoblastoma. HGF and KGF upregulated the levels of p21(cip), cyclins A, D, and E and cyclin-dependent kinases (CDK2 and CDK4) as well, but the KGF-mediated effect on the expression of these molecules lasted longer. Sustained effect of KGF on cell survival and proliferation could be attributed to its ability to inhibit p53, retinoblastoma, caspases, and p

  17. 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

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

    PubMed Central

    Kapoor, Radhika; Kakkar, Poonam

    2012-01-01

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

  19. Glycyrrhizic acid attenuates CCl4-induced hepatocyte apoptosis in rats via a p53-mediated pathway

    PubMed Central

    Guo, Xiao-Ling; Liang, Bo; Wang, Xue-Wei; Fan, Fu-Gang; Jin, Jing; Lan, Rui; Yang, Jing-Hui; Wang, Xiao-Chun; Jin, Lei; Cao, Qin

    2013-01-01

    AIM: To investigate the effect of glycyrrhizic acid (GA) on carbon tetrachloride (CCl4)-induced hepatocyte apoptosis in rats via a p53-dependent mitochondrial pathway. METHODS: Forty-five male Sprague-Dawley rats were randomly and equally divided into three groups, the control group, the CCl4 group, and the GA treatment group. To induce liver fibrosis in this model, rats were given a subcutaneous injection of a 40% solution of CCl4 in olive oil at a dose of 0.3 mL/100 g body weight biweekly for 8 wk, while controls received the same isovolumetric dose of olive oil by hypodermic injection, with an initial double-dose injection. In the GA group, rats were also treated with a 40% solution of CCl4 plus 0.2% GA solution in double distilled water by the intraperitoneal injection of 3 mL per rat three times a week from the first week following previously published methods, with modifications. Controls were given the same isovolumetric dose of double distilled water. Liver function parameters, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. Pathologic changes in the liver were detected by hematoxylin and eosin staining. Collagen fibers were evaluated by Sirius red staining. Hepatocyte apoptosis was investigated using the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) assay and the cleaved caspase-3 immunohistochemistry assay. The expression levels of p53 and apoptosis-related proteins were evaluated by immunohistochemistry or Western blotting analysis. RESULTS: After 8 wk of treatment, GA significantly reduced serum activity of ALT (from 526.7 ± 57.2 to 342 ± 44.8, P < 0.05) and AST (from 640 ± 33.7 to 462.8 ± 30.6, P < 0.05), attenuated the changes in liver histopathology and reduced the staging score (from 3.53 ± 0.74 to 3.00 ± 0.76, P < 0.05) in CCl4-treated rats. GA markedly reduced the positive area of Sirius red and the ratio of the hepatic fibrotic region (from 7

  20. CD147 promotes liver fibrosis progression via VEGF-A/VEGFR2 signalling-mediated cross-talk between hepatocytes and sinusoidal endothelial cells.

    PubMed

    Yan, Zhaoyong; Qu, Kai; Zhang, Jing; Huang, Qichao; Qu, Ping; Xu, Xinsen; Yuan, Peng; Huang, Xiaojun; Shao, Yongping; Liu, Chang; Zhang, Hongxin; Xing, Jinliang

    2015-10-01

    Although previous evidence indicates close involvement of CD147 in the pathogenesis of liver fibrosis, the underlying molecular mechanisms and its therapeutic value remain largely unknown. In the present study, we investigated the biological roles of CD147 in liver fibrosis and assessed its therapeutic value as a target molecule in the CCl4-induced liver fibrosis mouse model. We found that CD147 was highly expressed in both hepatocytes and SECs (sinusoidal endothelial cells) in fibrotic liver tissues. Additionally, it was significantly associated with the fibrosis stage. TGF-β1 (transforming growth factor β1) was found to be mainly responsible for the up-regulation of CD147. Bioinformatic and experimental data suggest a functional link between CD147 expression and VEGF-A (vascular endothelial growth factor A)/VEGR-2 (VEGF receptor 2) signalling-mediated angiogenesis in fibrotic liver tissues. Furthermore, we observed that the CD147-induced activation of the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway promotes the production of VEGF-A in hepatocytes and expression of VEGFR-2 in SECs, which was found to enhance the angiogenic capability of SECs. Finally, our data indicate that blocking of CD147 using an mAb (monoclonal antibody) attenuated liver fibrosis progression via inhibition of VEGF-A/VEGFR-2 signalling and subsequent amelioration of microvascular abnormality in the CCl4-induced mouse model. Our findings suggest a novel functional mechanism that CD147 may promote liver fibrosis progression via inducing the VEGF-A/VEGFR-2 signalling pathway-mediated cross-talk between hepatocytes and SECs. New strategies based on the intervention of CD147 can be expected for prevention of liver fibrosis.

  1. FGL2/fibroleukin mediates hepatic reperfusion injury by induction of sinusoidal endothelial cell and hepatocyte apoptosis in mice.

    PubMed

    Selzner, Nazia; Liu, Hao; Boehnert, Markus U; Adeyi, Oyedele A; Shalev, Itay; Bartczak, Agata M; Xue-Zhong, Max; Manuel, Justin; Rotstein, Ori D; McGilvray, Ian D; Grant, David R; Phillips, Melville J; Levy, Gary A; Selzner, Markus

    2012-01-01

    Sinusoidal endothelial cell (SEC) and hepatocyte death are early, TNF-α mediated events in ischemia and reperfusion of the liver (I/Rp). We previously reported that TNF-α induced liver injury is dependent on Fibrinogen like protein 2 (FGL2/Fibroleukin) and showed that FGL2 binding to its receptor, FcγRIIB, results in lymphocyte apoptosis. In this study we examine whether I/Rp is induced by specific binding of FGL2 to FcγRIIB expressed on SEC. Hepatic ischemia and reperfusion was induced in wild type (WT) mice and in mice with deletion or inhibition of FGL2 and FcRIIB. Liver injury was determined by AST release, necrosis and animal death. Apoptosis was evaluated with caspase 3 and TUNEL staining. FGL2 deletion or inhibition resulted in decreased liver injury as determined by a marked reduction in both levels of AST and ALT and hepatocyte necrosis. Caspase 3 staining of SEC (12% vs. 75%) and hepatocytes (12% vs. 45%) as well as TUNEL staining of SEC (13% vs. 60%, p=0.02) and hepatocytes (18% vs. 70%, p=0.03), markers of apoptosis, were lower in Fgl2(-/-) compared to WT mice. In vitro incubation of SEC with FGL2 induced apoptosis of SEC from WT mice, but not FcγRIIB(-/-) mice. Deletion of FcγRIIB fully protected mice against SEC and hepatocyte death in vivo. Survival of mice deficient in either Fgl2(-/-) (80%) or FcγRIIB(-/-) (100%) was markedly increased compared to WT mice (10%) which were subjected to 75min of total hepatic ischemia (p=0.001). FGL2 binding to the FcγRIIB receptor expressed on SEC is a critical event in the initiation of the hepatic reperfusion injury cascade through induction of SEC and hepatocyte death. Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  2. Association of the Hepatocyte Growth Factor Gene with Keratoconus in an Australian Population

    PubMed Central

    Sahebjada, Srujana; Schache, Maria; Richardson, Andrea J.; Snibson, Grant; Daniell, Mark; Baird, Paul N.

    2014-01-01

    Purpose A previous study has indicated suggestive association of the hepatocyte growth factor (HGF) gene with Keratoconus. We wished to assess this association in an independent Caucasian cohort as well as assess its association with corneal curvature. Participants Keratoconus patients were recruited from private and public clinics in Melbourne, Australia. Non-keratoconic individuals were identified from the Genes in Myopia (GEM) study from Australia. A total of 830 individuals were used for the analysis including 157 keratoconic and 673 non keratoconic subjects. Methods Tag single nucleotide polymorphisms (tSNPs) were chosen to encompass the hepatocyte growth factor gene as well as 2 kb upstream of the start codon through to 2 kb downstream of the stop codon. Logistic and linear regression including age and gender as covariates were applied in statistical analysis with subsequent Bonferroni correction. Results Ten tSNPs were genotyped. Following statistical analysis and multiple testing correction, a statistically significant association was found for the tSNP rs2286194 {p = 1.1×10-3 Odds Ratio 0.52, 95% CI - 0.35, 0.77} for keratoconus. No association was found between the 10 tSNPs and corneal curvature. Conclusions These findings provide additional evidence of significant association of the HGF gene with Keratoconus. This association does not appear to act through the corneal curvature route. PMID:24416191

  3. Role of Hepatocyte Growth Factor in the Immunomodulation Potential of Amniotic Fluid Stem Cells

    PubMed Central

    Maraldi, Tullia; Guida, Marianna; Zavatti, Manuela; De Pol, Anto

    2015-01-01

    Human amniotic fluid stem cells (hAFSCs) may be useful for regenerative medicine because of their potential to differentiate into all three germ layers and to modulate immune response with different types of secretion molecules. This last issue has not been completely elucidated. The aim of this study was to investigate the secretome profile of the hAFSC, focusing on the role of hepatocyte growth factor (HGF) in immunoregulation through short and long cocultures with human peripheral blood mononuclear cells. We found that HGF produced by hAFSCs exerts a cytoprotective role, inducing an increase in caspase-dependent apoptosis in human immune cells. This study provides evidence supporting the hypothesis that amniotic fluid is an ideal source of stem cells for expansion and banking properties for therapeutic use. hAFSCs not only are less immunogenic but also can secrete immunoregulatory factors that may be useful in autoimmune diseases or allogenic implants. Significance New information about the secretome pattern is reported in this paper. Human amniotic fluid stem cells (hAFSCs) possess immunomodulatory properties involving hepatocyte growth factor production. hAFSCs could be used in immunotherapies and might be able to avoid allogenic rejection. PMID:25873747

  4. The impairment of hepatocytes and sinusoidal endothelial cells during cold preservation in rat fatty liver induced by alcohol and the beneficial effect of hepatocyte growth factor.

    PubMed

    Takeda, Yoshihisa; Arii, Shigeki; Kaido, Toshimi; Imamura, Masayuki

    2003-04-01

    A fatty liver resulting from alcohol intake is often unattractive for grafting. In this study, we investigated the impairment of hepatocytes and sinusoidal endothelial cells (SECs) during cold preservation of alcohol-induced fatty liver and examined the efficacy of human recombinant hepatocyte growth factor (hrHGF). Rats were fed an alcohol diet. We performed histological examinations of the hepatocytes and observed the ultrastructural alteration of the SECs. Additionally, we measured hepatic transaminase and peroxidative lipids for hepatocellular injury and the hyaluronic acid uptake rate (HUR) to determine SEC injury. We added hrHGF to University of Wisconsin (UW) solution to assess the protective effect of the agent. Numerous fatty deposits were observed in ethanol-induced fatty livers. These grew with the duration of cold storage. Hepatic transaminases of the effluents increased during cold preservation in the livers of alcohol-treated rats. Additionally, peroxidative lipids in the effluents increased during cold preservation in the livers of alcohol-treated rats, whereas they were undetectable in non-alcohol-treated rat livers. The sinusoidal endothelium had severely deteriorated in the livers of alcohol-treated rats. Further, the HUR decreased with ethanol treatment and/or cold preservation. The addition of hrHGF suppressed the increase of hepatic transaminase in the effluent of cold-preserved alcohol-treated livers. Peroxidative lipids in the same effluents were undetectable. In fatty livers, both hepatocytes and SECs received severe damage during cold preservation. Furthermore, we demonstrated that hepatocellular injury was significantly inhibited by hrHGF.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  6. The specialized pro-resolving lipid mediator maresin 1 protects hepatocytes from lipotoxic and hypoxia-induced endoplasmic reticulum stress.

    PubMed

    Rius, Bibiana; Duran-Güell, Marta; Flores-Costa, Roger; López-Vicario, Cristina; Lopategi, Aritz; Alcaraz-Quiles, José; Casulleras, Mireia; Lozano, Juan José; Titos, Esther; Clària, Joan

    2017-08-02

    Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) are hallmarks of nonalcoholic fatty liver disease (NAFLD), which is the hepatic manifestation of the metabolic syndrome associated with obesity. The specialized pro-resolving lipid mediator maresin 1 (MaR1) preserves tissue homeostasis by exerting cytoprotective actions, dampening inflammation, and expediting its timely resolution. Here, we explored whether MaR1 protects liver cells from lipotoxic and hypoxia-induced ER stress. Mice were rendered obese by high-fat diet feeding, and experiments were performed in primary hepatocytes, Kupffer cells, and precision-cut liver slices (PCLSs). Palmitate-induced lipotoxicity increased ER stress and altered autophagy in hepatocytes, effects that were prevented by MaR1. MaR1 protected hepatocytes against lipotoxicity-induced apoptosis by activating the UPR prosurvival mechanisms and preventing the excessive up-regulation of proapoptotic pathways. Protective MaR1 effects were also seen in hepatocytes challenged with hypoxia and TNF-α-induced cell death. High-throughput microRNA (miRNA) sequencing revealed that MaR1 actions were associated with specific miRNA signatures targeting both protein folding and apoptosis. MaR1 also prevented lipotoxic-triggered ER stress and hypoxia-induced inflammation in PCLSs and enhanced Kupffer cell phagocytic capacity. Together, these findings describe the ability of MaR1 to oppose ER stress in liver cells under conditions frequently encountered in NAFLD.-Rius, B., Duran-Güell, M., Flores-Costa, R., López-Vicario, C., Lopategi, A., Alcaraz-Quiles, J., Casulleras, M., Lozano, J. J., Titos, E., Clària, J. The specialized pro-resolving lipid mediator maresin 1 protects hepatocytes from lipotoxic and hypoxia-induced endoplasmic reticulum stress. © FASEB.

  7. Apelin protects against liver X receptor-mediated steatosis through AMPK and PPARα in human and mouse hepatocytes.

    PubMed

    Huang, Jin; Kang, Saeromi; Park, Soo-Jin; Im, Dong-Soon

    2017-11-01

    Non-alcoholic fatty liver disease is the most commonly occurring chronic liver disease, and hepatic steatosis, a condition defined as extensive lipid accumulation in hepatocytes, is associated with liver dysfunction and metabolic diseases, such as, obesity and type II diabetes. Apelin is an adipokine that acts on a G protein-coupled receptor named APJ, and has been established to play pivotal roles in various physiological conditions. However, the function of apelin in hepatocytes has not been fully investigated. In order to assess the functional roles of apelin and APJ in hepatocytes, we used an in vitro model of liver X receptor (LXR)-mediated hepatocellular steatosis. In Hep3B human hepatoma cells, T0901317 (a specific LXR activator) induced lipid accumulation and this was inhibited by apelin. T0901317 also induced the expression of SREBP-1c, a key transcription factor for lipogenesis. Apelin not only inhibited SREBP-1c induction at the mRNA and protein levels but also induced lipolytic PPARα expression. Furthermore, these protective effects of apelin were inhibited by apelin F13A (a specific APJ antagonist). Furthermore, silencing of APJ by siRNA transfection also inhibited the actions of apelin. Specific inhibitors of cellular signaling components showed inhibition of lipid accumulation by apelin was mediated through Gi/o proteins, AMPK, and SREBP-1c suppression during the early stage and through AMPK, ERKs, and PPARα induction during the late stage. In addition, the protective effect of apelin was confirmed in mouse primary hepatocytes. Our observations suggest apelin-APJ signaling in hepatocytes functions to protect against lipid accumulation in liver through two signaling pathways, that is, via AMPK activation and PPARα induction. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Epidermal growth factor counteracts the glycogenic effect of insulin in parenchymal hepatocyte cultures.

    PubMed Central

    Chowdhury, M H; Agius, L

    1987-01-01

    Rat parenchymal hepatocytes in monolayer culture were used to study the metabolic effects of epidermal growth factor (EGF) and insulin on ketogenesis, gluconeogenesis and glycogen metabolism. EGF, unlike insulin, did not inhibit ketogenesis from palmitate or gluconeogenesis from pyruvate in hepatocyte cultures. It also had no effect on these pathways in the presence of insulin. In contrast, EGF potently counteracted the stimulation of [14C]pyruvate incorporation into glycogen by insulin, and also glycogen deposition from both gluconeogenic precursors and glucose. The EGF concentration causing half-maximal effect was about 0.1 nM. The anti-glycogenic effect of EGF was observed after both long-term (24 h) and short-term (1 h) exposure to EGF, and was more marked in the presence of insulin than in its absence. EGF did not displace bound insulin, suggesting that it neither competes for the insulin receptor nor affects the affinity of the receptor for insulin. EGF did not alter cellular cyclic AMP; and inhibition of cyclic AMP phosphodiesterase activity did not prevent the anti-glycogenic effect of EGF. In liver-derived dividing epithelial cells, Hep-G2 cells and fibroblasts, which have no capacity for gluconeogenesis, EGF did not counteract the stimulatory effect of insulin on [14C]glucose incorporation into glycogen, and in the epithelial cells EGF increased [14C]glucose incorporation into glycogen. The counter-effect of EGF on the glycogenic action of insulin in parenchymal hepatocytes may be due to a direct effect on glycogen metabolism or to an interaction with the post-receptor events in insulin action. PMID:2827626

  9. Perioperative hepatocyte growth factor (HGF) infusions improve hepatic regeneration following portal branch ligation (PBL) in rodents.

    PubMed

    Mangieri, Christopher W; McCartt, Jason C; Strode, Matthew A; Lowry, John E; Balakrishna, Prasad M

    2017-07-01

    As hepatic surgery has become safer and more commonly performed, the extent of hepatic resections has increased. When there is not enough expected hepatic reserve to facilitate primary resection of hepatic tumors, a clinical adjunct to facilitating primary resection is portal vein embolization (PVE). PVE allows the hepatic remnant to increase to an appropriate size prior to resection via hepatocyte regeneration; however, PVE is not always successful in facilitating adequate regeneration. One of the strongest trophic factors for hepatocyte regeneration is hepatocyte growth factor (HGF). The purpose of this study was to improve hepatic regeneration with perioperative HGF infusions in an animal model that mimics PVE. Portal branch ligation (PBL) in rodents is equivalent to PVE in humans. We performed left-sided PBL in Sprague-Dawley rodents with the experimental group receiving perioperative HGF infusions. Baseline and postoperative liver volumetrics were obtained with CT scanning methods as performed in clinical practice. Baseline and postoperative liver functions were assessed via indocyanine green (ICG) elimination testing. HGF infused rodents had statistically significant increase in all postoperative liver volumetrics. Most clinically relevant were increased right liver volumes (RLV), 14.10 versus 7.85 cm(3) (p value 0.0001), and increased degree of hypertrophy (DH %), 159.23 versus 47.11 % (p value 0.0079). HGF infused rodents also had a quick return to baseline liver function, 2.38 days compared to 6.13 days (p value 0.0001). Perioperative HGF infusions significantly increase hepatic regeneration following PBL in rodents. Perioperative HGF infusions following PVE are a possible adjunct to increase the amount of patients able to successfully undergo primary resection for hepatic tumors. Further basic science is warranted in examining the use of HGF infusions to increase hepatic regeneration and translating that basic science work to clinical practice.

  10. 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.

  11. Hepatocyte Hypoxia Inducible Factor-1 Mediates the Development of Liver Fibrosis in a Mouse Model of Nonalcoholic Fatty Liver Disease.

    PubMed

    Mesarwi, Omar A; Shin, Mi-Kyung; Bevans-Fonti, Shannon; Schlesinger, Christina; Shaw, Janet; Polotsky, Vsevolod Y

    2016-01-01

    Obstructive sleep apnea (OSA) is associated with the progression of non-alcoholic fatty liver disease (NAFLD) to steatohepatitis and fibrosis. This progression correlates with the severity of OSA-associated hypoxia. In mice with diet induced obesity, hepatic steatosis leads to liver tissue hypoxia, which worsens with exposure to intermittent hypoxia. Emerging data has implicated hepatocyte cell signaling as an important factor in hepatic fibrogenesis. We hypothesized that hepatocyte specific knockout of the oxygen sensing α subunit of hypoxia inducible factor-1 (HIF-1), a master regulator of the global response to hypoxia, may be protective against the development of liver fibrosis. Wild-type mice and mice with hepatocyte-specific HIF-1α knockout (Hif1a-/-hep) were fed a high trans-fat diet for six months, as a model of NAFLD. Hepatic fibrosis was evaluated by Sirius red stain and hydroxyproline assay. Liver enzymes, fasting insulin, and hepatic triglyceride content were also assessed. Hepatocytes were isolated from Hif1a-/-hep mice and wild-type controls and were exposed to sustained hypoxia (1% O2) or normoxia (16% O2) for 24 hours. The culture media was used to reconstitute type I collagen and the resulting matrices were examined for collagen cross-linking. Wild-type mice on a high trans-fat diet had 80% more hepatic collagen than Hif1a-/-hep mice (2.21 μg collagen/mg liver tissue, versus 1.23 μg collagen/mg liver tissue, p = 0.03), which was confirmed by Sirius red staining. Body weight, liver weight, mean hepatic triglyceride content, and fasting insulin were similar between groups. Culture media from wild-type mouse hepatocytes exposed to hypoxia allowed for avid collagen cross-linking, but very little cross-linking was seen when hepatocytes were exposed to normoxia, or when hepatocytes from Hif1a-/-hep mice were used in hypoxia or normoxia. Hepatocyte HIF-1 mediates an increase in liver fibrosis in a mouse model of NAFLD, perhaps due to liver tissue

  12. Hepatocyte Hypoxia Inducible Factor-1 Mediates the Development of Liver Fibrosis in a Mouse Model of Nonalcoholic Fatty Liver Disease

    PubMed Central

    Mesarwi, Omar A.; Shin, Mi-Kyung; Bevans-Fonti, Shannon; Schlesinger, Christina; Shaw, Janet; Polotsky, Vsevolod Y.

    2016-01-01

    Background Obstructive sleep apnea (OSA) is associated with the progression of non-alcoholic fatty liver disease (NAFLD) to steatohepatitis and fibrosis. This progression correlates with the severity of OSA-associated hypoxia. In mice with diet induced obesity, hepatic steatosis leads to liver tissue hypoxia, which worsens with exposure to intermittent hypoxia. Emerging data has implicated hepatocyte cell signaling as an important factor in hepatic fibrogenesis. We hypothesized that hepatocyte specific knockout of the oxygen sensing α subunit of hypoxia inducible factor-1 (HIF-1), a master regulator of the global response to hypoxia, may be protective against the development of liver fibrosis. Methods Wild-type mice and mice with hepatocyte-specific HIF-1α knockout (Hif1a-/-hep) were fed a high trans-fat diet for six months, as a model of NAFLD. Hepatic fibrosis was evaluated by Sirius red stain and hydroxyproline assay. Liver enzymes, fasting insulin, and hepatic triglyceride content were also assessed. Hepatocytes were isolated from Hif1a-/-hep mice and wild-type controls and were exposed to sustained hypoxia (1% O2) or normoxia (16% O2) for 24 hours. The culture media was used to reconstitute type I collagen and the resulting matrices were examined for collagen cross-linking. Results Wild-type mice on a high trans-fat diet had 80% more hepatic collagen than Hif1a-/-hep mice (2.21 μg collagen/mg liver tissue, versus 1.23 μg collagen/mg liver tissue, p = 0.03), which was confirmed by Sirius red staining. Body weight, liver weight, mean hepatic triglyceride content, and fasting insulin were similar between groups. Culture media from wild-type mouse hepatocytes exposed to hypoxia allowed for avid collagen cross-linking, but very little cross-linking was seen when hepatocytes were exposed to normoxia, or when hepatocytes from Hif1a-/-hep mice were used in hypoxia or normoxia. Conclusions Hepatocyte HIF-1 mediates an increase in liver fibrosis in a mouse model of

  13. 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

  14. Calcium flux and endogenous calcium content in isolated mammalian growth-plate chondrocytes, hyaline-cartilage chondrocytes, and hepatocytes.

    PubMed

    Iannotti, J P; Brighton, C T; Stambough, J L; Storey, B T

    1985-01-01

    The role of chondrocyte mitochondria in endochondral ossification has been the subject of intensive investigation and controversy. The purpose of this study was to quantitate the endogenous calcium content and the maximum capacity for calcium accumulation and release in isolated mammalian growth-plate chondrocytes and hyaline-cartilage chondrocytes. The results indicated that the mitochondria of the isolated growth-plate and hyaline-cartilage chondrocytes possess a greater endogenous calcium content, a greater capacity for calcium accumulation, and a larger labile Ca+2 pool than do the mitochondria of hepatocytes. Growth-plate and hyaline-cartilage mitochondria had an endogenous calcium content of 908 and 142 nanomoles of Ca+2 per milligram of mitochondrial protein. The growth-plate mitochondria had a maximum calcium capacity of 5249 nanomoles of Ca+2 per milligram of mitochondrial protein. In comparison, the mitochondria of hepatocytes had a much smaller endogenous-calcium content and a smaller maximum Ca+2 capacity: twenty-one and 3262 nanomoles of Ca+2 per milligram of mitochondrial protein, respectively. The mitochondrial labile-calcium pool in both growth-plate and hyaline-cartilage chondrocytes was twofold greater than that in the mitochondria of hepatocytes. Chondrocyte mitochondria released approximately 2400 nanomoles of Ca+2 per milligram of mitochondrial protein, whereas hepatocyte mitochondria released 1200 nanomoles of Ca+2 per milligram. These results suggest that the chondrocyte mitochondria are specialized for calcium transport and are important in the calcification of the extracellular matrix of the growth plate.

  15. Hepatocyte growth factor counteracts transforming growth factor-beta1, through attenuation of connective tissue growth factor induction, and prevents renal fibrogenesis in 5/6 nephrectomized mice.

    PubMed

    Inoue, Tsutomu; Okada, Hirokazu; Kobayashi, Tatsuya; Watanabe, Yusuke; Kanno, Yoshihiko; Kopp, Jeffrey B; Nishida, Takashi; Takigawa, Masaharu; Ueno, Munehisa; Nakamura, Toshikazu; Suzuki, Hiromichi

    2003-02-01

    We investigated the mechanism of the anti-fibrotic effects of hepatocyte growth factor (HGF) in the kidney, with respect to its effect on connective tissue growth factor (CTGF), a down-stream, profibrotic mediator of transforming growth factor-beta1 (TGF-beta1). In wild-type (WT) mice with 5/6 nephrectomy (Nx), HGF and TGF-beta1 mRNAs increased transiently in the remnant kidney by week 1 after the Nx, returned to baseline levels, and increased again at weeks 4 to 12. In contrast, CTGF and alpha1(I) procollagen (COLI) mRNAs increased in parallel with HGF and TGF-beta1 during the early stage, but did not re-increase during the late stage. In the case of TGF-beta1 transgenic (TG) mice with 5/6 Nx, excess TGF-beta1 derived from the transgene enhanced CTGF expression significantly in the remnant kidney, accordingly accelerating renal fibrogenesis. Administration of dHGF (5.0 mg/kg/day) to TG mice with 5/6 Nx for 4 weeks from weeks 2 to 6 suppressed CTGF expression in the remnant kidney, attenuating renal fibrosis and improving the survival rate. In an experiment in vitro, renal tubulointerstitial fibroblasts (TFB) were co-cultured with proximal tubular epithelial cells (PTEC). Pretreatment with HGF reduced significantly CTGF induction in PTEC by TGF-beta1, consequently suppressing COLI synthesis in TFB. In conclusion, HGF can block, at least partially, renal fibrogenesis promoted by TGF-beta1 in the remnant kidney, via attenuation of CTGF induction.

  16. 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.

  17. 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

  18. Carrier-mediated uptake of lucifer yellow in skate and rat hepatocytes: a fluid-phase marker revisited.

    PubMed

    Ballatori, N; Hager, D N; Nundy, S; Miller, D S; Boyer, J L

    1999-10-01

    Uptake of lucifer yellow (LY), a fluorescent disulfonic acid anionic dye, was studied in isolated skate (Raja erinacea) perfused livers and primary hepatocytes to evaluate its utility as a fluid-phase marker in these cells. However, our findings demonstrated that LY is transported across the plasma membrane of skate hepatocytes largely via carrier-mediated mechanisms. Isolated perfused skate livers cleared 50% of the LY from the recirculating perfusate within 1 h of addition of either 22 or 220 microM LY, with only 4.5 and 9% of the LY remaining in the perfusate after 7 h, respectively. Most of the LY was excreted into bile, resulting in high biliary LY concentrations (1 and 10 mM at the two doses, respectively), indicating concentrative transport into bile canalicular lumen. LY uptake by freshly isolated skate hepatocytes was temperature sensitive, exhibited saturation kinetics, and was inhibited by other organic anions. Uptake was mediated by both sodium-dependent [Michaelis-Menten constant (K(m)), 125 +/- 57 microM; maximal velocity (V(max)), 1.5 +/- 0.2 pmol. min(-1). mg cells(-1)] and sodium-independent (K(m), 207 +/- 55 microM; V(max), 1.7 +/- 0.2 pmol. min(-1). mg cells(-1)) mechanisms. Both of these uptake mechanisms were inhibited by various organic anions and transport inhibitors, including furosemide, bumetanide, sulfobromophthalein, rose bengal, probenecid, N-ethylmaleimide, taurocholate, and p-aminohippuric acid. Fluorescent imaging techniques showed intracellular vesicular compartmentation of LY in skate hepatocyte clusters. Studies in perfused rat livers also indicated that LY is taken up against a concentration gradient and concentrated in bile. LY uptake in isolated rat hepatocytes was saturable, but only at high concentrations, and demonstrated a K(m) of 3.7 +/- 1.0 mM and a V(max) of 1.75 +/- 0.16 nmol. min(-1). mg wet wt(-1). These results indicate that LY is transported into skate and rat hepatocytes and bile largely by carrier-mediated

  19. 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.

  20. Growth hormone-specific induction of the nuclear localization of porcine growth hormone receptor in porcine hepatocytes.

    PubMed

    Lan, H N; Hong, P; Li, R N; Shan, A S; Zheng, X

    2017-10-01

    The phenomenon of nuclear translocation of growth hormone receptor (GHR) in human, rat, and fish has been reported. To date, this phenomenon has not been described in a domestic animal (such as pig). In addition, the molecular mechanisms of GHR nuclear translocation have not been thoroughly elucidated. To this end, porcine hepatocytes were isolated and used as a cell model. We observed that porcine growth hormone (pGH) can induce porcine GHR's nuclear localization in porcine hepatocytes. Subsequently, the dynamics of pGH-induced pGHR's nuclear localization were analyzed and demonstrated that pGHR's nuclear localization occurs in a time-dependent manner. Next, we explored the mechanism of pGHR nuclear localization using different pGHR ligands, and we demonstrated that pGHR's nuclear translocation is GH(s)-dependent. We also observed that pGHR translocates into cell nuclei in a pGH dimerization-dependent fashion, whereas further experiments indicated that IMPα/β is involved in the nuclear translocation of the pGH-pGHR dimer. The pGH-pGHR dimer may form a pGH-GHR-JAK2 multiple complex in cell nuclei, which would suggest that similar to its function in the cell membrane, the nuclear-localized pGH-pGHR dimer might still have the ability to signal. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Hepatocyte Polarity

    PubMed Central

    Treyer, Aleksandr; Müsch, Anne

    2013-01-01

    Hepatocytes, like other epithelia, are situated at the interface between the organism’s exterior and the underlying internal milieu and organize the vectorial exchange of macromolecules between these two spaces. To mediate this function, epithelial cells, including hepatocytes, are polarized with distinct luminal domains that are separated by tight junctions from lateral domains engaged in cell-cell adhesion and from basal domains that interact with the underlying extracellular matrix. Despite these universal principles, hepatocytes distinguish themselves from other nonstriated epithelia by their multipolar organization. Each hepatocyte participates in multiple, narrow lumina, the bile canaliculi, and has multiple basal surfaces that face the endothelial lining. Hepatocytes also differ in the mechanism of luminal protein trafficking from other epithelia studied. They lack polarized protein secretion to the luminal domain and target single-spanning and glycosylphosphatidylinositol-anchored bile canalicular membrane proteins via transcytosis from the basolateral domain. We compare this unique hepatic polarity phenotype with that of the more common columnar epithelial organization and review our current knowledge of the signaling mechanisms and the organization of polarized protein trafficking that govern the establishment and maintenance of hepatic polarity. The serine/threonine kinase LKB1, which is activated by the bile acid taurocholate and, in turn, activates adenosine monophosphate kinase-related kinases including AMPK1/2 and Par1 paralogues has emerged as a key determinant of hepatic polarity. We propose that the absence of a hepatocyte basal lamina and differences in cell-cell adhesion signaling that determine the positioning of tight junctions are two crucial determinants for the distinct hepatic and columnar polarity phenotypes. PMID:23720287

  2. In vitro characterization of hepatocyte growth factor release from PHBV/PLGA microsphere scaffold.

    PubMed

    Zhu, Xin Hao; Wang, Chi-Hwa; Tong, Yen Wah

    2009-05-01

    Polymer scaffolds which can support cells to grow as well as deliver growth factors to the cells simultaneously have great potential for the successful regeneration of failed tissues. As popularly used vehicles to deliver anti-cancer drugs and growth factors, microspheres also show many advantages as substrates to guide the growth of cells. Therefore, we aimed to examine the feasibility of using microspheres as ideal scaffolds for liver tissue engineering. To determine the capabilities of previously used microsphere scaffold to deliver growth factors simultaneously, this work investigated a long-term (about three months) release of bovine serum albumin (BSA) from microsphere scaffolds fabricated by using two different polymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV, 8% PHV), poly(lactide-co-glycolide) acid (PLGA, 5050) and a blend of PLGA and PHBV. BSA served as a model for hepatocyte growth factor (HGF) since both proteins have similar molecular weights and hydrophilicity. Furthermore, HGF was encapsulated into the PLGA/PHBV composite microsphere with a core-shell structure, and sustained delivery of HGF with maintained bioactivity was achieved for at least 40 days. The moderate degradation rate (about 55% loss of the initial mass) and well-preserved structure after three months of incubation indicated that the PLGA/PHBV composite microspheres would therefore be more suitable than the pure PHBV or PLGA microspheres as a scaffold for engineering liver tissue.

  3. Epidermal growth factor and hepatocyte growth factor receptors collaborate to induce multiple biological responses in bovine mammary epithelial cells.

    PubMed

    Accornero, P; Martignani, E; Miretti, S; Starvaggi Cucuzza, L; Baratta, M

    2009-08-01

    The aim of this work was to explore whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) could increase the biological responses of a mammary epithelial cell line of bovine origin when added simultaneously. We also investigated a possible molecular mechanism underlying this cooperation. The development of mammary gland requires several circulating and locally produced hormones. Hepatocyte growth factor and its tyrosine kinase receptor, mesenchymal-epithelial transition factor (MET), are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor and its ligands have also been implicated in the growth and morphogenesis of the mammary epithelium. Both EGF and HGF seem to exert a morphogenic program in this tissue; therefore, we hypothesized that these cytokines could act cooperatively in bovine mammary epithelial cells. We have already shown that the bovine BME-UV cell line, a nontumorigenic mammary epithelial line, expresses both MET and EGF receptor. Simultaneous treatment with HGF and EGF elicited an increase in proliferation, dispersion, degradation of extracellular matrix, and motility. Following EGF treatment, BME-UV mammary cells exhibited an increase in MET expression at both the mRNA and protein levels. Long-term treatment of BME-UV cells with HGF and EGF together increased the level of activation of the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways when compared with HGF or EGF alone. These data outline a possible cooperative role of the EGF and HGF pathways and indicate that cross-talk between their respective receptors may modulate mammary gland development in the cow.

  4. 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.

  5. Hepatocyte growth factor/scatter factor is present in most pleural effusion fluids from cancer patients.

    PubMed Central

    Eagles, G.; Warn, A.; Ball, R. Y.; Baillie-Johnson, H.; Arakaki, N.; Daikuhara, Y.; Warn, R. M.

    1996-01-01

    Pleural effusion samples were obtained from 55 patients with malignant disease, including patients with primary lung cancers and those with a variety of other tumours metastatic to the pleura. The effusions were assayed for the presence of hepatocyte growth factor/scatter factor (HGF/SF), by both ELISA and bioassay. The presence of malignant cells in the effusions was also assessed. Detectable amounts of the factor, as judged by both criteria, were found in over 90% of all the effusions, including those from patients with a wide variety of carcinomas and also lymphomas. A wide range of HGF/SF levels were found for all tumour classes, some effusions containing high levels above 4 ng ml-1. It is concluded that tumours within the pleura and adjacent lung tissue are usually exposed to biologically significant levels of HGF/SF. PMID:8562345

  6. Establishment of human hair follicle mesenchymal stem cells with overexpressed human hepatocyte growth factor.

    PubMed

    Zhou, Dan; Cheng, Hongjing; Liu, Jinyu; Zhang, Lei

    2017-06-01

    Chronic liver disease has become a major health problem that causes serious damage to human health. Since the existing treatment effect was not ideal, we need to seek new treatment methods. We utilized the gene recombination technology to obtain the human hair mesenchymal stem cells which overexpression of human hepatocyte growth factor (hHGF). Furthermore, we verified the property of transfected cells through detecting surface marker by flow cytometry. We show here establishment of the hHGF-overexpressing lentivirus vector, and successfully transfection to human hair follicle mesenchymal stem cells. The verified experiments could demonstrate the human hair follicle mesenchymal stem cells which have been transfected still have the properties of stem cells. We successfully constructed human hair follicle mesenchymal stem cells which overexpression hHGF, and maintain the same properties compared with pro-transfected cells.

  7. Enhanced angiogenesis in grafted skins by gene transfer of human hepatocyte growth factor using laser-induced stress waves

    NASA Astrophysics Data System (ADS)

    Terakawa, Mitsuhiro; Sato, Shunichi; Saitoh, Daizoh; Tsuda, Hitoshi; Ashida, Hiroshi; Okano, Hideyuki; Obara, Minoru

    2007-02-01

    We delivered a therapeutic gene, hepatocyte growth factor (HGF), to skin grafts of rats using laser-induced stress waves (LISWs) with the objective of enhancing their adhesion. The density and uniformity of neovascularities were enhanced significantly in the grafted skins that were transfected using LISWs, suggesting the efficacy of this method to improve the outcome of skin transplantation.

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

    PubMed Central

    Jackson, Nancy M.; Kocarek, Thomas A.

    2008-01-01

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

  9. 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. © 2015 FEBS.

  10. The effect of hepatocyte growth factor on gene transcription during intestinal adaptation.

    PubMed

    Katz, Michael S; Thatch, Keith A; Schwartz, Marshall Z

    2011-02-01

    Previously, we investigated the physiologic effects of hepatocyte growth factor (HGF) on intestinal adaptation using a massive small bowel resection (MSBR) rat model. To correlate these altered physiologic changes with gene alterations, we used microarray technology at 7, 14, and 21 days after MSBR. Forty-five adult female rats were divided into 3 groups and underwent 70% MSBR, MSBR + HGF (intravenous 150 μg/kg per day), or sham operation (control). Five animals per group were killed at each time point. Ileal mucosa was harvested and RNA extracted. Rat Gene Chips and Expression Console software (Affymetrix, Santa Clara, CA) were used. Statistical analysis was done by analysis of variance using Partek Genomics Suite (Partek, Inc, St Louis, MO). Results were significant if fold change was more than 2 or less than -2, with P < .05. Compared with the control group, MSBR group had significant increases in up-regulated and down-regulated genes. The MSBR-HGF group had further increases in up-regulated and down-regulated genes compared with the MSBR group. At 7 days, 6 cellular hypertrophy families had 30 genes up-regulated, and HGF up-regulated an additional 14 genes. At 21 days, 5 hyperplasia gene families had 32 up-regulated genes. Hepatocyte growth factor up-regulated an additional 16 genes. Microarray analysis of intestinal adaptation identified an early emphasis on hypertrophy and later emphasis on hyperplasia. This is the first demonstration that the effect of HGF on intestinal adaptation is recruitment of more genes rather than an increase in the fold change of already up-regulated genes. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Hepatocytes display a compensatory survival response against cadmium toxicity by a mechanism mediated by EGFR and Src.

    PubMed

    Martínez Flores, K; Uribe Marín, B C; Souza Arroyo, V; Bucio Ortiz, L; López Reyes, A; Gómez-Quiroz, L E; Rojas del Castillo, E; Gutiérrez Ruiz, M C

    2013-04-01

    Although the liver is a cadmium-target organ, hepatocyte response involved in its toxicity is not yet elucidated. A link between this heavy metal treatment and Stat3 signaling pathways was examined in primary mouse hepatocytes. We provided evidence of a novel link among NADPH oxidase and Stat3 signaling, mediated by Src, EGFR, and Erk1/2. Cadmium activates NADPH oxidase. ROS produced by this oxidase activates Src, enable that in turn, transactivates EGFR that activates Stat3 in tyrosine, allowing its dimerization. Also, ROS from NADPH oxidase favors ERK1/2 activation that phosphorylates Stat3 in serine, resulting in a compensatory or adaptive survival response such as production of metallothionein-II in short Cd exposure times. However, after 12h CdCl2 treatment, cell viability diminished in 50%, accompanied by a drastic decrease of metallothionein-II production, and an increase in p53 activation and the pro-apoptotic protein Bax.

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

    PubMed Central

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

    1981-01-01

    We have constructed a toxic hybrid protein that is recognized by asialoglycoprotein (ASGP) receptors of cultured rat hepatocytes. The conjugate consists of fragment A of diphtheria toxin (DTA) linked by a disulfide bond to asialofetuin (ASF). This conjugate is highly toxic, inhibiting protein synthesis in primary rat hepatocytes at concentrations as low as 10 pM. The ASF-DTA conjugate was 600 and 1800 times as toxic as diphtheria toxin and DTA, respectively, on primary rat hepatocytes. The ASGP receptor recognizes galactose-terminated proteins. We tested a series of glycoproteins for their ability to block the action of the ASF-DTA conjugate. Fetuin and orosomucoid, two glycoproteins with terminal sialic acid on their oligosaccharide chains, did not block the action of the conjugate. Their galactose-terminated asialo derivatives, ASF and asialoorosomucoid, as expected, did block the action of the conjugate. The N-acetylglucosaminyl-terminated derivative (asialogalactoorsomucoid) had no appreciable effect on the activity of the conjugate. We tested the ASF-DTA conjugate on six cell types; except for primary rat hepatocytes, none of them were affected by a high concentration (10 nM) of ASF-DTA conjugate. A fetuin-DTA conjugate was less toxic by a factor of 300 than the ASF-DTA conjugate and exerted its effects primarily through non-receptor-mediated mechanisms. The highly toxic ASF-DTA conjugate is cell-type specific, and its action is mediated by a well-characterized receptor, whose mechanism of receptor-ligand internalization has been extensively investigated. Images PMID:6167984

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

    SciTech Connect

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

    1981-06-01

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

  14. Heparin induces dimerization and confers proliferative activity onto the hepatocyte growth factor antagonists NK1 and NK2

    PubMed Central

    1996-01-01

    Hepatocyte growth factor (HGF) is a potent epithelial mitogen whose actions are mediated through its receptor, the proto-oncogene c-Met. Two truncated variants of HGF known as NK1 and NK2 have been reported to be competitive inhibitors of HGF binding to c-Met, and to function as HGF antagonists (Lokker, N.A., and P.J. Godowski. 1993. J. Biol. Chem. 268: 17145-17150; Chan, A.M., J.S. Rubin, D.P. Bottaro, D.W. Hirschfield, M. Chedid, and S.A. Aaronson. 1991. Science (Wash. DC). 254:1382-1387). We show here, however, that NK1 acts as a partial agonist in mink lung cells. Interestingly, NK1, which is an HGF antagonist in hepatocytes in normal conditions, was converted to a partial agonist by adding heparin to the culture medium. The interaction of NK1 and heparin was further studied in BaF3 cells, which express little or no cell surface heparan sulfate proteoglycans. In BaF3 cells transfected with a plasmid encoding human c-Met, heparin and NK1 synergized to stimulate DNA synthesis and cell proliferation. There was no effect of heparin on the IL-3 sensitivity of BaF3-hMet cells, and no effect of NK1 plus heparin in control BaF3 cells, indicating that the response was specific and mediated through c-Met. The naturally occurring HGF splice variant NK2 also stimulated DNA synthesis in mink lung cells and exerted a heparin-dependent effect on BaF3-hMet cells, but not on BaF3-neo cells. The activating effect of heparin was mimicked by a variety of sulfated glycosaminoglycans. Mechanistic studies revealed that heparin increased the binding of NK1 to BaF3-hMet cells, stabilized NK1, and induced dimerization of NK1. Based on these studies, we propose that the normal agonist activity of NK1 and NK2 in mink lung cells is due to an activating interaction with an endogenous glycosaminoglycan. Consistent with that model, a large portion of the NK1 binding to mink lung cells could be blocked by heparin. Moreover, a preparation of glycosaminoglycans from the surface of mink lung

  15. Breast Cancer Cells Induce Cancer-Associated Fibroblasts to Secrete Hepatocyte Growth Factor to Enhance Breast Tumorigenesis

    PubMed Central

    Tyan, Shiaw-Wei; Kuo, Wen-Hung; Huang, Chun-Kai; Pan, Chi-Chun; Shew, Jin-Yuh; Chang, King-Jen; Lee, Eva Y.-H. P.; Lee, Wen-Hwa

    2011-01-01

    It has been well documented that microenvironment consisting of stroma affects breast cancer progression. However, the mechanisms by which cancer cells and fibroblasts, the major cell type in stroma, interact with each other during tumor development remains to be elucidated. Here, we show that the human cancer-associated fibroblasts (CAFs) had higher activity in enhancing breast tumorigenecity compared to the normal tissue-associated fibroblasts (NAFs) isolated from the same patients. The expression level of hepatocyte growth factor (HGF) in these fibroblasts was positively correlated with their ability to enhance breast tumorigenesis in mice. Deprivation of HGF using a neutralizing antibody reduced CAF-mediated colony formation of human breast cancer cells, indicating that CAFs enhanced cancer cell colony formation mainly through HGF secretion. Co-culture with human breast cancer MDA-MB-468 cells in a transwell system enhanced NAFs to secret HGF as well as promote tumorigenecity. The newly gained ability of these “educated” NAFs became irreversible after continuing this process till fourth passage. These results suggested that breast cancer cells could alter the nature of its surrounding fibroblasts to secrete HGF to support its own progression through paracrine signaling. PMID:21249190

  16. Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland

    PubMed Central

    1995-01-01

    We have examined the role of two mesenchymal ligands of epithelial tyrosine kinase receptors in mouse mammary gland morphogenesis. In organ cultures of mammary glands, hepatocyte growth factor (HGF, scatter factor) promoted branching of the ductal trees but inhibited the production of secretory proteins. Neuregulin (NRG, neu differentiation factor) stimulated lobulo-alveolar budding and the production of milk proteins. These functional effects are paralleled by the expression of the two factors in vivo: HGF is produced in mesenchymal cells during ductal branching in the virgin animal; NRG is expressed in the mesenchyme during lobulo-alveolar development at pregnancy. The receptors of HGF and NRG (c-met, c-erbB3, and c-erbB4), which are expressed in the epithelial cells, are not regulated. In organ culture, branching morphogenesis and lobulo-alveolar differentiation of the mammary gland could be abolished by blocking expression of endogenous HGF and NRG by the respective antisense oligonucleotides; in antisense oligonucleotide-treated glands, morphogenesis could again be induced by the addition of recombinant HGF and NRG. We thus show that two major postnatal morphogenic periods of mammary gland development are dependent on sequential mesenchymal- epithelial interactions mediated by HGF and NRG. PMID:7559778

  17. Hepatocyte growth factor secreted by bone marrow stem cell reduce ER stress and improves repair in alveolar epithelial II cells

    PubMed Central

    Nita, Izabela; Hostettler, Katrin; Tamo, Luca; Medová, Michaela; Bombaci, Giuseppe; Zhong, Jun; Allam, Ramanjaneyulu; Zimmer, Yitzhak; Roth, Michael; Geiser, Thomas; Gazdhar, Amiq

    2017-01-01

    Idiopathic Pulmonary Fibrosis (IPF) is a progressive, irreversible lung disease with complex pathophysiology. Evidence of endoplasmic reticulum (ER) stress has been reported in alveolar epithelial cells (AEC) in IPF patients. Secreted mediators from bone marrow stem cells (BMSC-cm) have regenerative properties. In this study we investigate the beneficial effects of BMSC-cm on ER stress response in primary AEC and ER stressed A549 cells. We hypothesize that BMSC-cm reduces ER stress. Primary AEC isolated from IPF patients were treated with BMSC-cm. To induce ER stress A549 cells were incubated with Tunicamycin or Thapsigargin and treated with BMSC-cm, or control media. Primary IPF-AEC had high Grp78 and CHOP gene expression, which was lowered after BMSC-cm treatment. Similar results were observed in ER stressed A549 cells. Alveolar epithelial repair increased in presence of BMSC-cm in ER stressed A549 cells. Hepatocyte growth factor (HGF) was detected in biologically relevant levels in BMSC-cm. Neutralization of HGF in BMSC-cm attenuated the beneficial effects of BMSC-cm including synthesis of surfactant protein C (SP-C) in primary AEC, indicating a crucial role of HGF in ER homeostasis and alveolar epithelial repair. Our data suggest that BMSC-cm may be a potential therapeutic option for treating pulmonary fibrosis. PMID:28157203

  18. Role of Phosphatidic Acid Phosphatase Domain Containing 2 in Squalestatin 1-Mediated Activation of the Constitutive Androstane Receptor in Primary Cultured Rat Hepatocytes.

    PubMed

    Pant, Asmita; Kocarek, Thomas A

    2016-03-01

    Farnesyl pyrophosphate (FPP) is a branch-point intermediate in the mevalonate pathway that is normally converted mainly to squalene by squalene synthase in the first committed step of sterol biosynthesis. Treatment with the squalene synthase inhibitor squalestatin 1 (SQ1) causes accumulation of FPP, its dephosphorylated metabolite farnesol, and several oxidized farnesol-derived metabolites. In addition, SQ1 treatment of primary cultured rat hepatocytes increases CYP2B expression through a mechanism that requires FPP synthesis and activation of the constitutive androstane receptor (CAR). Because direct farnesol treatment also increases CYP2B expression, it seems likely that SQ1-mediated CAR activation requires FPP dephosphorylation to farnesol. The lipid phosphatase, phosphatidic acid phosphatase domain containing 2 (PPAPDC2), was recently reported to catalyze FPP dephosphorylation. We therefore determined the effect of overexpressing or knocking down PPAPDC2 on SQ1-mediated CAR activation in primary cultured rat hepatocytes. Cotransfection of rat hepatocytes with a plasmid expressing rat or human PPAPDC2 enhanced SQ1-mediated activation of a CAR-responsive reporter by 1.7- or 2.4-fold over the SQ1-mediated activation that was produced when hepatocytes were cotransfected with empty expression plasmid. Similarly, transduction of rat hepatocytes with a recombinant adenovirus expressing PPAPDC2 enhanced SQ1-mediated CYP2B1 mRNA induction by 1.4-fold over the induction that was seen in hepatocytes transduced with control adenovirus. Cotransfection with a short hairpin RNA targeting PPAPDC2 reduced SQ1-mediated CAR activation by approximately 80% relative to the activation that occurred in hepatocytes transfected with nontargeting short hairpin RNA. These results indicate that PPAPDC2 plays an important role in SQ1-mediated CAR activation, most likely by catalyzing the conversion of FPP to farnesol. Copyright © 2016 by The American Society for Pharmacology and

  19. Integrin α6β4 Promotes Autocrine Epidermal Growth Factor Receptor (EGFR) Signaling to Stimulate Migration and Invasion toward Hepatocyte Growth Factor (HGF).

    PubMed

    Carpenter, Brittany L; Chen, Min; Knifley, Teresa; Davis, Kelley A; Harrison, Susan M W; Stewart, Rachel L; O'Connor, Kathleen L

    2015-11-06

    Integrin α6β4 is up-regulated in pancreatic adenocarcinomas where it contributes to carcinoma cell invasion by altering the transcriptome. In this study, we found that integrin α6β4 up-regulates several genes in the epidermal growth factor receptor (EGFR) pathway, including amphiregulin (AREG), epiregulin (EREG), and ectodomain cleavage protease MMP1, which is mediated by promoter demethylation and NFAT5. The correlation of these genes with integrin α6β4 was confirmed in The Cancer Genome Atlas Pancreatic Cancer Database. Based on previous observations that integrin α6β4 cooperates with c-Met in pancreatic cancers, we examined the impact of EGFR signaling on hepatocyte growth factor (HGF)-stimulated migration and invasion. We found that AREG and EREG were required for autocrine EGFR signaling, as knocking down either ligand inhibited HGF-mediated migration and invasion. We further determined that HGF induced secretion of AREG, which is dependent on integrin-growth factor signaling pathways, including MAPK, PI3K, and PKC. Moreover, matrix metalloproteinase activity and integrin α6β4 signaling were required for AREG secretion. Blocking EGFR signaling with EGFR-specific antibodies or an EGFR tyrosine kinase inhibitor hindered HGF-stimulated pancreatic carcinoma cell chemotaxis and invasive growth in three-dimensional culture. Finally, we found that EGFR was phosphorylated in response to HGF stimulation that is dependent on EGFR kinase activity; however, c-Met phosphorylation in response to HGF was unaffected by EGFR signaling. Taken together, these data illustrate that integrin α6β4 stimulates invasion by promoting autocrine EGFR signaling through transcriptional up-regulation of key EGFR family members and by facilitating HGF-stimulated EGFR ligand secretion. These signaling events, in turn, promote pancreatic carcinoma migration and invasion.

  20. Hepatocyte Growth Factor/Scatter Factor Released during Peritonitis Is Active on Mesothelial Cells

    PubMed Central

    Rampino, Teresa; Cancarini, Giovanni; Gregorini, Marilena; Guallini, Paola; Maggio, Milena; Ranghino, Andrea; Soccio, Grazia; Dal Canton, Antonio

    2001-01-01

    Peritonitis causes mesothelial detachment that may result in persistent peritoneal denudation and fibrosis. We investigated whether hepatocyte growth factor (HGF), a scatter factor that induces detachment from substrate and fibroblastic transformation of several cell types, is produced during peritonitis and is active on mesothelial cells. We studied 18 patients on peritoneal dialysis, 9 uncomplicated, 9 with peritonitis. HGF was measured in serum, peritoneal fluid, and supernatant of peripheral blood mononuclear cells and peritoneal mononuclear cells. Primary culture of human peritoneal mesothelial cells and the human mesothelial cell line MeT-5A were conditioned with recombinant HGF, serum, and peritoneal fluid. HGF levels were significantly higher in serum and peritoneal fluid of peritonitic than uncomplicated patients. Mononuclear cells of peritonitic patients produced more HGF than cells of uncomplicated patients. Recombinant HGF, serum, and peritoneal fluid of peritonitic patients caused mesothelial cell growth, detachment, transformation from epithelial to fibroblast-like shape, overexpression of vimentin, and synthesis of type I and III collagen. In conclusion, HGF released during peritonitis causes a change in mesothelial cell phenotype and function. HGF may affect the healing process facilitating repair through mesothelial cell growth, but may contribute to peritoneal fibrosis inducing cell detachment with mesothelial denudation and collagen synthesis. PMID:11583955

  1. 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.

  2. Therapeutic role of human hepatocyte growth factor (HGF) in treating hair loss

    PubMed Central

    2016-01-01

    Hepatocyte growth factor (HGF) is a paracrine hormone that plays an important role in epithelial-mesenchymal transition. HGF secreted by mesenchymal cells affects many properties of epithelial cells, such as proliferation, motility, and morphology. HGF has been reported to promote follicular growth. The purpose of the present study is to investigate the therapeutic role of HGF in hair loss treatment. A recombinant vector containing the human HGF (hHGF) gene (pTARGET-hHGF) was constructed, and the expression of hHGF in vitro was quantitatively and qualitatively evaluated. The effect of hHGF on hair growth was tested in mice, and results demonstrated that pTARGET-hHGF was successfully delivered into fibroblasts in vitro leading to a high expression of hHGF. Local injections of the pTARGET-hHGF recombinant vector into mice resulted in multiple beneficial effects compared to placebo, including faster hair regeneration, improved follicle development, and significantly increased HGF receptor (HGF-R). In conclusion, we have established a nonviral vector of hHGF which could be utilized to manipulate the sheath fibroblasts surrounding hair follicles (HF), thereby stimulating hair regeneration. PMID:27833804

  3. Hepatocyte growth factor plays a dual role in regulating skeletal muscle satellite cell proliferation and differentiation.

    PubMed

    Gal-Levi, R; Leshem, Y; Aoki, S; Nakamura, T; Halevy, O

    1998-03-12

    The role of hepatocyte growth factor (HGF) and its receptor, c-met, in proliferation and differentiation of satellite cells was studied in primary cultures of chicken skeletal muscle satellite cells and a myogenic C2 cell line. HGF mRNA was expressed mainly in the myotubes of both cultures. The addition of conditioned medium derived from those cultures had a scattering effect on the canine kidney epithelial cell line, MDCK. In contrast, c-met mRNA levels decreased during cell differentiation of C2 and primary satellite cells. Application of exogenous HGF to chicken myoblasts resulted in their enhanced DNA synthesis. Among several growth factors, HGF was the first to induce DNA synthesis in quiescent satellite cells, thereby driving them into the cell cycle. Ectopic expression of chicken HGF in primary satellite cells suppressed the activation of muscle-regulatory gene reporter constructs MCK-CAT, MRF4-CAT, MEF2-CAT and 4Rtk-CAT, as well as the gene expression of MyoD and myogenin, and MHC protein expression. Ectopic MyoD reversed HGF's inhibitory effect on MCK transactivation. These data suggest that HGF inhibits cell differentiation by inhibiting the activity of basic helix-loop-helix (bHLH)/E protein heterodimers, thus inhibiting myogenic determination factor activity and subsequent muscle-specific protein expression. During muscle growth and regeneration, HGF plays a dual role in satellite-cell myogenesis, affecting both the proliferation and differentiation of these cells in a paracrine fashion.

  4. Therapeutic role of human hepatocyte growth factor (HGF) in treating hair loss.

    PubMed

    Qi, Yonghao; Li, Miao; Xu, Lian; Chang, Zhijing; Shu, Xiong; Zhou, Lijun

    2016-01-01

    Hepatocyte growth factor (HGF) is a paracrine hormone that plays an important role in epithelial-mesenchymal transition. HGF secreted by mesenchymal cells affects many properties of epithelial cells, such as proliferation, motility, and morphology. HGF has been reported to promote follicular growth. The purpose of the present study is to investigate the therapeutic role of HGF in hair loss treatment. A recombinant vector containing the human HGF (hHGF) gene (pTARGET-hHGF) was constructed, and the expression of hHGF in vitro was quantitatively and qualitatively evaluated. The effect of hHGF on hair growth was tested in mice, and results demonstrated that pTARGET-hHGF was successfully delivered into fibroblasts in vitro leading to a high expression of hHGF. Local injections of the pTARGET-hHGF recombinant vector into mice resulted in multiple beneficial effects compared to placebo, including faster hair regeneration, improved follicle development, and significantly increased HGF receptor (HGF-R). In conclusion, we have established a nonviral vector of hHGF which could be utilized to manipulate the sheath fibroblasts surrounding hair follicles (HF), thereby stimulating hair regeneration.

  5. Chemokines induce axon outgrowth downstream of Hepatocyte Growth Factor and TCF/β-catenin signaling

    PubMed Central

    Bhardwaj, Deepshikha; Náger, Mireia; Camats, Judith; David, Monica; Benguria, Alberto; Dopazo, Ana; Cantí, Carles; Herreros, Judit

    2013-01-01

    Axon morphogenesis is a complex process regulated by a variety of secreted molecules, including morphogens and growth factors, resulting in the establishment of the neuronal circuitry. Our previous work demonstrated that growth factors [Neurotrophins (NT) and Hepatocyte Growth Factor (HGF)] signal through β-catenin during axon morphogenesis. HGF signaling promotes axon outgrowth and branching by inducing β-catenin phosphorylation at Y142 and transcriptional regulation of T-Cell Factor (TCF) target genes. Here, we asked which genes are regulated by HGF signaling during axon morphogenesis. An array screening indicated that HGF signaling elevates the expression of chemokines of the CC and CXC families. In line with this, CCL7, CCL20, and CXCL2 significantly increase axon outgrowth in hippocampal neurons. Experiments using blocking antibodies and chemokine receptor antagonists demonstrate that chemokines act downstream of HGF signaling during axon morphogenesis. In addition, qPCR data demonstrates that CXCL2 and CCL5 expression is stimulated by HGF through Met/b-catenin/TCF pathway. These results identify CC family members and CXCL2 chemokines as novel regulators of axon morphogenesis downstream of HGF signaling. PMID:23641195

  6. Microscopic analysis of the cellular events during scatter factor/hepatocyte growth factor-induced epithelial tubulogenesis.

    PubMed

    Williams, M J; Clark, P

    2003-11-01

    Scatter factor/hepatocyte growth factor (SF/HGF), a large multifunctional polypeptide growth and motility factor, is known to play important roles during embryonic development, adult tissue growth and repair. In an established three-dimensional type I collagen model, SF/HGF induces Madin-Darby canine kidney (MDCK) epithelial cysts to form long, branching tubules (tubulogenesis). In addition, the composition of the surrounding extracellular matrix (ECM) has been shown to modulate SF/HGF-induced morphogenesis, where tubulogenesis was completely abrogated in Matrigel basement membrane. Many cellular events that occur during SF/HGF-mediated remodelling, and its modulation by the ECM, remain unclear. We have investigated these mechanisms through microscopic examination of the time-course of SF/HGF-induced responses in MDCK cysts cultured in type I collagen or Matrigel. We found that early responses to SF/HGF were matrix-independent. Changes included increased paracellular spacing between normally closely apposed lateral membranes, and the formation of filopodial processes, indicating a partial motile response. Cell-cell contact was maintained, with the persistence of cell junctions. Therefore, while one or a number of ECM components are preventing SF/HGF-primed cells from undergoing an invasive and/or migratory programme, non-permissive matrices are not preventing SF/HGF signalling to the cell. Later matrix-dependent responses, which occurred in type I collagen but not Matrigel, included the formation of basal protrusions that comprise two or more neighbouring cells, which extend to form nascent tubules. Modified polarity of cells comprising the basal protrusions was evident, with a marker for the apical membrane being found in the same region as adherens junctions and desmosomes, typically localized at lateral membranes. We propose a model for SF/HGF-induced tubulogenesis in which tubules form from basal protrusions of adjacent cells. This mechanism of in vitro tubule

  7. 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.

  8. 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

  9. The biosynthesis of ascorbate protects isolated rat hepatocytes from cumene hydroperoxide-mediated oxidative stress.

    PubMed

    Chan, Tom S; Shangari, Nandita; Wilson, John X; Chan, Helen; Butterworth, Roger F; O'Brien, Peter J

    2005-04-01

    Most animals synthesize ascorbate. It is an essential enzymatic cofactor for the synthesis of a variety of biological molecules and also a powerful antioxidant. There is, however, little direct evidence supporting an antioxidant role for endogenously produced ascorbate. Recently, we demonstrated that incubation of rat hepatocytes with 1-bromoheptane or phorone simultaneously depleted glutathione (GSH) and triggered rapid ascorbate synthesis. The present study investigates the hypothesis that endogenous ascorbate synthesis can confer protection against oxidative stress. Rat and guinea pig hepatocytes were depleted of GSH with 1-bromoheptane and subsequently treated with the oxidative stressor cumene hydroperoxide (CHP) in the presence or absence of the ascorbate synthesis inhibitor sorbinil. In rat hepatocytes, ascorbate content increased linearly (from 15.1 to 35.8 nmol/10(6) cells) over a 105-min incubation. Prior depletion of GSH increased CHP-induced cellular reactive oxygen species (ROS) production, lipid peroxidation, and cell death in rat and guinea pig hepatocytes. Inhibiting ascorbate synthesis, however, further elevated ROS production (2-fold), lipid peroxidation (1.5-fold), and cell death (2-fold) in rat hepatocytes only. This is the first time that endogenous ascorbate synthesis has been shown to decrease cellular susceptibility to oxidative stress. Protection by endogenously produced ascorbate may therefore need to be addressed when extrapolating data to humans from experiments using rodents capable of synthesizing ascorbate.

  10. Heparin-binding epidermal growth factor-like growth factor and hepatocyte growth factor inhibit cholestatic liver injury in mice through different mechanisms

    PubMed Central

    Sakamoto, Kouichi; Khai, Ngin Cin; Wang, Yuqing; Irie, Rie; Takamatsu, Hideo; Matsufuji, Hiroshi; Kosai, Ken-Ichiro

    2016-01-01

    In contrast to hepatocyte growth factor (HGF), the therapeutic potential and pathophysiologic roles of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in liver diseases remain relatively unknown. To address the lack of effective pharmacologic treatments for cholestatic liver injuries, as well as to clarify the biologic features of these growth factors, we explored the effects of HB-EGF and HGF in mice with cholestatic liver injury induced by bile duct ligation (BDL). The mice were assessed 3, 5 and/or 14 days after BDL (acute, subacute and/or chronic phases, respectively) and intravenous injection of adenoviral vector expressing LacZ (control), HB-EGF, HGF, or HB-EGF and HGF. HB-EGF, HGF, or a combination of the growth factors exerted potent antioncotic (antinecrotic), antiapoptotic, anticholestatic, and regenerative effects on hepatocytes in vivo, whereas no robust antiapoptotic or regenerative effects were detected in interlobular bile ducts. Based on serum transaminase levels, the acute protective effects of HB-EGF on hepatocytes were greater than those of HGF. On the other hand, liver fibrosis and cholestasis during the chronic phase were more potently inhibited by HGF compared with HB-EGF. Compared with either growth factor alone, combining HB-EGF and HGF produced greater anticholestatic and regenerative effects during the chronic phase. Taken together, these findings suggest that HB-EGF and HGF inhibited BDL-induced cholestatic liver injury, predominantly by exerting acute cytoprotective and chronic antifibrotic effects, respectively; combining the growth factors enhanced the anticholestatic effects and liver regeneration during the chronic phase. Our results contribute to a better understanding of the pathophysiologic roles of HB-EGF and HGF, as well as to the development of novel effective therapies for cholestatic liver injuries. PMID:27779646

  11. Glycycoumarin inhibits hepatocyte lipoapoptosis through activation of autophagy and inhibition of ER stress/GSK-3-mediated mitochondrial pathway

    PubMed Central

    Zhang, Enxiang; Yin, Shutao; Song, Xinhua; Fan, Lihong; Hu, Hongbo

    2016-01-01

    Herbal medicine as an alternative approach in the treatment of disease has drawn growing attention. Identification of the active ingredient is needed for effective utilization of the herbal medicine. Licorice is a popular herbal plant that is widely used to treat various diseases including liver diseases. Glycycoumarin (GCM) is a representative of courmarin compounds isolated from licorice. In the present study, the protective effect of GCM on hepatocyte lipoapoptosis has been evaluated using both cell culture model of palmitate-induced lipoapoptosis and animal model of non-alcoholic steatohepatitis (NASH). The results demonstrated for the first time that GCM was highly effective in suppressing hepatocyte lipoapoptosis in both in vitro and in vivo. Mechanistically, GCM was able to re-activate the impaired autophagy by lipid metabolic disorders. In line with the activation of autophagy, ER stress-mediated JNK and mitochondrial apoptotic pathway activation was inhibited by GCM both in vitro and in vivo. In addition, inactivation of GSK-3 might also contribute to the protective effect of GCM on hepatocyte lipoapoptosis. Our findings supported GCM as a novel active component of licorice against non-alcoholic fatty liver disease (NAFLD). PMID:27901086

  12. Hepatocyte growth factor incorporated chitosan nanoparticles augment the differentiation of stem cell into hepatocytes for the recovery of liver cirrhosis in mice

    PubMed Central

    2011-01-01

    Background Short half-life and low levels of growth factors in the niche of injured microenvironment necessitates the exogenous and sustainable delivery of growth factors along with stem cells to augment the regeneration of injured tissues. Methods Here, recombinant human hepatocyte growth factor (HGF) was incorporated into chitosan nanoparticles (CNP) by ionic gelation method and studied for its morphological and physiological characteristics. Cirrhotic mice received either hematopoietic stem cells (HSC) or mesenchymal stemcells (MSC) with or without HGF incorporated chitosan nanoparticles (HGF-CNP) and saline as control. Biochemical, histological, immunostaining and gene expression assays were carried out using serum and liver tissue samples. One way analysis of variance was used for statics application Results Serum levels of selected liver protein and enzymes were significantly increased in the combination of MSC and HGF-CNP (MSC+HGF-CNP) treated group. Immunopositive staining for albumin (Alb) and cytokeratin 18 (CK18), and reverse transcription-polymerase chain reaction (RT-PCR) for Alb, alpha fetoprotein (AFP), CK18, cytokeratin 19 (CK19) ascertained that MSC-HGF-CNP treatment could be an effective combination to repopulate liver parenchymal cells in the liver cirrhosis. Zymogram and western blotting for matrix metalloproteinases 2 and 9 (MMP2 and MMP9) revealed that MMP2 actively involved in the fibrolysis of cirrhotic tissue. Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment. These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM). Conclusion Bone marrow stem cells were isolated, characterized and transplanted in mice model. Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem

  13. The potential lymphangiogenic effects of hepatocyte growth factor/scatter factor in vitro and in vivo.

    PubMed

    Jiang, Wen G; Davies, Gaynor; Martin, Tracey A; Parr, Christian; Watkins, Gareth; Mansel, Robert E; Mason, Malcolm D

    2005-10-01

    Lymphangiogenesis is key to the lymphatic spread of cancer cells. The current study examined the potential effect of hepatocyte growth factor (HGF), a factor known to have strong biological effects on endothelial cells, on the lymphangiogenic function of endothelial cells and the formation of lymphatic vessels using both in vitro and in vivo models. Human endothelial cells that have lymphatic characteristics, human prostate and breast cancer cells PC-3 and MDA MB 231, were used. Expression of lymphatic markers, podoplanin, Prox-1, vascular endothelial growth factor receptor 3 (VEGF-R3) and LYVE-1 was determined using reverse transcription polymerase reaction and quantitative PCR. In nude mice prostate and breast xenograft tumour models, either HGF or an HGF-producing fibroblast cell line MRC-5 was given with or without the HGF antagonist, NK4. The lymphangiogenic marker and lymphatic vessels in tumour tissues were also assessed using quantitative PCR and immunohistochemistry, respectively. In the mice tumour models, infusion of rhHGF significantly increased the levels of podoplanin and LYVE-1 in the tumour (p=0.05 for podoplanin and p<0.05 for LYVE-1 vs. without HGF in the prostate tumour model, p<0.05 for podoplanin and p<0.01 for LYVE-1 vs. without HGF for the breast tumour model; p<0.05 for podoplanin and p<0.01 for LYVE-1 vs. without HGF in the breast tumour model). The increased level of LYVE-1 transcript was supported by an increase in the number of LYVE-1-positive lymphatic vessels in tumours, using immunohistochemical analysis. Co-injection of MRC5 cells also increased the levels of LYVE-1 and number of LYVE-1-positive vessels in tumour tissues. The effects of HGF and MRC5 were significantly reduced by the HGF antagonist, NK4. In the in vitro models, rhHGF significantly increased the level of both podoplanin and LYVE-1, as shown by quantitative PCR analysis. Hepatocyte growth factor has potential lymphangiogenic activities, and this may have important

  14. 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.

  15. Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

    PubMed Central

    Ozen, Evin; Gozukizil, Aysim; Erdal, Esra; Uren, Aykut; Bottaro, Donald P.; Atabey, Nese

    2012-01-01

    The Hepatocyte Growth Factor (HGF)/c-Met signaling pathway regulates hepatocyte proliferation, and pathway aberrations are implicated in the invasive and metastatic behaviors of hepatocellular carcinoma (HCC). In addition to c-Met, heparin acts as a co-receptor to modulate pathway activity. Recently, anti-metastatic and anti-cancer effects of heparin have been reported. However, the role of heparin in the regulation of HGF signaling remains controversial and the effects of heparin on HGF-induced biological responses during hepatocarcinogenesis is not yet defined. In this study we determined the effects of heparin on HGF-induced activities of HCC cells and the underlying molecular mechanisms. Here, we report for the first time that heparin inhibits HGF-induced adhesion, motility and invasion of HCC cells. In addition, heparin reduced HGF-induced activation of c-Met and MAPK in a dose-dependent manner, as well as decreased transcriptional activation and expression of Early growth response factor 1 (Egr1). HGF-induced MMP-2 and MMP-9 activation, and MT1-MMP expression, also were inhibited by heparin. Stable knockdown of Egr1 caused a significant decrease in HGF-induced invasion, as well as the activation and expression of MMPs. Parallel to these findings, the overexpression of Egr1 increased the invasiveness of HCC cells. Our results suggest that Egr1 activates HGF-induced cell invasion through the regulation of MMPs in HCC cells and heparin inhibits HGF-induced cellular invasion via the downregulation of Egr1. Therefore, heparin treatment might be a therapeutic approach to inhibit invasion and metastasis of HCC, especially for patients with active HGF/c-Met signaling. PMID:22912725

  16. Basic fibroblast growth factor-treated adipose tissue-derived mesenchymal stem cell infusion to ameliorate liver cirrhosis via paracrine hepatocyte growth factor.

    PubMed

    Tang, Wei-Ping; Akahoshi, Tomohiko; Piao, Jing-Shu; Narahara, Sayoko; Murata, Masaharu; Kawano, Takahito; Hamano, Nobuhito; Ikeda, Tetsuo; Hashizume, Makoto

    2015-06-01

    Recent studies show that adipose tissue-derived mesenchymal stem cells have potential clinical applications. However, the mechanism has not been fully elucidated yet. Here, we investigated the effect of basic fibroblast growth factor-treated adipose tissue-derived mesenchymal stem cells infusion on a liver fibrosis rat model and elucidated the underlying mechanism. Adipose tissue-derived mesenchymal stem cells were infused into carbon tetrachloride-induced hepatic fibrosis rats through caudal vein. Liver functions and pathological changes were assessed. A co-culture model was used to clarify the potential mechanism. Basic fibroblast growth factor treatment markedly improved the proliferation, differentiation, and hepatocyte growth factor expression ability of adipose tissue-derived mesenchymal stem cells. Although adipose tissue-derived mesenchymal stem cells infusion alone slightly ameliorated liver functions and suppressed fibrosis progression, basic fibroblast growth factor-treatment significantly enhanced the therapeutic effect in association with elevated hepatocyte growth factor expression. Moreover, double immunofluorescence staining confirmed that the infused cells located in fibrosis area. Furthermore, co-culture with adipose tissue-derived mesenchymal stem cell led to induction of hepatic stellate cell apoptosis and enhanced hepatocyte proliferation. However, these effects were significantly weakened by knockdown of hepatocyte growth factor. Mechanism investigation revealed that co-culture with adipose tissue-derived mesenchymal stem cells activated c-jun N-terminal kinase-p53 signaling in hepatic stellate cell and promoted apoptosis. Basic fibroblast growth factor treatment enhanced the therapeutic effect of adipose tissue-derived mesenchymal stem cells, and secretion of hepatocyte growth factor from adipose tissue-derived mesenchymal stem cells plays a critical role in amelioration of liver injury and regression of fibrosis. © 2015 Journal of

  17. Lenvatinib in combination with golvatinib overcomes hepatocyte growth factor pathway-induced resistance to vascular endothelial growth factor receptor inhibitor

    PubMed Central

    Nakagawa, Takayuki; Matsushima, Tomohiro; Kawano, Satoshi; Nakazawa, Youya; Kato, Yu; Adachi, Yusuke; Abe, Takanori; Semba, Taro; Yokoi, Akira; Matsui, Junji; Tsuruoka, Akihiko; Funahashi, Yasuhiro

    2014-01-01

    Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several tumor types; however, some tumors show intrinsic resistance to VEGFR inhibitors, and some patients develop acquired resistance to these inhibitors. Therefore, a strategy to overcome VEGFR inhibitor resistance is urgently required. Recent reports suggest that activation of the hepatocyte growth factor (HGF) pathway through its cognate receptor, Met, contributes to VEGFR inhibitor resistance. Here, we explored the effect of the HGF/Met signaling pathway and its inhibitors on resistance to lenvatinib, a VEGFR inhibitor. In in vitro experiments, addition of VEGF plus HGF enhanced cell growth and tube formation of HUVECs when compared with stimulation by either factor alone. Lenvatinib potently inhibited the growth of HUVECs induced by VEGF alone, but cells induced by VEGF plus HGF showed lenvatinib resistance. This HGF-induced resistance was cancelled when the Met inhibitor, golvatinib, was added with lenvatinib. Conditioned medium from tumor cells producing high amounts of HGF also conferred resistance to inhibition by lenvatinib. In s.c. xenograft models based on various tumor cell lines with high HGF expression, treatment with lenvatinib alone showed weak antitumor effects, but treatment with lenvatinib plus golvatinib showed synergistic antitumor effects, accompanied by decreased tumor vessel density. These results suggest that HGF from tumor cells confers resistance to tumor endothelial cells against VEGFR inhibitors, and that combination therapy using VEGFR inhibitors with Met inhibitors may be effective for overcoming resistance to VEGFR inhibitors. Further evaluation in clinical trials is warranted. PMID:24689876

  18. Lenvatinib in combination with golvatinib overcomes hepatocyte growth factor pathway-induced resistance to vascular endothelial growth factor receptor inhibitor.

    PubMed

    Nakagawa, Takayuki; Matsushima, Tomohiro; Kawano, Satoshi; Nakazawa, Youya; Kato, Yu; Adachi, Yusuke; Abe, Takanori; Semba, Taro; Yokoi, Akira; Matsui, Junji; Tsuruoka, Akihiko; Funahashi, Yasuhiro

    2014-06-01

    Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several tumor types; however, some tumors show intrinsic resistance to VEGFR inhibitors, and some patients develop acquired resistance to these inhibitors. Therefore, a strategy to overcome VEGFR inhibitor resistance is urgently required. Recent reports suggest that activation of the hepatocyte growth factor (HGF) pathway through its cognate receptor, Met, contributes to VEGFR inhibitor resistance. Here, we explored the effect of the HGF/Met signaling pathway and its inhibitors on resistance to lenvatinib, a VEGFR inhibitor. In in vitro experiments, addition of VEGF plus HGF enhanced cell growth and tube formation of HUVECs when compared with stimulation by either factor alone. Lenvatinib potently inhibited the growth of HUVECs induced by VEGF alone, but cells induced by VEGF plus HGF showed lenvatinib resistance. This HGF-induced resistance was cancelled when the Met inhibitor, golvatinib, was added with lenvatinib. Conditioned medium from tumor cells producing high amounts of HGF also conferred resistance to inhibition by lenvatinib. In s.c. xenograft models based on various tumor cell lines with high HGF expression, treatment with lenvatinib alone showed weak antitumor effects, but treatment with lenvatinib plus golvatinib showed synergistic antitumor effects, accompanied by decreased tumor vessel density. These results suggest that HGF from tumor cells confers resistance to tumor endothelial cells against VEGFR inhibitors, and that combination therapy using VEGFR inhibitors with Met inhibitors may be effective for overcoming resistance to VEGFR inhibitors. Further evaluation in clinical trials is warranted.

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

    PubMed

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

    2010-07-08

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

  20. The potential roles of hepatocyte growth factor (HGF)-MET pathway inhibitors in cancer treatment.

    PubMed

    Parikh, Rahul A; Wang, Peng; Beumer, Jan H; Chu, Edward; Appleman, Leonard J

    2014-01-01

    MET is located on chromosome 7q31 and is a proto-oncogene that encodes for hepatocyte growth factor (HGF) receptor, a member of the receptor tyrosine kinase (RTK) family. HGF, also known as scatter factor (SF), is the only known ligand for MET. MET is a master regulator of cell growth and division (mitogenesis), mobility (motogenesis), and differentiation (morphogenesis); it plays an important role in normal development and tissue regeneration. The HGF-MET axis is frequently dysregulated in cancer by MET gene amplification, translocation, and mutation, or by MET or HGF protein overexpression. MET dysregulation is associated with an increased propensity for metastatic disease and poor overall prognosis across multiple tumor types. Targeting the dysregulated HGF-MET pathway is an area of active research; a number of monoclonal antibodies to HGF and MET, as well as small molecule inhibitors of MET, are under development. This review summarizes the key biological features of the HGF-MET axis, its dysregulation in cancer, and the therapeutic agents targeting the HGF-MET axis, which are in development.

  1. PET of c-Met in cancer with 64Cu-labeled Hepatocyte Growth Factor

    PubMed Central

    Luo, Haiming; Hong, Hao; Slater, Michael R.; Graves, Stephen A.; Shi, Sixiang; Yang, Yunan; Nickles, Robert J.; Fan, Frank; Cai, Weibo

    2015-01-01

    The hepatocyte growth factor (HGF) and its receptor, c-Met, are actively involved in tumor progression/metastasis and associated closely with poor prognostic outcome of cancer patients. Thus developing positron emission tomography (PET) agents for assessing c-Met expression would be extremely useful for diagnosis of cancer and subsequent monitoring of responses to c-Met-targeted therapies. Here we report the characterization of recombinant human hepatocyte growth factor (rh-HGF) as a PET tracer for detection of c-Met expression in vivo. Methods rh-HGF was expressed in human embryonic kidney (HEK) 293 cells and purified by nickel-nitrilogriacetic acid (Ni-NTA) affinity chromatography. The concentrated rh-HGF was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with 64Cu. c-Met binding evaluation by flow cytometry was performed in both U87MG and MDA-MB-231 cell lines, which have high and low level of c-Met, respectively. PET imaging and biodistribution studies were performed in nude mice bearing U87MG and MDA-MB-231 xenografted tumors. Results The rh-HGF expression yield was 150–200 µg protein per 5 × 106 cells after 48 h transfection with purity of 85% ~ 90%. Flow cytometry examination confirmed strong and specific binding capacity of rh-HGF to c-Met. After labeled with 64Cu, PET imaging revealed specific and prominent uptake of 64Cu-NOTA-rh-HGF in c-Met positive U87MG tumors (6.7 ± 1.8 %ID/g at 9 h post-injection) and significantly lower uptake in c-Met negative MDA-MB-231 tumors (1.8 ± 0.6 %ID/g at 9 h post-injection). The fact that sonicated-denatured rh-HGF (termed as dnrh-HGF) had significantly lower uptake in U87MG tumors, along with histology analysis, confirmed the c-Met specificity of 64Cu-NOTA-rh-HGF. Conclusion The study provided the initial evidence to confirm that 64Cu-NOTA-rh-HGF is applicable for visualizing c-Met expression in vivo, which may also find potential applications in

  2. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26111969

  4. Hepatocyte growth factor plays a key role in insulin resistance-associated compensatory mechanisms.

    PubMed

    Araújo, Tiago G; Oliveira, Alexandre G; Carvalho, Bruno M; Guadagnini, Dioze; Protzek, André O P; Carvalheira, Jose B C; Boschero, Antonio C; Saad, Mario J A

    2012-12-01

    Insulin resistance is present in obesity and in type 2 diabetes and is associated with islet cell hyperplasia and hyperinsulinemia, but the driving forces behind this compensatory mechanism are incompletely understood. Previous data have suggested the involvement of an unknown circulating insulin resistance-related β-cell growth factor. In this context, looking for candidates to be a circulating factor, we realized that hepatocyte growth factor (HGF) is a strong candidate as a link between insulin resistance and increased mass of islets/hyperinsulinemia. Our approach aimed to show a possible cause-effect relationship between increase in circulating HGF levels and compensatory islet hyperplasia/hyperinsulinemia by showing the strength of the association, whether or not is a dose-dependent response, the temporality, consistency, plausibility, and reversibility of the association. In this regard, our data showed: 1) a strong and consistent correlation between HGF and the compensatory mechanism in three animal models of insulin resistance; 2) HGF increases β-cell mass in a dose-dependent manner; 3) blocking HGF shuts down the compensatory mechanisms; and 4) an increase in HGF levels seems to precede the compensatory response associated with insulin resistance, indicating that these events occur in a sequential mode. Additionally, blockages of HGF receptor (Met) worsen the impaired insulin-induced insulin signaling in liver of diet-induced obesity rats. Overall, our data indicate that HGF is a growth factor playing a key role in islet mass increase and hyperinsulinemia in diet-induced obesity rats and suggest that the HGF-Met axis may have a role on insulin signaling in the liver.

  5. Hepatocyte growth factor exerts multiple biological functions on bovine mammary epithelial cells.

    PubMed

    Accornero, P; Martignani, E; Macchi, E; Baratta, M

    2007-09-01

    The met proto-oncogene product Met is a member of the family of tyrosine kinase growth factor receptors, and hepatocyte growth factor/scatter factor (HGF/SF) has been identified as its only ligand. Bovine Met and HGF/SF have been recently cloned and their expression has been characterized in the mammary gland, but no data regarding the biological effects of this ligand/receptor couple in bovine mammary cells are yet available. We examined the role of HGF/SF and its receptor in a bovine mammary epithelial cell line (BME-UV). Expression of Met at the mRNA level in BME-UV mammary epithelial cells evaluated by real-time PCR was similar to the expression in MDCK cells, a widely used model for Met biology. Met expression in BME-UV at the protein level was confirmed by western blot. The analysis of some signal transductional pathways downstream from the Met receptor revealed that HGF/SF addition to BME-UV cells induced activation of the extracellular signal-regulated kinase 1/2 proliferative pathway and the Akt antiapoptotic pathway. The BME-UV cells treated with HGF responded with increased proliferation, cell scatter, and motility. Met activation by HGF induced degradation of the extracellular matrix and migration through matrigel coated transwells. Moreover, BME-UV cells included in a 3-dimensional matrix of collagen and treated with HGF developed tubular structures, reminiscent of the mammary gland ducts. These data indicate that HGF and Met might be important regulators of mammary gland growth, morphogenesis, and development in the bovine.

  6. An inhibitor of cyclin-dependent kinase, stress-induced p21Waf-1/Cip-1, mediates hepatocyte mito-inhibition during the evolution of cirrhosis.

    PubMed

    Lunz, John G; Tsuji, Hirokazu; Nozaki, Isao; Murase, Noriko; Demetris, Anthony J

    2005-06-01

    During the evolution of cirrhosis, there is a relative decrease in volume percentage of hepatocytes and a relative increase in biliary epithelial cells and myofibroblasts. This is recognized histopathologically as a ductular reaction and leads to gradual distortion of the normal hepatic architecture. The final or decompensated stage of cirrhosis is characterized by a further decline in hepatocyte proliferation and loss of functional liver mass that manifests clinically as ascites, encephalopathy, and other signs of liver failure. In this report, we tested the hypothesis that p21-mediated hepatocyte mito-inhibition accelerates the evolution of cirrhosis using an established mouse model of decompensated biliary cirrhosis, p21-deficient mice, and liver tissue from humans awaiting liver replacement. Despite the same insult of long-term (12-week) bile duct ligation, mice prone to decompensation showed significantly more oxidative stress and hepatocyte nuclear p21 expression, which resulted in less hepatocyte proliferation, an exaggerated ductular reaction, and more advanced disease compared with compensation-prone controls. Mice deficient in p21 were better able than wild-type controls to compensate for long-term bile duct ligation because of significantly greater hepatocyte proliferation, which led to a larger liver mass and less architectural distortion. Mito-inhibitory hepatocyte nuclear p21 expression in humans awaiting liver replacement directly correlated with pathological disease stage and model of end-stage liver disease scoring. In conclusion, stress-induced upregulation of hepatocyte p21 inhibits hepatocyte proliferation during the evolution of cirrhosis. These findings have implications for understanding the evolution of cirrhosis and associated carcinogenesis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

  7. 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.

  8. High concentration but low activity of hepatocyte growth factor in periodontitis.

    PubMed

    Lönn, Johanna; Johansson, Carin Starkhammar; Nakka, Sravya; Palm, Eleonor; Bengtsson, Torbjörn; Nayeri, Fariba; Ravald, Nils

    2014-01-01

    High levels of hepatocyte growth factor (HGF), a healing factor with regenerative and cytoprotective effects, are associated with inflammatory diseases, including periodontitis. HGF biologic activity requires binding to its receptors, the proto-oncogene c-Met and heparan sulfate proteoglycan (HSPG). This study investigates HGF expression and its relationship to subgingival microbiota in medically healthy individuals with and without periodontitis. Saliva, gingival crevicular fluid (GCF), and blood samples from 30 patients with severe periodontitis and 30 healthy controls were analyzed for HGF concentration using enzyme-linked immunosorbent assay and binding affinity for HSPG and c-Met using surface plasmon resonance. The regenerative effects of saliva from three patients and controls were analyzed in an in vitro model of cell injury. Subgingival plaques were analyzed for the presence of 18 bacterial species. Patients with periodontitis showed higher HGF concentrations in saliva, GCF, and serum (P <0.001); however, the binding affinities for HSPG and c-Met were reduced in GCF and saliva (P <0.002). In contrast to the controls, saliva from patients showed no significant regenerative effect over time on gingival epithelial cells. Compared with controls, patients had a higher prevalence of periodontally related bacteria. Higher circulatory HGF levels indicate a systemic effect of periodontitis. However, the HGF biologic activity at local inflammation sites was reduced, and this effect was associated with the amount of periodontal bacteria. Loss of function of healing factors may be an important mechanism in degenerative processes in periodontally susceptible individuals.

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

    SciTech Connect

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

    2006-11-17

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

  10. 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.

  11. Homogenizing cellular tension by hepatocyte growth factor in expanding epithelial monolayer

    PubMed Central

    Jang, Hwanseok; Notbohm, Jacob; Gweon, Bomi; Cho, Youngbin; Park, Chan Young; Kee, Sun-Ho; Fredberg, Jeffrey J.; Shin, Jennifer H.; Park, Yongdoo

    2017-01-01

    Hepatocyte growth factor (HGF) induces cell migration and scattering by mechanisms that are thought to tip a local balance of competing physical forces; cell-to-cell and cell-to-substrate forces. In this local process, HGF is known to attenuate local cadherin-dependent adhesion forces for cell-cell junction development and enhance local integrin-dependent contractile forces for pulling neighboring cells apart. Here we use an expanding island of confluent Madin-Darby canine kidney (MDCK) cells as a model system to quantify the collective cell migration. In the absence of HGF, cell trajectories are highly tortuous whereas in the presence of HGF, they become far less so, resembling free expansion of a gas. At the level of cell-to-cell junctions, HGF attenuates the linkage of stress fibers to cell-to-cell junctions with concomitant decrease in intercellular stress. At the level of cell-to-substrate junctions, HGF augments the linkage of stress fibers to cell-to-substrate junctions with no apparent effect on traction. Together, HGF induces both structural changes in the actin-bound junctional protein complex and physical forces spanning multicellular clusters, which further promotes the expansion of confluent cellular layer. PMID:28374776

  12. Homogenizing cellular tension by hepatocyte growth factor in expanding epithelial monolayer.

    PubMed

    Jang, Hwanseok; Notbohm, Jacob; Gweon, Bomi; Cho, Youngbin; Park, Chan Young; Kee, Sun-Ho; Fredberg, Jeffrey J; Shin, Jennifer H; Park, Yongdoo

    2017-04-04

    Hepatocyte growth factor (HGF) induces cell migration and scattering by mechanisms that are thought to tip a local balance of competing physical forces; cell-to-cell and cell-to-substrate forces. In this local process, HGF is known to attenuate local cadherin-dependent adhesion forces for cell-cell junction development and enhance local integrin-dependent contractile forces for pulling neighboring cells apart. Here we use an expanding island of confluent Madin-Darby canine kidney (MDCK) cells as a model system to quantify the collective cell migration. In the absence of HGF, cell trajectories are highly tortuous whereas in the presence of HGF, they become far less so, resembling free expansion of a gas. At the level of cell-to-cell junctions, HGF attenuates the linkage of stress fibers to cell-to-cell junctions with concomitant decrease in intercellular stress. At the level of cell-to-substrate junctions, HGF augments the linkage of stress fibers to cell-to-substrate junctions with no apparent effect on traction. Together, HGF induces both structural changes in the actin-bound junctional protein complex and physical forces spanning multicellular clusters, which further promotes the expansion of confluent cellular layer.

  13. Human biliary tree stem/progenitor cells immunomodulation: Role of hepatocyte growth factor.

    PubMed

    Maraldi, Tullia; Guida, Marianna; Beretti, Francesca; Resca, Elisa; Carpino, Guido; Cardinale, Vincenzo; Gentile, Raffaele; Ardizzoni, Andrea; Murgia, Alba; Alvaro, Domenico; Gaudio, Eugenio; De Pol, Anto

    2017-04-01

    Human biliary tree stem/progenitor cells (hBTSC) are multipotent epithelial stem cells with the potential for allogenic transplant in liver, biliary tree, and pancreatic diseases. Human mesenchymal stem cells, but also epithelial stem cells, are able to modulate immune responses with different types of secretion molecules. The initial aim of the present study was to develop for the first time a culture protocol in order to expand hBTSC in vitro through passages, allowing to maintain a similar stem cell and secretome profile. Furthermore, we investigated the secretome profile of the hBTSC to assess the production of molecules capable of affecting immune feedback. We found that hepatocyte growth factor produced by hBTSC exerts its cytoprotective role inducing apoptosis in human immune cells, such as lymphocytes. The present study, therefore, supports the hypothesis that hBTSC can be useful for the purpose of regenerative medicine, as they can be banked and expanded, and they can secrete immunoregulatory factors. © 2016 The Japan Society of Hepatology.

  14. Changes in serum human hepatocyte growth factor levels after transcatheter arterial embolization and partial hepatectomy.

    PubMed

    Kaneko, A; Hayashi, N; Tanaka, Y; Ito, T; Kasahara, A; Kubo, M; Mukuda, T; Fusamoto, H; Kamada, T

    1992-08-01

    We examined the changes in serum human hepatocyte growth factor (hHGF), also called "scatter factor," levels after transcatheter arterial embolization (TAE) and partial hepatectomy (PH) in patients with hepatocellular carcinoma and metastatic liver tumor. In most cases, the serum hHGF levels increased transiently 1-3 days after TAE or PH, and then decreased nearly to the basal levels in 1 wk, suggesting that hHGF may play an important role in liver regeneration in humans. The mean amount of increase in serum hHGF levels after PH was 0.38 ng/ml, which was greater than that after TAE (0.16 ng/ml). In three cases of TAE followed by PH, two showed a greater increase in serum hHGF levels with PH than with TAE, but the third showed the reverse result. Because the rate of increase in serum ALT levels did not affect that of serum hHGF levels, the degree of liver injury induced by TAE or PH does not seem to be a determinant in serum hHGF elevation.

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

    PubMed Central

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

    2011-01-01

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

  16. Administration of antisense DNA for hepatocyte growth factor causes an depressive and anxiogenic response in rats.

    PubMed

    Wakatsuki, Masatoshi; Akiyoshi, Jotaro; Ichioka, Shugo; Tanaka, Yoshihiro; Tsuru, Jusen; Matsushita, Hirotaka; Hanada, Hiroaki; Isogawa, Koichi

    2007-12-01

    Hepatocyte growth factor (HGF) is induced in neurons during ischemia and is neuroprotective against post-ischemic delayed neuronal death in the hippocampus. HGF might play an important role in the maturation and functioning of these neurons in the hippocampus. Our aim was to determine what effect HGF antisense has on depression and anxiety in rats. HGF antisense was infused at a constant rate into cerebral lateral ventricles and its effect on anxiety in rats was monitored. In forced swimming test, rats that received antisense DNA increased the length of time that they were immobile in the water. In the elevated plus maze test, the black and white box test and conditioned fear test, HGF antisense administration caused all indicators of anxiety to increase. Number of HGF-positive cells in C1 of hippocampus was significantly decreased in the HGF antisense-infused group compared to the vehicle- and scrambled oligonucleotide-treated group. No significant effect on general locomotor activity was seen. These results indicate that inhibition of HGF induces an increase in depression and anxiety-related behaviors suggesting a depressive and anxiogenic-like effect.

  17. Effect of hepatocyte growth factor and angiotensin II on rat cardiomyocyte hypertrophy

    PubMed Central

    Chen, Ai-Lan; Ou, Cai-Wen; He, Zhao-Chu; Liu, Qi-Cai; Dong, Qi; Chen, Min-Sheng

    2012-01-01

    Angiotensin II (Ang II) plays an important role in cardiomyocyte hypertrophy. The combined effect of hepatocyte growth factor (HGF) and Ang II on cardiomyocytes is unknown. The present study was designed to determine the effect of HGF on cardiomyocyte hypertrophy and to explore the combined effect of HGF and Ang II on cardiomyocyte hypertrophy. Primary cardiomyocytes were isolated from neonatal rat hearts and cultured in vitro. Cells were treated with Ang II (1 µM) alone, HGF (10 ng/mL) alone, and Ang II (1 µM) plus HGF (10 ng/mL) for 24, 48, and 72 h. The amount of [3H]-leucine incorporation was then measured to evaluate protein synthesis. The mRNA levels of β-myosin heavy chain and atrial natriuretic factor were determined by real-time PCR to evaluate the presence of fetal phenotypes of gene expression. The cell size of cardiomyocytes was also studied. Ang II (1 µM) increased cardiomyocyte hypertrophy. Similar to Ang II, treatment with 1 µM HGF promoted cardiomyocyte hypertrophy. Moreover, the combination of 1 µM Ang II and 10 ng/mL HGF clearly induced a combined pro-hypertrophy effect on cardiomyocytes. The present study demonstrates for the first time a novel, combined effect of HGF and Ang II in promoting cardiomyocyte hypertrophy. PMID:23044624

  18. 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.

  19. 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

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

    PubMed

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

    2011-01-01

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

  1. Receptor binding and cell-mediated metabolism of (/sup 125/I)monoiodoglucagon by isolated canine hepatocytes

    SciTech Connect

    Hagopian, W.A.; Tager, H.S.

    1984-07-25

    A reverse-phase HPLC method has been developed to purify /sup 125/I-labeled products resulting from the chloramine-T-based iodination of glucagon. In addition the products ((/sup 125/I)iodoTyr/sup 10/ /sup 13/)glucagon, ((/sup 125/I)iodoTyr/sup 13/)glucagon, and ((/sup 125/I)iodoTyr/sup 10/)glucagon) have been used to study the receptor binding of glucagon and the cell-mediated metabolism of the hormone by isolated canine hepatocytes. It was concluded that (a) not withstanding apparent differences in affinities exhibited by the three peptides, the interactions with the glucagon receptor are functionally equivalent, and (b) the cell-mediated metabolism of receptor-bound glucagon involves the formation of hormone-derived peptides in which the biologically important NH/sub 2/-terminal region of the hormone has been modified by limited proteolytic cleavage.

  2. Gene transfection with human hepatocyte growth factor complementary DNA plasmids attenuates cardiac remodeling after acute myocardial infarction in goat hearts implanted with ventricular assist devices.

    PubMed

    Shirakawa, Yukitoshi; Sawa, Yoshiki; Takewa, Yoshiaki; Tatsumi, Eisuke; Kaneda, Yasufumi; Taenaka, Yoshiyuki; Matsuda, Hikaru

    2005-09-01

    Although a left ventricular assist device is often used to provide circulatory support until transplantation in severe heart failure, the mortality of long-term use of left ventricular assist devices remains high. We have shown that hepatocyte growth factor causes angiogenesis, antifibrosis, and antiapoptosis in the myocardium. Therefore, gene therapy with hepatocyte growth factor-complementary DNA plasmids may enhance the chance of "bridge to recovery." In this study, we performed gene therapy with hepatocyte growth factor in the impaired goat heart with a left ventricular assist device. Cardiac impairment was induced in 6 adult goats (56-65 kg) by ligation of the coronary artery, and ventricular assist devices were installed. The hepatocyte growth factor group (HGF; n = 3) was administered human hepatocyte growth factor-complementary DNA plasmid (2.0 mg) in the myocardium. The control group (n = 3) was similarly administered beta-galactosidase plasmid. Four weeks after gene transfection, we attempted to wean all goats from the ventricular assist device. The myocardia transfected with human hepatocyte growth factor-complementary DNA contained human hepatocyte growth factor protein at levels as high as 1.0 +/- 0.3 ng/g tissue 3 days after transfection. After weaning from the ventricular assist device, the HGF group showed good hemodynamics, whereas the control group showed deterioration. The percentage of fractional shortening was significantly higher in the HGF group than the control group (HGF vs control, 37.9% +/- 1.7% vs 26.4% +/- 0.3%, respectively; P < .01). Left ventricular dilatation associated with myocyte hypertrophy and fibrotic changes was detected in the control group but not in the HGF group. Vascular density was markedly increased in the HGF group. These results suggest that gene therapy with human hepatocyte growth factor may enhance the chance of bridge to recovery in the impaired heart supported with a ventricular assist device.

  3. 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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Levels of hepatocyte growth factor in serum correlate with quality of life in hemodialysis patients.

    PubMed

    Baum, Ewa; Pawlaczyk, Krzysztof; Maćkowiak, Beata; Sosinska, Patrycja; Matecka, Monika; Kolodziejczak, Barbara; Musielak, Michał; Breborowicz, Andrzej

    2015-01-01

    Patients with end stage renal failure (ESRD) report low quality of life and inflammation may be one of the contributing factors. We studied if the hemodialysis induced inflammation correlates with the patients quality of life. Study was performed in 76 (35 males and 41 females) ESRD patients treated with hemodialysis. Effect of one dialysis session on blood concentration of Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), Interleukin 6 (IL6) and Monocyte Chemoattractant Protein-1 (MCP-1) was studied. Results were correlated with answers given by patients to a short questionnaire composed of questions from Kidney Disease Quality of Life Short Form (KDQoL-SF) questionnaire. Hemodialysis induced increase of serum level of HGF (+117%) and IL-6 (+17%). Declared by patients health status correlated with their age, GFR, kt/V and hemodialysis induced change in serum IL6 and HGF level (R(2) = 0469, P < 0.001). Physical activity correlated with age, serum IL-6 and hemodialysis induced change in serum HGF and VEGF (R(2) = 0.362, P < 0.001). Presence of social/mental problems during previous 4 weeks correlated with age, serum HGF and hemodialysis induced changes in serum HGF and VEGF levels (R(2) = 0.333, P < 0.001). Interference of the kidney disease with daily life activities correlated with age, serum VEGF and hemodialysis induced change in serum HGF and IL6 levels (R(2) = 0.422, P < 0.001). Inflammation correlates with reduced quality of life in ESRD. Low hemodialysis-induced release of the anti-inflammatory cytokine HGF correlates with impaired quality of life in that group of patients.

  5. HSP27 is required for invasion and metastasis triggered by hepatocyte growth factor.

    PubMed

    Pavan, Simona; Musiani, Daniele; Torchiaro, Erica; Migliardi, Giorgia; Gai, Marta; Di Cunto, Ferdinando; Erriquez, Jessica; Olivero, Martina; Di Renzo, Maria Flavia

    2014-03-15

    The hepatocyte growth factor (HGF) also known as scatter factor activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27 kDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the remodeling of actin filaments induced by HGF and that motility in vitro depended on the p38MAPK-MK2 axis. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired the ability of circulating ovarian cancer cells to home to the lungs and to form experimental hematogenous metastases and the capability of cancer cells to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the proinvasive and prometastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy.

  6. Hepatocyte growth factor/scatter factor modulates cell motility, proliferation, and proteoglycan synthesis of chondrocytes.

    PubMed

    Takebayashi, T; Iwamoto, M; Jikko, A; Matsumura, T; Enomoto-Iwamoto, M; Myoukai, F; Koyama, E; Yamaai, T; Matsumoto, K; Nakamura, T

    1995-06-01

    Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that promotes proliferation, motility, and morphogenesis in epithelial cells. Recently the HGF receptor, c-met protooncogene product, has been shown to be expressed in developing limb buds (Sonnenberg, E., D. Meyer, M. Weidner, and C. Birchmeiyer, 1993. J. Cell Biol. 123: 223-235), suggesting that some populations of mesenchymal cells in limb buds respond to HGF/SF. To test the possibility that HGF/SF is involved in regulation of cartilage development, we isolated chondrocytes from knee joints and costal cartilages of 23-d embryonic and 4-wk-old rabbits, and analyzed the effects of HGF/SF on migration and proliferation of these cells. We found that HGF/SF stimulated migration of cultured articular chondrocytes but did not scatter limb mesenchymal fibroblasts or synovial fibroblasts in culture. HGF/SF also stimulated proliferation of chondrocytes; a maximum three-fold stimulation in DNA synthesis was observed at the concentration of 3 ng/ml of HGF/SF. Moreover, HGF/SF had the ability to enhance proteoglycan synthesis in chondrocytes. The responsiveness of chondrocytes to HGF/SF was also supported by the observation that they expressed the HGF/SF receptor. Addition of the neutralizing antibody to rat HGF/SF affected neither DNA synthesis nor proteoglycan synthesis in rat chondrocytes, suggesting a paracine mechanism of action of HGF/SF on these cells. In situ hybridization analysis showed that HGF/SF mRNA was restrictively expressed in the areas of future joint regions in developing limb buds and in the intercostal spaces of developing costal cartilages. These findings suggest that HGF/SF plays important roles in cartilage development through its multiple activities.

  7. Hepatocyte growth factor/scatter factor modulates cell motility, proliferation, and proteoglycan synthesis of chondrocytes

    PubMed Central

    1995-01-01

    Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that promotes proliferation, motility, and morphogenesis in epithelial cells. Recently the HGF receptor, c-met protooncogene product, has been shown to be expressed in developing limb buds (Sonnenberg, E., D. Meyer, M. Weidner, and C. Birchmeiyer, 1993. J. Cell Biol. 123: 223-235), suggesting that some populations of mesenchymal cells in limb buds respond to HGF/SF. To test the possibility that HGF/SF is involved in regulation of cartilage development, we isolated chondrocytes from knee joints and costal cartilages of 23-d embryonic and 4-wk-old rabbits, and analyzed the effects of HGF/SF on migration and proliferation of these cells. We found that HGF/SF stimulated migration of cultured articular chondrocytes but did not scatter limb mesenchymal fibroblasts or synovial fibroblasts in culture. HGF/SF also stimulated proliferation of chondrocytes; a maximum three-fold stimulation in DNA synthesis was observed at the concentration of 3 ng/ml of HGF/SF. Moreover, HGF/SF had the ability to enhance proteoglycan synthesis in chondrocytes. The responsiveness of chondrocytes to HGF/SF was also supported by the observation that they expressed the HGF/SF receptor. Addition of the neutralizing antibody to rat HGF/SF affected neither DNA synthesis nor proteoglycan synthesis in rat chondrocytes, suggesting a paracine mechanism of action of HGF/SF on these cells. In situ hybridization analysis showed that HGF/SF mRNA was restrictively expressed in the areas of future joint regions in developing limb buds and in the intercostal spaces of developing costal cartilages. These findings suggest that HGF/SF plays important roles in cartilage development through its multiple activities. PMID:7775584

  8. Hepatocyte Growth Factor Gene-Modified Mesenchymal Stem Cells Augment Sinonasal Wound Healing

    PubMed Central

    Li, Jing; Li, Yong; Yang, Chen; Lin, Hai; Duan, Hong-Gang

    2015-01-01

    This study was designed to investigate the effects of hepatocyte growth factor (HGF) transgenic mesenchymal stem cells (HGF-MSCs) on wound healing in the sinonasal mucosa and nasal epithelial cells (NECs). We also sought to determine whether HGF-MSCs and MSCs can migrate into the injured mucosa and differentiate into ciliated cells. Human HGF-overexpressing umbilical cord MSCs (hHGF-UCMSCs) were established, and upregulation of hHGF expression was confirmed by real-time PCR (RT-PCR) and enzyme-linked immunosorbant assay (ELISA). To investigate the paracrine effect of human MSCs (hMSCs) on nasal epithelial repair, hMSC- and HGF-MSC-conditioned media (CM) were used in NEC proliferation assays and in an in vitro scratch-wound repair model. The in vivo sinonasal wound-healing model was established, and all enrolled rabbits were randomly assigned to four groups: the GFP-MSC group, the HGF-MSC group, the Ad-HGF group, and the surgery control group. The average decreased diameter was recorded, and the medial wall of the maxillary sinus was removed for histological analysis and scanning electron microscopy. Collagen deposition in the wound tissue was detected via Masson trichrome (M&T) staining. The distribution of MSCs and HGF-MSCs was observed by immunofluorescence. MSCs improved nasal wound healing both in vivo and in vitro. HGF overexpression in MSCs augmented the curative effects. Reduced collagen deposition and transforming growth factor beta1 (TGF-β1) expression were detected in the HGF-MSC group compared with the MSC-, Ad-HGF-, and phosphate-buffered saline-treated groups based on M&T staining and ELISA. The enhanced therapeutic effects of HGF-MSCs were accompanied by decreased level of the fibrogenic cytokine TGF-β1. In addition, both HGF-MSCs and MSCs can migrate to the injured mucosa and epithelial layer. PMID:25835956

  9. Defect of hepatocyte growth factor production by fibroblasts in human pulmonary emphysema.

    PubMed

    Plantier, Laurent; Marchand-Adam, Sylvain; Marchal-Sommé, Joëlle; Lesèche, Guy; Fournier, Michel; Dehoux, Monique; Aubier, Michel; Crestani, Bruno

    2005-04-01

    Pulmonary emphysema results from an excessive degradation of lung parenchyma associated with a failure of alveolar repair. Secretion by pulmonary fibroblasts of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) is crucial to an effective epithelial repair after lung injury. We hypothesized that abnormal HGF or KGF secretion by pulmonary fibroblasts could play a role in the development of emphysema. We measured in vitro production of HGF and KGF by human fibroblasts cultured from emphysematous and normal lung samples. HGF and KGF production was quantified at basal state and after stimulation. Intracellular content of HGF was lower in emphysema (1.52 pg/mug, range of 0.15-7.40 pg/mug) than in control fibroblasts (14.16 pg/mug, range of 2.50-47.62 pg/mug; P = 0.047). HGF production by emphysema fibroblasts (19.3 pg/mug protein, range of 10.4-39.2 pg/mug) was lower than that of controls at baseline (57.5 pg/mug, range of 20.4-116 pg/mug; P = 0.019) and after stimulation with interleukin-1beta or prostaglandin E(2). Neither retinoic acids (all-trans and 9-cis) nor N-acetylcysteine could reverse this abnormality. KGF production by emphysema fibroblasts (5.3 pg/mug, range of 2.2-9.3 pg/mug) was similar to that of controls at baseline (2.6 pg/mug, range of 1-6.1 pg/mug; P = 0.14) but could not be stimulated with interleukin-1beta. A decreased secretion of HGF by pulmonary fibroblasts could contribute to the insufficient alveolar repair in pulmonary emphysema.

  10. 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

  11. Hepatocyte growth factor modification promotes the amelioration effects of human umbilical cord mesenchymal stem cells on rat acute kidney injury.

    PubMed

    Chen, Yuan; Qian, Hui; Zhu, Wei; Zhang, Xu; Yan, Yongmin; Ye, Shengqin; Peng, Xiujuan; Li, Wei; Xu, Wenrong

    2011-01-01

    Human umbilical cord-derived mesenchymal stem cells (hucMSCs) are particularly attractive cells for cellular and gene therapy in acute kidney injury (AKI). Adenovirus-mediated gene therapy has been limited by immune reaction and target genes selection. However, in the present study, we investigated the therapeutic effects of hepatocyte growth factor modified hucMSCs (HGF-hucMSCs) in ischemia/reperfusion-induced AKI rat models. In vivo animal models were generated by subjecting to 60 min of bilateral renal injury by clamping the renal pedicles and then introduced HGF-hucMSCs via the left carotid artery. Our results revealed that serum creatinine and urea nitrogen levels decreased to the baseline more quickly in HGF-hucMSCs-treated group than that in hucMSCs- or green fluorescent protein-hucMSCs-treated groups at 72 h after injury. The percent of proliferating cell nuclear antigen-positive cells in HGF-hucMSCs-treated group was higher than that in the hucMSCs or green fluorescent protein-hucMSCs-treated groups. Moreover, injured renal tissues treated with HGF-hucMSCs also exhibited less hyperemia and renal tubule cast during the recovery process. Immunohistochemistry and living body imaging confirmed that HGF-hucMSCs localize to areas of renal injury. Real-time polymerase chain reaction result showed that HGF-hucMSCs also inhibited caspase-3 and interleukin-1β mRNA expression in injured renal tissues. Western blot also showed HGF-hucMSCs-treated groups had lower expression of interleukin-1β. Terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate (dUTP) nick end labeling method indicated that HGF-hucMSCs-treated group had the least apoptosis cells. In conclusion, our findings suggest that HGF modification promotes the amelioration of ischemia/reperfusion-induced rat renal injury via antiapoptotic and antiinflammatory mechanisms; thus, providing a novel therapeutic application for hucMSCs in AKI.

  12. Structure of pleiotrophin- and hepatocyte growth factor-binding sulfated hexasaccharide determined by biochemical and computational approaches.

    PubMed

    Li, Fuchuan; Nandini, Chilkunda D; Hattori, Tomohide; Bao, Xingfeng; Murayama, Daisuke; Nakamura, Toshikazu; Fukushima, Nobuhiro; Sugahara, Kazuyuki

    2010-09-03

    Endogenous pleiotrophin and hepatocyte growth factor (HGF) mediate the neurite outgrowth-promoting activity of chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains isolated from embryonic pig brain. CS/DS hybrid chains isolated from shark skin have a different disaccharide composition, but also display these activities. In this study, pleiotrophin- and HGF-binding domains in shark skin CS/DS were investigated. A high affinity CS/DS fraction was isolated using a pleiotrophin-immobilized column. It showed marked neurite outgrowth-promoting activity and strong inhibitory activity against the binding of pleiotrophin to immobilized CS/DS chains from embryonic pig brain. The inhibitory activity was abolished by chondroitinase ABC or B, and partially reduced by chondroitinase AC-I. A pentasulfated hexasaccharide with a novel structure was isolated from the chondroitinase AC-I digest using pleiotrophin affinity and anion exchange chromatographies. It displayed a potent inhibitory effect on the binding of HGF to immobilized shark skin CS/DS chains, suggesting that the pleiotrophin- and HGF-binding domains at least partially overlap in the CS/DS chains involved in the neuritogenic activity. Computational chemistry using molecular modeling and calculations of the electrostatic potential of the hexasaccharide and two pleiotrophin-binding octasaccharides previously isolated from CS/DS hybrid chains of embryonic pig brain identified an electronegative zone potentially involved in the molecular recognition of the oligosaccharides by pleiotrophin. Homology modeling of pleiotrophin based on a related midkine protein structure predicted the binding pocket of pleiotrophin for the oligosaccharides and provided new insights into the molecular mechanism of the interactions between the oligosaccharides and pleiotrophin.

  13. 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.

  14. Sustained Expression of Naked Plasmid DNA Encoding Hepatocyte Growth Factor in Mice Promotes Liver and Overall Body Growth

    PubMed Central

    Yang, Junwei; Chen, Shiping; Huang, Leaf; Michalopoulos, George K.; Liu, Youhua

    2007-01-01

    To understand the physiological functions of exogenous hepatocyte growth factor (HGF) on normal adult animals, we delivered human HGF gene into mice by a hydrodynamics-based in vivo gene transfection approach using a naked plasmid vector. Systemic administration of naked plasmid containing HGF cDNA driven under cytomegalovirus promoter (pCMV-HGF) by rapid injection via the tail vein produced a remarkable level of human HGF protein in the circulation, beginning to appear at 4 hours and peaking at 12 hours following injection. Tissue distribution studies identified the liver as the organ with the highest level of transgene expression. Through weekly repeated injections of plasmid vector, we achieved sustained, long-term, high levels of exogenous HGF expression in mice for 8 weeks. Increases of more than 31% and 16% in liver and body weights were found, respectively, in the mice that received pCMV-HGF plasmid compared with that given the control vector for 8 weeks. Expression of exogenous HGF in vivo activated mitogen-activated protein kinases and induced proliferating cell nuclear antigen expression in normal adult liver and kidneys. These data suggest that systemic administration of naked plasmid vector is a convenient, safe, and highly efficient approach to introduce and maintain exogenous HGF gene expression in vivo in a controllable fashion. Our results also indicate that long-term expression of human HGF in mice markedly activates growth-related signal transduction events, promotes cell proliferation, and leads to liver and overall body growth in whole adult animals. PMID:11283849

  15. Hepatitis C virus NS5A and core proteins induce oxidative stress-mediated calcium signalling alterations in hepatocytes.

    PubMed

    Dionisio, Natalia; Garcia-Mediavilla, Maria V; Sanchez-Campos, Sonia; Majano, Pedro L; Benedicto, Ignacio; Rosado, Juan A; Salido, Gines M; Gonzalez-Gallego, Javier

    2009-05-01

    The hepatitis C virus (HCV) structural core and non-structural NS5A proteins induce in liver cells a series of intracellular events, including elevation of reactive oxygen and nitrogen species (ROS/RNS). Since oxidative stress is associated to altered intracellular Ca(2+) homeostasis, we aimed to investigate the effect of these proteins on Ca(2+) mobilization in human hepatocyte-derived transfected cells, and the protective effect of quercetin treatment. Ca(2+) mobilization and actin reorganization were determined by spectrofluorimetry. Production of ROS/RNS was determined by flow cytometry. Cells transfected with NS5A and core proteins showed enhanced ROS/RNS production and resting cytosolic Ca(2+) concentration, and reduced Ca(2+) concentration into the stores. Phenylephrine-evoked Ca(2+) release, Ca(2+) entry and extrusion by the plasma membrane Ca(2+)-ATPase were significantly reduced in transfected cells. Similar effects were observed in cytokine-activated cells. Phenylephrine-evoked actin reorganization was reduced in the presence of core and NS5A proteins. These effects were significantly prevented by quercetin. Altered Ca(2+) mobilization and increased calpain activation were observed in replicon-containing cells. NS5A and core proteins induce oxidative stress-mediated Ca(2+) homeostasis alterations in human hepatocyte-derived cells, which might underlie the effects of both proteins in the pathogenesis of liver disorders associated to HCV infection.

  16. Sab (Sh3bp5) dependence of JNK mediated inhibition of mitochondrial respiration in palmitic acid induced hepatocyte lipotoxicity

    PubMed Central

    Win, Sanda; Than, Tin Aung; Le, Bao Han Allison; García-Ruiz, Carmen; Fernandez-Checa, Jose C; Kaplowitz, Neil

    2015-01-01

    Background & Aims Sustained JNK activation by saturated fatty acids plays a role in lipotoxicity and the pathogenesis of NASH. We have reported that the interaction of JNK with mitochondrial Sab leads to inhibition of respiration, increased ROS, cell death and hepatotoxicity. We tested whether this pathway underlies palmitic acid (PA)-induced lipotoxicity in hepatocytes. Methods Primary mouse hepatocytes from adeno-shlacZ or adeno-shSab treated mice and Huh7 cells were used. Results In PMH, PA dose dependently up to 1mM stimulated oxygen consumption rate (OCR) due to mitochondrial β-oxidation. At ≥ 1.5mM, PA gradually reduced OCR, followed by cell death. Inhibition of JNK, caspases or treatment with antioxidant butylated hydroxyanisole (BHA) protected PMH against cell death. Sab knockdown or a membrane permeable Sab blocking peptide prevented PA-induced mitochondrial impairment, but inhibited only the late phase of both JNK activation (beyond 4 hours) and cell death. PA increased P-PERK and downstream target CHOP in PMH but failed to activate the IRE-1α arm of the UPR. However, Sab silencing did not affect PA-induced PERK activation. Conversely, specific inhibition of PERK prevented JNK activation and cell death, indicating a major role upstream of JNK activation. Conclusions The effect of P-JNK on mitochondria plays a key role in PA-mediated lipotoxicity. The interplay of P-JNK with mitochondrial Sab leads to impaired respiration, ROS production, sustained JNK activation, and apoptosis. PMID:25666017

  17. Prediction of drug-induced immune-mediated hepatotoxicity using hepatocyte-like cells derived from human embryonic stem cells.

    PubMed

    Kim, Dong Eon; Jang, Mi-Jin; Kim, Young Ran; Lee, Joo-Young; Cho, Eun Byul; Kim, Eunha; Kim, Yeji; Kim, Mi Young; Jeong, Won-Il; Kim, Seyun; Han, Yong-Mahn; Lee, Seung-Hyo

    2017-07-15

    Drug-induced liver injury (DILI) is a leading cause of liver disease and a key safety factor during drug development. In addition to the initiation events of drug-specific hepatotoxicity, dysregulated immune responses have been proposed as major pathological events of DILI. Thus, there is a need for a reliable cell culture model with which to assess drug-induced immune reactions to predict hepatotoxicity for drug development. To this end, stem cell-derived hepatocytes have shown great potentials. Here we report that hepatocyte-like cells derived from human embryonic stem cells (hES-HLCs) can be used to evaluate drug-induced hepatotoxic immunological events. Treatment with acetaminophen significantly elevated the levels of inflammatory cytokines by hES-HLCs. Moreover, three human immune cell lines, Jurkat, THP-1, and NK92MI, were activated when cultured in conditioned medium obtained from acetaminophen-treated hES-HLCs. To further validate, we tested thiazolidinedione (TZD) class, antidiabetic drugs, including troglitazone withdrawn from the market because of severe idiosyncratic drug hepatotoxicity. We found that TZD drug treatment to hES-HLCs resulted in the production of pro-inflammatory cytokines and eventually associated immune cell activation. In summary, our study demonstrates for the first time the potential of hES-HLCs as an in vitro model system for assessment of drug-induced as well as immune-mediated hepatotoxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Inhibition of hepatocyte nuclear factor 1b induces hepatic steatosis through DPP4/NOX1-mediated regulation of superoxide.

    PubMed

    Long, Zi; Cao, Meng; Su, Shuhao; Wu, Guangyuan; Meng, Fansen; Wu, Hao; Liu, Jiangzheng; Yu, Weihua; Atabai, Kamran; Wang, Xin

    2017-09-21

    Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder that is closely associated with insulin resistance and type 2 diabetes. Previous studies have suggested that hepatocyte nuclear factor 1b (HNF1b) ameliorates insulin resistance. However, the role of HNF1b in the regulation of lipid metabolism and hepatic steatosis remains poorly understood. We found that HNF1b expression was decreased in steatotic livers. We injected mice with lentivirus (LV) expressing HNF1b shRNA to generate mice with hepatic knockdown of HNF1b. We also injected high fat (HF) diet-induced obese and db/db diabetic mice with LV expressing HNF1b to overexpress HNF1b. Knockdown of HNF1b increased hepatic lipid contents and induced insulin resistance in mice and in hepatocytes. Knockdown of HNF1b worsened HF diet-induced increases in hepatic lipid contents, liver injury and insulin resistance in mice and PA-induced lipid accumulation and impaired insulin signaling in hepatocytes. Moreover, overexpression of HNF1b alleviated HF diet-induced increases in hepatic lipid content and insulin resistance in mice. Knockdown of HNF1b increased expression of genes associated with lipogenensis and endoplasmic reticulum (ER) stress. DPP4 and NOX1 expression was increased by knockdown of HNF1b and HNF1b directly bound with the promoters of DPP4 and NOX1. Overexpression of DPP4 or NOX1 was associated with an increase in lipid droplets in hepatocytes and decreased expression of DPP4 or NOX1 suppressed the effects of knockdown of HNF1b knockdown on triglyceride (TG) formation and insulin signaling. Knockdown of HNF1b increased superoxide level and decreased glutathione content, which was inhibited by downregulation of DPP4 and NOX1. N-acetylcysteine (NAC) suppressed HNF1b knockdown-induced ER stress, TG formation and insulin resistance. Palmitic acid (PA) decreased HNF1b expression which was inhibited by NAC. Taken together, these studies demonstrate that HNF1b plays an essential role

  19. Regeneration of Aged Rat Vocal Folds using Hepatocyte Growth Factor Therapy

    PubMed Central

    Ohno, Tsunehisa; Yoo, Mi Jin; Swanson, Erik R; Hirano, Shigeru; Ossoff, Robert H; Rousseau, Bernard

    2011-01-01

    Objectives/Hypothesis We investigated acute changes in extracellular matrix gene expression and histologic changes in the deposition of collagen and hyaluronan (HA) from hepatocyte growth factor (HGF) treatment of the aged rat vocal fold. We hypothesized that: 1) HGF induces matrix metalloproteinase gene expression, which may contribute to the downregulation of collagen; and 2) HGF induces hyaluronan synthase (HAS) gene expression, which may play a role in the upregulation of ECM HA. Study Design prospective animal study. Methods Fifteen, 18-month old, Sprague-Dawley rats were involved in this study. For gene expression analyses, ten rats were divided into two groups and received serial injections of sham (saline) or HGF (2ng/µL) and sacrificed 2 weeks after the initial injection to investigate acute changes in extracellular matrix gene expression. A separate group of five animals received the above treatment and were sacrificed 4 weeks after the initial injection to investigate histologic changes in the deposition of collagen and HA. Results Real-time polymerase chain reaction revealed significantly upregulated MMP-2, -9, and HAS-3 messenger RNA (mRNA) expression and significantly downregulated procollagen type I mRNA expression in the HGF-treatment group, compared to the sham-treatment group. Histologic staining revealed significantly reduced collagen deposition and increased deposition of HA in the HGF-treated vocal fold, compared to the sham-treated vocal fold. Conclusions HGF induced the upregulation of MMP-2, -9, and HAS-3, and downregulated the expression of procollagen type I. Histologically, aged vocal folds treated with HGF revealed decreased collagen deposition, and increased deposition of HA, compared to sham-treated vocal folds. PMID:19507223

  20. Hepatocyte growth factor attenuates pancreatic damage in caerulein-induced pancreatitis in rats.

    PubMed

    Warzecha, Z; Dembiński, A; Konturek, P C; Ceranowicz, P; Konturek, S J; Tomaszewska, R; Schuppan, D; Stachura, J; Nakamura, T

    2001-10-26

    Hepatocyte growth factor (HGF) overexpression was reported in experimental and clinical acute pancreatitis. These observations prompted us to determine the effect of HGF administration on the development of caerulein-induced pancreatitis in rats. Acute pancreatitis was induced by s.c. infusion of caerulein (10 microg/kg/h) for 5 h. HGF was administrated twice (30 min before caerulein or saline infusion and 3 h later) at the doses: 0.4, 2, 10 or 50 microg/kg s.c. Immediately after cessation of caerulein or saline infusion, the pancreatic blood flow, plasma amylase and lipase activity, plasma cytokines concentration, cell proliferation, and morphological signs of pancreatitis were examined. Caerulein administration induced acute edematous pancreatitis manifested by 41% decrease in DNA synthesis, 53% inhibition of pancreatic blood flow, a significant increase in plasma amylase and lipase activity, plasma interleukin-1beta and interleukin-6 concentration, as well as, the development of the histological signs of pancreatic damage (edema, leukocyte infiltration, and vacuolization). Administration of HGF without induction of pancreatitis increased plasma interleukin-10. Treatment with HGF, during induction of pancreatitis, increased plasma interleukin-10 and attenuated the pancreatic damage, what was manifested by histological improvement of pancreatic integrity, the partial reversion of the drop in DNA synthesis and pancreatic blood flow, and the reduction in pancreatitis evoked increase in plasma amylase, lipase, and interleukin-1beta and interleukin-6 levels. HGF administrated at the dose 2 microg/kg exhibited a similar beneficial effect as administration of HGF at the doses 10 or 50 microg/kg. Treatment with HGF at the dose 0.4 microg/kg was less effective. We conclude that: (1) administration of HGF attenuates pancreatic damage in caerulein-induced pancreatitis; (2) this effect seems to be related to the increase in production of interleukin-10, the reduction in

  1. Development of angiotensin IV analogs as hepatocyte growth factor/Met modifiers.

    PubMed

    Kawas, Leen H; McCoy, Alene T; Yamamoto, Brent J; Wright, John W; Harding, Joseph W

    2012-03-01

    The 6-AH family [D-Nle-X-Ile-NH-(CH(2))(5)-CONH(2); where X = various amino acids] of angiotensin IV (Ang IV) analogs binds directly to hepatocyte growth factor (HGF) and inhibit HGF's ability to form functional dimers. The metabolically stabilized 6-AH family member, D-Nle-Tyr-Ile-NH-(CH(2))(5)-CONH(2,) had a t(1/2) in blood of 80 min compared with the parent compound norleual [Nle-Tyr-Leu-Ψ-(CH(2)-NH(2))(3-4)-His-Pro-Phe], which had a t(1/2) in blood of <5 min. 6-AH family members were found to act as mimics of the dimerization domain of HGF (hinge region) and inhibited the interaction of an HGF molecule with a (3)H-hinge region peptide resulting in an attenuated capacity of HGF to activate its receptor Met. This interference translated into inhibition of HGF-dependent signaling, proliferation, and scattering in multiple cell types at concentrations down into the low picomolar range. We also noted a significant correlation between the ability of the 6-AH family members to block HGF dimerization and inhibition of the cellular activity. Furthermore, a member of the 6-AH family with cysteine at position 2, was a particularly effective antagonist of HGF-dependent cellular activities. This compound suppressed pulmonary colonization by B16-F10 murine melanoma cells, which are characterized by an overactive HGF/Met system. Together, these data indicate that the 6-AH family of Ang IV analogs exerts its biological activity by modifying the activity of the HGF/Met system and offers the potential as therapeutic agents in disorders that are dependent on or possess an overactivation of the HGF/Met system.

  2. Hepatocyte growth factor overexpression in the nervous system enhances learning and memory performance in mice.

    PubMed

    Kato, Takashi; Funakoshi, Hiroshi; Kadoyama, Keiichi; Noma, Satsuki; Kanai, Masaaki; Ohya-Shimada, Wakana; Mizuno, Shinya; Doe, Nobutaka; Taniguchi, Taizo; Nakamura, Toshikazu

    2012-09-01

    Hepatocyte growth factor (HGF) and its receptor, c-Met, play pivotal roles in the nervous system during development and in disease states. However, the physiological roles of HGF in the adult brain are not well understood. In the present study, to assess its role in learning and memory function, we used transgenic mice that overexpress HGF in a neuron-specific manner (HGF-Tg) to deliver HGF into the brain without injury. HGF-Tg mice displayed increased alternation rates in the Y-maze test compared with age-matched wild-type (WT) controls. In the Morris water maze (MWM) test, HGF-Tg mice took less time to find the platform on the first day, whereas the latency to escape to the hidden platform was decreased over training days compared with WT mice. A transfer test revealed that the incidence of arrival at the exact location of the platform was higher for HGF-Tg mice compared with WT mice. These results demonstrate that overexpression of HGF leads to an enhancement of both short- and long-term memory. Western blot analyses revealed that the levels of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, but not NR1, were increased in the hippocampus of HGF-Tg mice compared with WT controls, suggesting that an upregulation of NR2A and NR2B could represent one mechanism by which HGF enhances learning and memory performance. These results demonstrate that modulation of learning and memory performance is an important physiological function of HGF that contributes to normal CNS plasticity, and we propose HGF as a novel regulator of higher brain functions.

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

    PubMed Central

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

    2013-01-01

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

  4. 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

  5. Hepatocyte growth factor-like protein is a positive regulator of early mammary gland ductal morphogenesis.

    PubMed

    Gurusamy, Devikala; Ruiz-Torres, Sasha J; Johnson, Abby L; Smith, Dana A; Waltz, Susan E

    2014-08-01

    The Ron receptor tyrosine kinase regulates multiple cellular processes and is important during mammary gland development and tumor progression. Hepatocyte growth factor-like protein [HGFL] is the only known ligand for the Ron receptor and recent studies have identified major roles for HGFL during breast cancer metastasis. Understanding the functional importance HGFL during mammary gland development will provide significant insights onto its contribution during tumor development and metastasis. In this study, we assessed the role of HGFL during postnatal mammary gland development using mice that were either proficient [HGFL +/+] or deficient [HGFL-/-] for HGFL. Postnatal ductal morphology and stromal cell associations were analyzed at multiple time points through puberty until adulthood. HGFL deficiency resulted in several mammary gland developmental defects including smaller terminal end buds [TEBs], significantly fewer TEBs, and delayed ductal outgrowth during early puberty. Additionally, HGFL deficient animals exhibited significantly altered TEB epithelial cell turnover with decreased proliferation and increased apoptosis coupled with decreased TEB diameter. Macrophage recruitment to the TEBs was also significantly decreased in the HGFL-/- mice compared to controls. Moreover, the levels of STAT3 mRNA as well as the phosphorylation status of this protein were lower in the HGFL-/- mammary glands compared to controls. Taken together, our data provide the first evidence for HGFL as a positive regulator of mammary gland ductal morphogenesis by controlling overall epithelial cell turnover, macrophage recruitment, and STAT3 activation in the developing mammary gland. With a function in early mammary gland development, HGFL represents a potential target for the development of novel breast cancer therapies. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Treatment of Femoral Head Necrosis With Bone Marrow Mesenchymal Stem Cells Expressing Inducible Hepatocyte Growth Factor.

    PubMed

    Pan, Zhi-Min; Zhang, Yu; Cheng, Xi-Gao; Gao, Gui-Cheng; Wang, Xiang-Rui; Cao, Kai

    Our study assessed the effect of bone marrow mesenchymal stem cells (BMSCs) expressing inducible hepatocyte growth factor (HGF) on the recovery of femoral head necrosis (FHN). BMSCs were isolated by density gradient centrifugation. A recombinant AdTRE-HGF was constructed as the response plasmid and Adeno-X Tet-on as the regulator vector. The regulator and the response vectors were coinfected into BMSCs and induced at 0, 200, 500, 1000, and 1200 ng/mL doxycycline (Dox). After 3 days, the concentration of HGF was determined using enzyme-linked immunosorbent assay. Forty rabbits were selected to establish the FHN model and divided into 4 experimental groups. After the rabbits were killed by ketamine overdose, the restoration of FHN was assessed. The distribution of HGF-positive cells was observed by immunohistochemical method. Enzyme-linked immunosorbent assay results showed that 1000 ng/mL Dox induced the highest HGF expression level, even higher than the 1200 ng/mL Dox induction. The highest osteonecrosis incidence and empty lacunae percentage were found in group A compared with all the other groups (all P < 0.05). Furthermore, dramatically lower osteonecrosis incidence and empty lacunae percentage were found in group C compared with those of groups B and D (all P < 0.05). A significantly higher level of HGF protein was detected in group C compared with the other groups (all P < 0.05). Our study successfully developed the AdTRE-HGF, a recombinant adenovirus carrying HGF gene, for high expression of HGF in BMSCs. Importantly, introduction of BMSCs expressing HGF successfully produced the desired therapeutic effect in reversing FHN, in a Dox-dependent manner.

  7. The low levels of circulating hepatocyte growth factor in nephrolithiasis cases: independent from gene polymorphism.

    PubMed

    Ozturk, Nurinnisa; Aksoy, Hulya; Aksoy, Yilmaz; Yildirim, Abdulkadir; Akcay, Fatih; Yanmaz, Vefa

    2015-10-01

    Environmental and genetic factors are important in development of nephrolithiasis. In a recent study, it has been demonstrated that hepatocyte growth factor (HGF) has an anti-apoptotic effect and thus can reduce the adhesion of calcium oxalate monohydrate crystals to renal epithelial cells. The aim of this study was to evaluate the HGF serum levels and its two gene polymorphisms and possible association of the two in patients with nephrolithiasis. One hundred and five patients with nephrolithiasis and 70 healthy volunteers with similar demographic features were included in this study. Serum HGF levels were measured, and HGF intron 13 C>A (in 102 stone patients and 68 healthy subjects) and intron 14 T>C (in 99 stone patients and 56 healthy subjects) polymorphisms were determined using real-time polymerase chain reaction with TaqMan allelic discrimination method. There were no statistically significant differences in HGF intron 13 C>A and intron 14 T>C polymorphisms between the control and patient groups (X (2) = 1.72 df = 2; p = 0.42, and X (2) = 0.68 df = 2; p = 0.71, respectively). Mean serum HGF concentration was significantly lower in the stone disease patients than in the control subjects (1.05 ± 0.63 pg/mL and 1.35 ± 0.58 ng/mL respectively, p = 0.0001). When allele distribution frequency between stone patients and healthy subjects was compared, there were no significant differences in intron 13 and intron 14 allele distributions between two groups (p = 0.43 and p = 0.44, respectively). It may be concluded from the findings that decrease in HGF levels may play a role in renal stone formation, independent from gene polymorphisms.

  8. Growth hormone acutely increases glucose output by hepatocytes isolated from hypophysectomized rats.

    PubMed

    Blake, W L; Clarke, S D

    1989-08-01

    A series of experiments using isolated rat hepatocytes was carried out to establish rat liver cells in suspension as a physiological model for examining GH responses, and to determine whether acute recombinant bovine GH (rbGH) treatment of rat liver cells increased glucose output and/or suppressed fatty acid synthesis from lactate. Rat liver cells were isolated by collagenase perfusion and incubated in short-term (less than 60 min) suspension. The amount of insulin, glucagon or vasopressin required to elicit a half-maximal response was within the physiological range of the circulating hormone. When hepatocytes from normal rats were acutely (less than 60 min) treated with 0, 0.1, 10, 100 or 1000 nmol rbGH/l, rates of hepatocyte glucose output and fatty acid synthesis were unaltered. In addition, acute rbGH treatment (1000 nmol/l) did not alter hepatocyte responsiveness to insulin or vasopressin. However, acute rbGH treatment of hepatocytes isolated from hypophysectomized rats significantly (P less than 0.05) increased the rate of glucose output twofold and moderately (P less than 0.10) enhanced fatty acid synthesis. The accelerated rate of glucose production was not accompanied by an increase in the amount of glycogen phosphorylase-a. The observations with liver cells from hypophysectomized rats are not consistent with a GH receptor-transducing mechanism which is like that for glucagon (adenylate cyclase-linked) or insulin (tyrosine kinase-linked).

  9. Hepatocyte growth factor (HGF) inhibits skeletal muscle cell differentiation: a role for the bHLH protein twist and the cdk inhibitor p27.

    PubMed

    Leshem, Y; Spicer, D B; Gal-Levi, R; Halevy, O

    2000-07-01

    Hepatocyte growth factor (HGF) plays a crucial role in regulating the differentiation of both fetal and adult skeletal myoblasts. This study aimed at defining the intracellular factors that mediate the effect of HGF on adult myoblast differentiation. HGF increased Twist expression while decreasing p27(kip1) protein levels and not affecting the induction of p21(Cip1/Waf1) in satellite cells. Like HGF, overexpression of Twist did not affect p21 expression while inhibiting muscle-specific proteins. Both ectopic Twist-antisense (Twist-AS) and p27 partially rescued the effects of HGF on bromodeoxyuridine (BrdU) incorporation and myosin heavy chain (MHC) expression in muscle satellite cells; the two plasmids together effected full rescue, suggesting that HGF independently regulates these two factors to mediate its effects. Ectopic p27 promoted differentiation in the presence of HGF by blocking the induction of Twist. Using Twist-AS to lower Twist levels restored the HGF-dependent reduction of p27 and MHC. In the presence of ectopic HGF, satellite cells formed thin mononuclear myotubes. Neither ectopic p27, Twist-AS, or their combination reversed this change in cell morphology, suggesting that HGF acts through additional mediators to inhibit downstream events during myogenesis. Taken together, the results suggest that the effects of HGF on muscle cell proliferation and differentiation are mediated through changes in the expression levels of the myogenic-inhibitory basic helix-loop-helix (bHLH) protein Twist and the cell-cycle inhibitor p27.

  10. Effects of growth factors on hormonal stimulation of amino acid transport in primary cultures of rat hepatocytes.

    PubMed Central

    Auberger, P; Samson, M; Le Cam, A

    1983-01-01

    In primary cultures of rat hepatocytes, epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and foetal-calf serum (FCS) prevented the stimulation of amino acid transport by glucagon (cyclic AMP-dependent) and by catecholamines (cyclic AMP-independent), but not by insulin. The insulin effect, as well as the effect of other hormones, were totally inhibited by thrombin through a mechanism independent of its proteolytic activity. The inhibitory effect of growth factors, not found in freshly isolated hepatocytes, was expressed very early in culture (4h). Induction of tyrosine aminotransferase by glucagon or dexamethasone, which, like stimulation of transport, represents a late hormonal effect, was not affected by EGF, PDGF or FCS, but was inhibited by thrombin. In contrast, none of the rapid changes in protein phosphorylation caused by hormones was altered by growth factors. Thus the inhibition by growth factors of hormonal stimulation of transport presumably involves late step(s) in the cascade of events implicated in this hormonal effect. Images Fig. 6. PMID:6134522

  11. Effects of growth hormone, insulin-like growth factor I, triiodothyronine, thyroxine, and cortisol on gene expression of carbohydrate metabolic enzymes in sea bream hepatocytes.

    PubMed

    Leung, L Y; Woo, Norman Y S

    2010-11-01

    The present study investigated the regulatory effects of growth hormone (GH), human insulin-like growth factor I (hIGF-I), thyroxine (T(4)), triiodothyronine (T(3)) and cortisol, on mRNA expression of key enzymes involved in carbohydrate metabolism, including glucokinase (GK), glucose-6-phosphatase (G6Pase), glycogen synthase (GS), glycogen phosphorylase (GP) and glucose-6-phosphate dehydrogenase (G6PDH) in hepatocytes isolated from silver sea bream. Genes encoding GK, G6Pase, GS and GP were partially cloned and characterized from silver sea bream liver and real-time PCR assays were developed for the quantification of the mRNA expression profiles of these genes in order to evaluate the potential of these carbohydrate metabolic pathways. GK mRNA level was elevated by GH and hIGF-I, implying that GH-induced stimulation of GK expression may be mediated via IGF-I. GH was found to elevate GS and G6Pase expression, but reduce G6PDH mRNA expression. However, hIGF-I did not affect mRNA levels of GS, G6Pase and G6PDH, suggesting that GH-induced modulation of GS, G6Pase and G6PDH expression levels is direct, and occurs independently of the action of IGF-I. T(3) and T(4) directly upregulated transcript abundance of GK, G6Pase, GS and GP. Cortisol significantly increased transcript amounts of G6Pase and GS but markedly decreased transcript abundance of GK and G6PDH. These changes in transcript abundance indicate that (1) the potential of glycolysis is stimulated by GH and thyroid hormones, but attenuated by cortisol, (2) gluconeogenic and glycogenic potential are augmented by GH, thyroid hormones and cortisol, (3) glycogenolytic potential is upregulated by thyroid hormones but not affected by GH or cortisol, and (4) the potential of the pentose phosphate pathway is attenuated by GH and cortisol but unaffected by thyroid hormones.

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

    PubMed Central

    Crljen, Vladiana; Volinia, Stefano; Banfic, Hrvoje

    2002-01-01

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

  13. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. 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

  15. Aryl hydrocarbon receptor nuclear translocator in hepatocytes is required for aryl hydrocarbon receptor-mediated adaptive and toxic responses in liver.

    PubMed

    Nukaya, Manabu; Walisser, Jacqueline A; Moran, Susan M; Kennedy, Gregory D; Bradfield, Christopher A

    2010-12-01

    The aryl hydrocarbon receptor (AHR) plays a central role in the toxic responses to halogenated dibenzo-p-dioxins ("dioxins"), in the metabolic adaptation to polycyclic aromatic hydrocarbons, and in the development of the mature vascular system. A number of lines of evidence support the idea that the regulation of adaptive metabolism requires an AHR partnership with the aryl hydrocarbon receptor nuclear translocator (ARNT). Yet, for AHR-dependent vascular development and dioxin toxicity, the role of ARNT is less certain. In fact, numerous models have been proposed over the years to suggest that the AHR signals in important ways via ARNT-independent events. In an effort to clarify the role of ARNT in AHR-mediated dioxin hepatotoxicity, we generated a conditional Arnt mouse model. Such a model was essential because global inactivation of Arnt results in embryonic lethality presumably due to this protein's role as a heterodimeric partner for the hypoxia-inducible factors (HIFs). Using a hepatocyte-specific Arnt deletion, we were able to demonstrate that hepatocyte ARNT is required for major aspects of AHR-mediated dioxin toxicity in the liver. Results from this conditional Arnt allele are also consistent with a model where hepatocyte ARNT is unrelated to AHR-mediated hepatovascular development. In sum, these data suggest that AHR-ARNT dimers within the hepatocyte direct the toxic and adaptive and developmental functions associated with the AHR and that developmental vascular events arise due to signaling in a distinct cell type expressing this dimeric pair.

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

    PubMed

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

    2015-11-01

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

  17. 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

  18. PET of c-Met in Cancer with ⁶⁴Cu-Labeled Hepatocyte Growth Factor.

    PubMed

    Luo, Haiming; Hong, Hao; Slater, Michael R; Graves, Stephen A; Shi, Sixiang; Yang, Yunan; Nickles, Robert J; Fan, Frank; Cai, Weibo

    2015-05-01

    The hepatocyte growth factor (HGF) and its receptor, c-Met, are actively involved in tumor progression and metastasis and are closely associated with a poor prognostic outcome for cancer patients. Thus, the development of PET agents that can assess c-Met expression would be extremely useful for diagnosing cancer and subsequently monitoring response to c-Met-targeted therapies. Here, we report the characterization of recombinant human HGF (rh-HGF) as a PET tracer for detection of c-Met expression in vivo. rh-HGF was expressed in human embryonic kidney 293 cells and purified by nickel-nitrilotriacetic acid affinity chromatography. The concentrated rh-HGF was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid and labeled with (64)Cu. c-Met binding evaluation by flow cytometry was performed on both U87MG and MDA-MB-231 cell lines, which have a high level and a low level, respectively, of c-Met. PET imaging and biodistribution studies were performed on nude mice bearing U87MG and MDA-MB-231 xenografted tumors. The rh-HGF expression yield was 150-200 μg of protein per 5 × 10(6) cells after a 48-h transfection, with purity of approximately 85%-90%. Flow cytometry examination confirmed that rh-HGF had a strong and specific capacity to bind to c-Met. After (64)Cu labeling, PET imaging revealed specific and prominent uptake of (64)Cu-NOTA-rh-HGF in c-Met-positive U87MG tumors (percentage injected dose per gram, 6.8 ± 1.8 at 9 h after injection) and significantly lower uptake in c-Met-negative MDA-MB-231 tumors (percentage injected dose per gram, 1.8 ± 0.6 at 9 h after injection). The fact that sonication-denatured rh-HGF had significantly lower uptake in U87MG tumors, along with histology analysis, confirmed the c-Met specificity of (64)Cu-NOTA-rh-HGF. This study provided initial evidence that (64)Cu-NOTA-rh-HGF visualizes c-Met expression in vivo, an application that may prove useful for c-Met-targeted cancer therapy. © 2015 by the

  19. Individual cell-based models of cell scatter of ARO and MLP-29 cells in response to hepatocyte growth factor.

    PubMed

    Scianna, Marco; Merks, Roeland M H; Preziosi, Luigi; Medico, Enzo

    2009-09-07

    The different behaviors of colonies of two cell lines, ARO (thyroid carcinoma-derived cells) and MLP-29 (mouse liver progenitor cells), in response to hepatocyte growth factor (HGF) are described deducing suitable cellular Potts models (CPM). It is shown how increased motility and decreased adhesiveness are responsible for cell-cell dissociation and tissue invasion in the ARO cells. On the other hand, it is shown that, in addition to the biological mechanisms above, it is necessary to include directional persistence in cell motility and HGF diffusion to describe the scattering and the branching processes characteristic of MLP-29 cells.

  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. 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.

  2. Initial heme uptake from albumin by short-term cultured rat hepatocytes is mediated by a transport mechanism differing from that of other organic anions.

    PubMed

    Noyer, C M; Immenschuh, S; Liem, H H; Muller-Eberhard, U; Wolkoff, A W

    1998-07-01

    Although it is known that circulating heme accumulates in liver cells, the process by which heme enters hepatocytes is only partly understood. Hemopexin and a putative hemopexin receptor on hepatocyte membranes may mediate the uptake process. However, whether there are sufficient hemopexin receptors on rat hepatocytes to account for the bulk of heme entering cells is unknown. It is likely that heme may be transferred directly from albumin with the help of a plasma membrane heme transporter. To clarify the transport mechanism of heme into liver cells, we studied the uptake by short-term cultured rat hepatocytes of 55Fe-heme incubated with rat serum albumin. In these cells, the initial uptake of 55Fe-heme at 37 degrees C was five- to eightfold higher than that at 4 degrees C, linear for at least 5 minutes, and saturable. The Km of heme uptake was 0.95 +/- 0.27 micromol/L, and the Vmax was 0.12 +/- 0.01 pmol/min/mg protein (n = 3). Neither isosmotic substitution of sucrose for NaCl in the medium nor adenosine triphosphate (ATP) depletion, perturbations that are known to reduce uptake of bilirubin, sulfobromophthalein (BSP), and taurocholate, had any influence on 55Fe-heme uptake. In addition, heme uptake was not reduced in the presence of a greater than 500-fold molar excess of BSP. These results indicate that hepatocytes take up heme by a process that is distinct from that of these other organic anions.

  3. An Inducible Transgenic Mouse Model for Immune Mediated Hepatitis Showing Clearance of Antigen Expressing Hepatocytes by CD8+ T Cells

    PubMed Central

    Cebula, Marcin; Ochel, Aaron; Hillebrand, Upneet; Pils, Marina C.; Schirmbeck, Reinhold; Hauser, Hansjörg; Wirth, Dagmar

    2013-01-01

    The liver has the ability to prime immune responses against neo antigens provided upon infections. However, T cell immunity in liver is uniquely modulated by the complex tolerogenic property of this organ that has to also cope with foreign agents such as endotoxins or food antigens. In this respect, the nature of intrahepatic T cell responses remains to be fully characterized. To gain deeper insight into the mechanisms that regulate the CD8+ T cell responses in the liver, we established a novel OVA_X_CreERT2 mouse model. Upon tamoxifen administration OVA antigen expression is observed in a fraction of hepatocytes, resulting in a mosaic expression pattern. To elucidate the cross-talk of CD8+ T cells with antigen-expressing hepatocytes, we adoptively transferred Kb/OVA257-264-specific OT-I T cells to OVA_X_CreERT2 mice or generated triple transgenic OVA_X CreERT2_X_OT-I mice. OT-I T cells become activated in OVA_X_CreERT2 mice and induce an acute and transient hepatitis accompanied by liver damage. In OVA_X_CreERT2_X_OT-I mice, OVA induction triggers an OT-I T cell mediated, fulminant hepatitis resulting in 50% mortality. Surviving mice manifest a long lasting hepatitis, and recover after 9 weeks. In these experimental settings, recovery from hepatitis correlates with a complete loss of OVA expression indicating efficient clearance of the antigen-expressing hepatocytes. Moreover, a relapse of hepatitis can be induced upon re-induction of cured OVA_X_CreERT2_X_OT-I mice indicating absence of tolerogenic mechanisms. This pathogen-free, conditional mouse model has the advantage of tamoxifen inducible tissue specific antigen expression that reflects the heterogeneity of viral antigen expression and enables the study of intrahepatic immune responses to both de novo and persistent antigen. It allows following the course of intrahepatic immune responses: initiation, the acute phase and antigen clearance. PMID:23869228

  4. An inducible transgenic mouse model for immune mediated hepatitis showing clearance of antigen expressing hepatocytes by CD8+ T cells.

    PubMed

    Cebula, Marcin; Ochel, Aaron; Hillebrand, Upneet; Pils, Marina C; Schirmbeck, Reinhold; Hauser, Hansjörg; Wirth, Dagmar

    2013-01-01

    The liver has the ability to prime immune responses against neo antigens provided upon infections. However, T cell immunity in liver is uniquely modulated by the complex tolerogenic property of this organ that has to also cope with foreign agents such as endotoxins or food antigens. In this respect, the nature of intrahepatic T cell responses remains to be fully characterized. To gain deeper insight into the mechanisms that regulate the CD8+ T cell responses in the liver, we established a novel OVA_X_CreER(T2) mouse model. Upon tamoxifen administration OVA antigen expression is observed in a fraction of hepatocytes, resulting in a mosaic expression pattern. To elucidate the cross-talk of CD8+ T cells with antigen-expressing hepatocytes, we adoptively transferred K(b)/OVA257-264-specific OT-I T cells to OVA_X_CreER(T2) mice or generated triple transgenic OVA_X CreER(T2)_X_OT-I mice. OT-I T cells become activated in OVA_X_CreER(T2) mice and induce an acute and transient hepatitis accompanied by liver damage. In OVA_X_CreER(T2)_X_OT-I mice, OVA induction triggers an OT-I T cell mediated, fulminant hepatitis resulting in 50% mortality. Surviving mice manifest a long lasting hepatitis, and recover after 9 weeks. In these experimental settings, recovery from hepatitis correlates with a complete loss of OVA expression indicating efficient clearance of the antigen-expressing hepatocytes. Moreover, a relapse of hepatitis can be induced upon re-induction of cured OVA_X_CreER(T2)_X_OT-I mice indicating absence of tolerogenic mechanisms. This pathogen-free, conditional mouse model has the advantage of tamoxifen inducible tissue specific antigen expression that reflects the heterogeneity of viral antigen expression and enables the study of intrahepatic immune responses to both de novo and persistent antigen. It allows following the course of intrahepatic immune responses: initiation, the acute phase and antigen clearance.

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

    PubMed Central

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

    2015-01-01

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

  6. 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

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

    PubMed

    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.

  8. 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

  9. Expression and release of the latent transforming growth factor beta binding protein by hepatocytes from rat liver.

    PubMed

    Roth, S; Schurek, J; Gressner, A M

    1997-06-01

    In very recent studies it was established that transforming growth factor beta (TGF-beta), likely to be the most relevant fibrogenic cytokine and regulator of cell proliferation, differentiation, and matrix metabolism, is expressed by hepatocytes (parenchymal cell [PC]) and secreted from cultured PC in a latent form incapable of receptor binding. The structural composition of the latent TGF-beta complex secreted by cultured PC is unknown. In some TGF-beta expressing cell types this cytokine is released as a large molecular weight complex containing in addition to the TGF-beta latency associated peptide (LAP) a disulfide bonded latent TGF-beta binding protein (LTBP), of which the existence and function in liver is hitherto unknown. This study is directed to the identification of LTBP expression in rat PC. Cells were isolated from rat liver with the collagenase method and analyzed for LTBP before and during culture under standard conditions using alkaline phosphatase anti-alkaline phosphatase (APAAP) immunostainings, metabolic labeling, messenger RNA (mRNA) detection (reverse-transcription polymerase chain reaction [RT-PCR]) and sequencing, and immunoblotting of gel chromatographically separated cell extracts and conditioned media, respectively. APAAP immunostainings applying a specific polyclonal LTBP-antiserum (ab 39) indicated expression of LTBP in PC of liver in situ and freshly isolated PC but a strong expression in cultured PC. Transcripts of LTBP-1 were detected by RT-PCR and confirmed by sequence analyses. Metabolic labeling of PC with [35S]-Met/Cys followed by immunoprecipitation of cell lysates with LTBP antiserum confirmed the synthesis of the high molecular mass complex of 250 kd containing LTBP with a molecular mass of 160 kd. Latent TGF-beta complexes, associated with LTBP related proteins, could be separated from both extracts and conditioned media of PC by gel filtration chromatography. They confirmed the release of the large latent TGF-beta complex

  10. Hepatocyte growth factor inhibition: a novel therapeutic approach in pancreatic cancer

    PubMed Central

    Pothula, Srinivasa P; Xu, Zhihong; Goldstein, David; Biankin, Andrew V; Pirola, Romano C; Wilson, Jeremy S; Apte, Minoti V

    2016-01-01

    Background: Pancreatic stellate cells (PSCs, which produce the stroma of pancreatic cancer (PC)) interact with cancer cells to facilitate PC growth. A candidate growth factor pathway that may mediate this interaction is the HGF–c-MET pathway. Methods: Effects of HGF inhibition (using a neutralising antibody AMG102) alone or in combination with gemcitabine were assessed (i) in vivo using an orthotopic model of PC, and (ii) in vitro using cultured PC cells (AsPC-1) and human PSCs. Results: We have shown that human PSCs (hPSCs) secrete HGF but do not express the receptor c-MET, which is present predominantly on cancer cells. HGF inhibition was as effective as standard chemotherapy in inhibiting local tumour growth but was significantly more effective than gemcitabine in reducing tumour angiogenesis and metastasis. HGF inhibition has resulted in reduced metastasis; however, interestingly this antimetastatic effect was lost when combined with gemcitabine. This suggests that gemcitabine treatment selects out a subpopulation of cancer cells with increased epithelial–mesenchymal transition (EMT) and stem-cell characteristics, as supported by our findings of increased expression of EMT and stem-cell markers in tumour sections from our animal model. In vitro studies showed that hPSC secretions induced proliferation and migration, but inhibited apoptosis, of cancer cells. These effects were countered by pretreatment of hPSC secretions with a HGF-neutralising antibody but not by gemcitabine, indicating a key role for HGF in PSC–PC interactions. Conclusions: Our studies suggest that targeted therapy to inhibit stromal–tumour interactions mediated by the HGF–c-MET pathway may represent a novel therapeutic approach in PC that will require careful modelling for optimal integration with existing treatment modalities. PMID:26766740

  11. Differential Mitogenic Effects of Single Chain Hepatocyte Growth Factor (HGF)/Scatter Factor and HGF/NK1 following Cleavage by Factor Xa*

    PubMed Central

    Pediaditakis, Peter; Monga, Satdarshan P. S.; Mars, Wendy M.; Michalopoulos, George K.

    2007-01-01

    Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that is involved in many normal as well as pathological conditions. HGF/NK1, a splice variant of HGF/SF, has been reported to have either antagonistic or agonistic effects with regard to c-Met signaling depending on the cell type. In these experiments, we have determined that HGF/NK1 is a potent mitogen for rat hepatocytes in culture. Furthermore, we have found that coagulation factor Xa (fXa) is capable of cleaving HGF/NK1 and single chain HGF/SF (scHGF/SF). The products resulting from cleavage of HGF/NK1 or scHGF/SF by fXa appear as single bands under non-reducing conditions. The reaction products from the digestion of HGF/NK1 by fXa were separated under reducing conditions, and the cleavage site, as determined by N-terminal sequencing, was located C-terminal to arginine 134. Previous work established that the heparin-binding domain for HGF/SF is located in the N domain of HGF/SF. Additionally, the dimerization of the HGF/SF receptor (c-Met) by the ligand HGF/NK1 is facilitated by heparin and related sulfonated sugars on the cell surface, whereas heparin is not required for HGF/SF-mediated dimerization. Cleavage of single chain HGF/SF or HGF/NK1 by factor Xa does not alter the affinity of the respective molecules for heparin, but it did variably affect the associated mitogenic activity of these factors. The associated mitogenic activity of HGF/NK1 was reduced by more than 90%, whereas the mitogenic activity of scHGF/SF was unaffected. This suggests mandatory maintenance of a steric interaction of the N domain and the first kringle domain for HGF/NK1 to act as an agonist for rat hepatocyte growth but is not required by full-length HGF/SF. PMID:11832492

  12. The Role of Constitutive Androstane Receptor in Oxazaphosphorine-mediated Induction of Drug-metabolizing Enzymes in Human Hepatocytes

    PubMed Central

    Wang, Duan; Li, Linhao; Fuhrman, Jennifer; Ferguson, Stephen; Wang, Hongbing

    2013-01-01

    Purpose The objective of this study was to investigate the roles of the constitutive androstane receptor (CAR) in cyclophosphamide (CPA)- and ifosfamide (IFO)-mediated induction of hepatic drug-metabolizing enzymes (DME). Methods Induction of DMEs was evaluated using real-time RT-PCR and Western blotting analysis in human primary hepatocyte (HPH) cultures. Activation of CAR, pregnane X receptor (PXR), and aryl hydrocarbon receptor by CPA and IFO was assessed in cell-based reporter assays in HepG2 cells and/or nuclear translocation assays in HPHs. Results CYP2B6 reporter activity was significantly enhanced by CPA and IFO in HepG2 cells co-transfected with CYP2B6 reporter plasmid and a chemical-responsive human CAR variant (CAR1+A) construct. Real-time RT-PCR and Western blotting analysis in HPHs showed that both CPA and IFO induced the expressions of CYP2B6 and CYP3A4. Notably, treatment of HPHs with CPA but not IFO resulted in significant nuclear accumulation of CAR, which represents the initial step of CAR activation. Further studies in HPHs demonstrated that selective inhibition of PXR by sulforaphane preferentially repressed IFO- over CPA-mediated induction of CYP2B6. Conclusion These results provide novel insights into the differential roles of CAR in the regulation of CPA- and IFO-induced DME expression and potential drug-drug interactions. PMID:21487929

  13. Effects of hepatocyte growth factor in myocarditis rats induced by immunization with porcine cardiac myosin

    PubMed Central

    Nakano, Jota; Marui, Akira; Muranaka, Hiroyuki; Masumoto, Hidetoshi; Noma, Hisashi; Tabata, Yasuhiko; Ido, Akio; Tsubouchi, Hirohito; Ikeda, Tadashi; Sakata, Ryuzo

    2014-01-01

    OBJECTIVES Myocarditis is considered one of the major causes of dilated cardiomyopathy. Hepatocyte growth factor (HGF) has pleiotropic activities that promote tissue regeneration and facilitate functional improvement of injured tissue. We investigated whether the epicardial sustained-release of HGF, using gelatin hydrogel sheets, improves cardiac function in a chronic myocarditis rat model. METHODS Six weeks after Lewis rats were immunized with porcine cardiac myosin to establish autoimmune myocarditis, HGF- or normal saline (NS)-incorporated gelatin hydrogel sheets were applied to the epicardium (G-HGF and G-NS, respectively). At either 2 or 4 weeks after treatment, these were compared with the Control myocarditis group. Cardiac function was evaluated by echocardiography and cardiac catheterization. Development of fibrosis was determined by histological study and expression of transforming growth factor-β1 (TGF-β1). Bax and Bcl-2 levels were measured to evaluate apoptotic activity. RESULTS At both points, fractional shortening and end-systolic elastance were higher in the G-HGF group than in the Control and G-NS groups (P < 0.01). Fractional shortening at 2 weeks of each group were as follows: 31.0 ± 0.9%, 24.8 ± 2.7% and 48.6 ± 2.6% (Control, G-NS and G-HGF, respectively). The ratio of the fibrotic area of the myocardium was lower in the G-HGF group than in the Control and G-NS groups at 2 weeks (G-HGF, 8.8 ± 0.9%; Control, 17.5 ± 0.2%; G-NS, 15.6 ± 0.7%; P < 0.01). The ratio at 4 weeks was lower in the G-HGF group than in the G-NS group (10.9 ± 1.4% vs 18.5 ± 1.3%; P < 0.01). The mRNA expression of TGF-β1 in the G-HGF group was lower than in the Control group at 2 weeks (0.6 ± 0.1 vs 1.1 ± 0.2) and lower than that in the G-NS group at 4 weeks (0.7 ± 0.1 vs 1.3 ± 0.2). The Bax-to-Bcl-2 ratios at both points were lower in the G-HGF group than in the Control group. CONCLUSIONS Sustained-released HGF markedly improves cardiac function in chronic

  14. miR-181a mediates TGF-β-induced hepatocyte EMT and is dysregulated in cirrhosis and hepatocellular cancer.

    PubMed

    Brockhausen, Jennifer; Tay, Szun S; Grzelak, Candice A; Bertolino, Patrick; Bowen, David G; d'Avigdor, William M; Teoh, Narcy; Pok, Sharon; Shackel, Nick; Gamble, Jennifer R; Vadas, Mathew; McCaughan, Geoff W

    2015-01-01

    Epithelial-mesenchymal transition (EMT) has been implicated in the processes of embryogenesis, tissue fibrosis and carcinogenesis. Transforming growth factor-β (TGF-β) has been identified as a key driver of EMT and plays a key role in the pathogenesis of cirrhosis and hepatocellular carcinoma (HCC). The aim was to identify microRNA (miR) expression in TGF-β-induced hepatocyte EMT. We treated a human hepatocyte cell line PH5CH8 with TGF-β to induce an EMT-like change in phenotype and then identified dysregulated miRs using TaqMan Low Density Arrays. MiR expression was altered using miR-181a mimic and inhibitor in the same system and gene changes were identified using TaqMan gene arrays. MiR-181a gene expression was measured in human and mouse cirrhotic or HCC liver tissue samples. Gene changes were identified in rAAV-miR-181a-expressing mouse livers using TaqMan gene arrays. We identified miR-181a as a miR that was significantly up-regulated in response to TGF-β treatment. Over-expression of a miR-181a mimic induced an in vitro EMT-like change with a phenotype similar to that seen with TGF-β treatment alone and was reversed using a miR-181a inhibitor. MiR-181a was shown to be up-regulated in experimental and human cirrhotic and HCC tissue. Mouse livers expressing rAAV-miR-181a showed genetic changes associated with TGF-β signalling and EMT. MiR-181a had a direct effect in inducing hepatocyte EMT and was able to replace TGF-β-induced effects in vitro. MiR-181a was over-expressed in cirrhosis and HCC and is likely to play a role in disease pathogenesis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    PubMed Central

    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; Germain, Stéphane

    2016-01-01

    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. Significance The results from the present study show that fibroblast growth factor-2 (FGF-2) priming is more

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

    PubMed Central

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

    2014-01-01

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

  17. A mutation of MET, encoding hepatocyte growth factor receptor, is associated with human DFNB97 hearing loss

    PubMed Central

    Mujtaba, Ghulam; Schultz, Julie M; Imtiaz, Ayesha; Morell, Robert J; Friedman, Thomas B; Naz, Sadaf

    2015-01-01

    Background Hearing loss is a heterogeneous neurosensory disorder. Mutations of 56 genes are reported to cause recessively inherited nonsyndromic deafness. Objective We sought to identify the genetic lesion causing hearing loss segregating in a large consanguineous Pakistani family. Methods and Results Mutations of GJB2 and all other genes reported to underlie recessive deafness were ruled out as the cause of the phenotype in the affected members of the participating family. Homozygosity mapping with a dense array of one million SNP markers allowed us to map the gene for recessively inherited severe hearing loss to chromosome 7q31.2, defining a new deafness locus designated DFNB97 (maximum LOD score of 4.8). Whole-exome sequencing revealed a novel missense mutation c.2521T>G (p.F841V) in MET, which encodes the receptor for hepatocyte growth factor. The mutation co-segregated with the hearing loss phenotype in the family and was absent from 800 chromosomes of ethnically matched control individuals as well as from 136,602 chromosomes in public databases of nucleotide variants. Analyses by multiple prediction programs indicated that p.F841V is likely damaging to MET function. Conclusion We identified a missense mutation of MET, encoding the hepatocyte growth factor receptor, as a likely cause of hearing loss in humans. PMID:25941349

  18. Hepatocyte growth factor, vascular endothelial growth factor, glial cell-derived neurotrophic factor and nerve growth factor are differentially affected by early chronic ethanol or red wine intake.

    PubMed

    Fiore, Marco; Mancinelli, Rosanna; Aloe, Luigi; Laviola, Giovanni; Sornelli, Federica; Vitali, Mario; Ceccanti, Mauro

    2009-08-10

    Ethanol intake during pregnancy and lactation induces severe changes in brain and liver throughout mechanisms involving growth factors. These are signaling molecules regulating survival, differentiation, maintenance and connectivity of brain and liver cells. Ethanol is an element of red wine which contains also compounds with antioxidant properties. Aim of the study was to investigate differences in hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) in brain areas and liver by ELISA of 1-month-old male mice exposed perinatally to ethanol at 11 vol.% or to red wine at same ethanol concentration. Ethanol was administered before and during pregnancy up to pups' weaning. Ethanol per se elevated HGF in liver and cortex, potentiated liver VEGF, reduced GDNF in the liver and decreased NGF content in hippocampus and cortex in the offspring. We did not find changes in HGF or NGF due to red wine exposure. However, we revealed elevation in VEGF levels in liver and reduced GDNF in the cortex of animals exposed to red wine but the VEGF liver increase was more marked in animals exposed to ethanol only compared to the red wine group. In conclusion the present findings in the mouse show differences in ethanol-induced toxicity when ethanol is administered alone or in red wine that may be related to compounds with antioxidant properties present in the red wine.

  19. 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.

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

    PubMed

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

    2015-12-25

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

  1. Requirement of gene fadD33 for the growth of Mycobacterium tuberculosis in a hepatocyte cell line.

    PubMed

    Rindi, L; Bonanni, D; Lari, N; Garzelli, C

    2004-04-01

    Gene fadD33 of Mycobacterium tuberculosis, one of the 36 homologues of gene fadD of Escherichia coli identified in the M. tuberculosis genome, predictively encodes an acyl-CoA synthase, an enzyme involved in fatty acids metabolism. The gene is underexpressed in the attenuated strain M. tuberculosis H37Ra relative to virulent H37Rv and plays a role in M. tuberculosis virulence in BALB/c mice by supporting mycobacterial replication in the liver. In the present paper, we investigated the role of fadD33 expression in bacterial growth within the hepatocyte cell line HepG2, as well as in human monocyte-derived THP-1 cells and peripheral blood mononuclear cells. M. tuberculosis H37Rv proved able to grow within HepG2 cells, while the intracellular replication of M. tuberculosis H37Ra was markedly impaired; complementation of strain H37Ra with gene fadD33 restored its replication to the levels of H37Rv. Moreover, disruption of gene fadD33 by allelic exchange mutagenesis reduced the intracellular growth of M. tuberculosis H37Rv, and complementation of the fadD33-disrupted mutant with gene fadD33 restored bacterial replication. Conversely, fadD33 expression proved unable to influence M. tuberculosis growth in human phagocytes, as fadD33-disrupted M. tuberculosis H37Rv mutant, as well as fadD33-complemented M. tuberculosis H37Ra, grew within THP-1 cells and peripheral monocytes basically at the same rates as parent H37Rv and H37Ra strains. The results of these experiments indicate that gene fadD33 expression confers growth advantage to M. tuberculosis in immortalized hepatocytes, but not in macrophages, thus emphasizing the importance of fadD33 in liver-specific replication of M. tuberculosis.

  2. Actions of activin A, connective tissue growth factor, hepatocyte growth factor and teratocarcinoma-derived growth factor 1 on the development of the bovine preimplantation embryo.

    PubMed

    Kannampuzha-Francis, Jasmine; Tribulo, Paula; Hansen, Peter J

    2016-05-17

    The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. In the present study we tested four growth factors expressed in the endometrium for their ability to modify the development of the bovine embryo to the blastocyst stage and alter the expression of genes found to be upregulated (bone morphogenetic protein 15 (BMP15) and keratin 8, type II (KRT8)) or downregulated (NADH dehydrogenase 1 (ND1) and S100 calcium binding protein A10 (S100A10)) in embryos competent to develop to term. Zygotes were treated at Day 5 with 0.01, 0.1 or 1.0 nM growth factor. The highest concentration of activin A increased the percentage of putative zygotes that developed to the blastocyst stage. Connective tissue growth factor (CTGF) increased the number of cells in the inner cell mass (ICM), decreased the trophectoderm : ICM ratio and increased blastocyst expression of KRT8 and ND1. The lowest concentration of hepatocyte growth factor (HGF) reduced the percentage of putative zygotes becoming blastocysts. Teratocarcinoma-derived growth factor 1 increased total cell number at 0.01 nM and expression of S100A10 at 1.0 nM, but otherwise had no effects. Results confirm the prodevelopmental actions of activin A and indicate that CTGF may also function as an embryokine by regulating the number of ICM cells in the blastocyst and altering gene expression. Low concentrations of HGF were inhibitory to development.

  3. Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide.

    PubMed Central

    Karlsson, J O; Cravalho, E G; Borel Rinkes, I H; Tompkins, R G; Yarmush, M L; Toner, M

    1993-01-01

    A three-part, coupled model of cell dehydration, nucleation, and crystal growth was used to study intracellular ice formation (IIF) in cultured hepatocytes frozen in the presence of dimethyl sulfoxide (DMSO). Heterogeneous nucleation temperatures were predicted as a function of DMSO concentration and were in good agreement with experimental data. Simulated freezing protocols correctly predicted and explained experimentally observed effects of cooling rate, warming rate, and storage temperature on hepatocyte function. For cells cooled to -40 degrees C, no IIF occurred for cooling rates less than 10 degrees C/min. IIF did occur at faster cooling rates, and the predicted volume of intracellular ice increased with increasing cooling rate. Cells cooled at 5 degrees C/min to -80 degrees C were shown to undergo nucleation at -46.8 degrees C, with the consequence that storage temperatures above this value resulted in high viability independent of warming rate, whereas colder storage temperatures resulted in cell injury for slow warming rates. Cell damage correlated positively with predicted intracellular ice volume, and an upper limit for the critical ice content was estimated to be 3.7% of the isotonic water content. The power of the model was limited by difficulties in estimating the cytosol viscosity and membrane permeability as functions of DMSO concentration at low temperatures. Images FIGURE 1 PMID:8312489

  4. 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.

  5. 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.

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

    PubMed Central

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

    1998-01-01

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

  7. Impact of MET inhibition on small-cell lung cancer cells showing aberrant activation of the hepatocyte growth factor/MET pathway.

    PubMed

    Taniguchi, Hirokazu; Yamada, Tadaaki; Takeuchi, Shinji; Arai, Sachiko; Fukuda, Koji; Sakamoto, Shuichi; Kawada, Manabu; Yamaguchi, Hiroyuki; Mukae, Hiroshi; Yano, Seiji

    2017-07-01

    Small-cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers, and is characterized as extremely aggressive, often displaying rapid tumor growth and multiple organ metastases. In addition, the clinical outcome of SCLC patients is poor due to early relapse and acquired resistance to standard chemotherapy treatments. Hence, novel therapeutic strategies for the treatment of SCLC are urgently required. Accordingly, several molecular targeted therapies were evaluated in SCLC; however, they failed to improve the clinical outcome. The receptor tyrosine kinase MET is a receptor for hepatocyte growth factor (HGF), and aberrant activation of HGF/MET signaling is known as one of the crucial mechanisms enabling cancer progression and invasion. Here, we found that the HGF/MET signaling was aberrantly activated in chemoresistant or chemorelapsed SCLC cell lines (SBC-5, DMS273, and DMS273-G3H) by the secretion of HGF and/or MET copy number gain. A cell-based in vitro assay revealed that HGF/MET inhibition, induced either by MET inhibitors (crizotinib and golvatinib), or by siRNA-mediated knockdown of HGF or MET, constrained growth of chemoresistant SCLC cells through the inhibition of ERK and AKT signals. Furthermore, treatment with either crizotinib or golvatinib suppressed the systemic metastasis of SBC-5 cell tumors in natural killer cell-depleted SCID mice, predominantly through cell cycle arrest. These findings reveal the therapeutic potential of targeting the HGF/MET pathway for inhibition, to constrain tumor progression of SCLC cells showing aberrant activation of HGF/MET signaling. We suggest that it would be clinically valuable to further investigate HGF/MET-mediated signaling in SCLC cells. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  8. Hepatocyte growth factor regulates cyclooxygenase-2 expression via β-catenin, Akt, and p42/p44 MAPK in human bronchial epithelial cells

    PubMed Central

    Lee, Young H.; Suzuki, Yuichiro J.; Griffin, Autumn J.; Day, Regina M.

    2008-01-01

    Hepatocyte growth factor (HGF) is upregulated in response to lung injury and has been implicated in tissue repair through its antiapoptotic and proliferative activities. Cyclooxygenase-2 (COX-2) is an inducible enzyme in the biosynthetic pathway of prostaglandins, and its activation has been shown to play a role in cell growth. Here, we report that HGF induces gene transcription of COX-2 in human bronchial epithelial cells (HBEpC). Treatment of HBEpC with HGF resulted in phosphorylation of the HGF receptor (c-Met), activation of Akt, and upregulation of COX-2 mRNA. Adenovirus-mediated gene transfer of a dominant negative (DN) Akt mutant revealed that HGF increased COX-2 mRNA in an Akt-dependent manner. COX-2 promoter analysis in luciferase reporter constructs showed that HGF regulation required the β-catenin-responsive T cell factor-4 binding element (TBE). The HGF activation of the COX-2 gene transcription was blocked by DN mutant of β-catenin or by inhibitors that blocked activation of Akt. Inhibition of p42/p44 MAPK pathway blocked HGF-mediated activation of β-catenin gene transcription but not Akt activation, suggesting that p42/p44 MAPK acts in a parallel mechanism for β-catenin activation. We also found that inhibition of COX-2 with NS-398 blocked HGF-induced growth in HBEpC. Together, the results show that the HGF increases COX-2 gene expression via an Akt-, MAPK-, and β-catenin-dependent pathway in HBEpC. PMID:18245266

  9. Hepatocyte growth factor is the most potent endogenous stimulant of rabbit gastric epithelial cell proliferation and migration in primary culture.

    PubMed Central

    Takahashi, M; Ota, S; Shimada, T; Hamada, E; Kawabe, T; Okudaira, T; Matsumura, M; Kaneko, N; Terano, A; Nakamura, T

    1995-01-01

    Various growth factors are suggested to be involved in gastric mucosal repair. Our previous studies have shown that exogenous hepatocyte growth factor (HGF) has a proliferative effect on gastric epithelial cells. In the present study, comparison of the maximum proliferative effects and the optimum concentrations of several growth factors revealed that HGF was the most potent mitogen for gastric epithelial cells, as is the case for hepatocytes. Restitution of gastric epithelial cell monolayers was assessed using a round wound restitution model. HGF was the most effective agent for facilitating gastric epithelial restitution among those tested. A binding assay revealed specific binding of HGF to its receptor on gastric epithelial cells. Northern blot analysis confirmed the expression of specific HGF receptor mRNA (c-met) by gastric epithelial cells but not by gastric fibroblasts. To investigate endogenous HGF production, we determined the effect of gastric fibroblast-conditioned medium on epithelial proliferation and restitution. The conditioned medium produced similar effects to HGF and its activity was neutralized by an anti-HGF antibody. In addition, expression of HGF mRNA was detected in gastric fibroblasts but not in gastric epithelial cells. Our immunohistochemical study confirmed these in vitro data by means of demonstrating the existence and localization of HGF at human native gastric mucosa. HGF was localized at fibroblasts under the epithelial cell layer around gastric ulcers. These results suggest that HGF may be a potent endogenous promotor of gastric epithelial cell proliferation and migration, and may contribute to gastric mucosal repair through a paracrine mechanism. Images PMID:7738166

  10. Development and Characterization of a Novel Anti-idiotypic Monoclonal Antibody to Growth Hormone, Which Can Mimic Physiological Functions of Growth Hormone in Primary Porcine Hepatocytes.

    PubMed

    Lan, Hai-Nan; Jiang, Hai-Long; Li, Wei; Wu, Tian-Cheng; Hong, Pan; Li, Yu Meng; Zhang, Hui; Cui, Huan-Zhong; Zheng, Xin

    2015-04-01

    B-32 is one of a panel of monoclonal anti-idiotypic antibodies to growth hormone (GH) that we developed. To characterize and identify its potential role as a novel growth hormone receptor (GHR) agonist, we determined that B-32 behaved as a typical Ab2β based on a series of enzyme-linked immunosorbent assay assays. The results of fluorescence-activated cell sorting, indirect immunofluorescence and competitive receptor binding assays demonstrated that B-32 specifically binds to the GHR expressed on target cells. Next, we examined the resulting signal transduction pathways triggered by this antibody in primary porcine hepatocytes. We found that B-32 can activate the GHR and Janus kinase (2)/signal transducers and activators of transcription (JAK2/STAT5) signalling pathways. The phosphorylation kinetics of JAK2/STAT5 induced by either GH or B-32 were analysed in dose-response and time course experiments. In addition, B32 could also stimulate porcine hepatocytes to secrete insulin-like growth factors-1. Our work indicates that a monoclonal anti-idiotypic antibody to GH (B-32) can serve as a GHR agonist or GH mimic and has application potential in domestic animal (pig) production.

  11. 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.

  12. Quantitative analysis of individual hepatocyte growth factor receptor clusters in influenza A virus infected human epithelial cells using localization microscopy.

    PubMed

    Wang, Qiaoyun; Dierkes, Rüdiger; Kaufmann, Rainer; Cremer, Christoph

    2014-04-01

    In this report, we applied a special localization microscopy technique (Spectral Precision Distance/Spatial Position Determination Microscopy/SPDM) to quantitatively analyze the effect of influenza A virus (IAV) infection on the spatial distribution of individual HGFR (Hepatocyte Growth Factor Receptor) proteins on the membrane of human epithelial cells at the single molecule resolution level. We applied this SPDM method to Alexa 488 labeled HGFR proteins with two different ligands. The ligands were either HGF (Hepatocyte Growth Factor), or IAV. In addition, the HGFR distribution in a control group of mock-incubated cells without any ligands was investigated. The spatial distribution of 1×10(6) individual HGFR proteins localized in large regions of interest on membranes of 240 cells was quantitatively analyzed and found to be highly non-random. Between 21% and 24% of the HGFR molecules were located in 44,304 small clusters with an average diameter of 54nm. The mean density of HGFR molecule signals per individual cluster was very similar in control cells, in cells with ligand only, and in IAV infected cells, independent of the incubation time. From the density of HGFR molecule signals in the clusters and the diameter of the clusters, the number of HGFR molecule signals per cluster was estimated to be in the range between 4 and 11 (means 5-6). This suggests that the membrane bound HGFR clusters form small molecular complexes with a maximum diameter of few tens of nm, composed of a relatively low number of HGFR molecules. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. 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.

  14. Effects of hepatocyte growth factor on MMP-2 expression in scleral fibroblasts from a guinea pig myopia model

    PubMed Central

    Li, Xiu-Juan; Yang, Xiao-Peng; Wan, Guang-Ming; Wang, Yu-Ying; Zhang, Jin-Song

    2014-01-01

    AIM To investigate the effects of hepatocyte growth factor (HGF) on MMP-2 expression in scleral fibroblasts from guinea pig with LIM. METHODS Sixty 1-week-old guinea pigs were chosen for the study. The right eyes were treated with -10.0 D lenses as the LIM group; the left eyes remained untreated as the control group. The refraction and axial length were measured by streak retinoscopy and A-scan ultrasonography respectively prior to and 4 weeks after the experiment. Four weeks later, the guinea pigs were sacrificed and primary scleral fibroblasts were taken for tissue culture. The 3rd-5th generation scleral fibroblasts were chosen for the experiments. The expression levels of HGF and MMP-2 protein in the scleral fibroblasts were analyzed by Western blotting. After HGF with different doses acted on the scleral fibroblasts of the control group, MMP-2 protein expression in the scleral fibroblasts was analyzed by Western blotting. HGF siRNA was transfected into the scleral fibroblasts of the LIM group and the protein expressions of HGF and MMP-2 were analyzed by Western blotting. RESULTS The LIM group became myopic with a significant increase in axial length (7.97±0.29 mm vs 7.01±0.26 mm, P<0.05), and a significant decrease in refraction (-5.06±0.31 D vs 0.55±0.25 D, P<0.05) compared with the control group. The protein expression of HGF in the scleral fibroblasts of the LIM group was significantly higher compared with the control group ( 1.26±0.04 vs 0.32 ±0.04, P<0.05). The protein expression of MMP-2 in the scleral fibroblasts of the LIM group was significantly higher compared with the control group (0.89±0.06 vs 0.42±0.05, P<0.05). In the scleral fibroblasts of the control group, HGF(0, 0.1, 1, 10 ng/mL) upregulated MMP-2 protein expression in a dose-dependent manner (0.35±0.03, 0.44±0.02, 0.91±0.03, 1.33±0.04, all P<0.05). In the scleral fibroblasts of the LIM group transfected with HGF siRNA, MMP-2 protein expressions were significantly decreased

  15. L-Methionine Toxicity in Freshly-Isolated Mouse Hepatocytes is Gender-Dependent and Mediated in Part by Transamination

    PubMed Central

    Dever, Joseph T.; Elfarra, Adnan A.

    2008-01-01

    L-methionine (Met) has been implicated in parenteral nutrition-associated cholestasis in infants and, at high levels, causes liver toxicity by mechanisms that are not clear. In this study, Met toxicity was characterized in freshly-isolated male and female mouse hepatocytes incubated with 5-30 mM Met for 0 to 5 h. In male hepatocytes, 20 mM Met was cytotoxic at 4 h as indicated by trypan blue exclusion and LDH leakage assays. Cytotoxicity was preceded by GSH depletion at 3 h without GSSG formation. Exposure to 30 mM Met resulted in increased cytotoxicity and GSH depletion. Interestingly, female hepatocytes were resistant to Met-induced cytotoxicity at these concentrations and showed increased cellular GSH levels compared to hepatocytes exposed to medium alone. The effects of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, and 3-deazaadenosine (3-DA), an inhibitor of the Met transmethylation pathway enzyme S-adenosylhomocysteine hydrolase on Met toxicity in male hepatocytes were then examined. Addition of 0.2 mM AOAA partially blocked Met-induced GSH depletion and cytotoxicity whereas 0.1 mM 3-DA potentiated Met-induced toxicity. Exposure of male hepatocytes to 0.3 mM 3-methylthiopropionic acid (3-MTP), a known Met transamination metabolite, resulted in cytotoxicity and cellular GSH depletion similar to that observed with 30 mM Met whereas incubations with D-methionine resulted in no toxicity. Female hepatocytes were less sensitive to 3-MTP toxicity than males which may partially explain their resistance to Met toxicity. Collectively, these results suggest that Met transamination and not transmethylation plays a major role in Met toxicity in male mouse hepatocytes. PMID:18552130

  16. L-methionine toxicity in freshly isolated mouse hepatocytes is gender-dependent and mediated in part by transamination.

    PubMed

    Dever, Joseph T; Elfarra, Adnan A

    2008-09-01

    L-methionine (Met) has been implicated in parenteral nutrition-associated cholestasis in infants and, at high levels, it causes liver toxicity by mechanisms that are not clear. In this study, Met toxicity was characterized in freshly isolated male and female mouse hepatocytes incubated with 5 to 30 mM Met for 0 to 5 h. In male hepatocytes, 20 mM Met was cytotoxic at 4 h as indicated by trypan blue exclusion and lactate dehydrogenase leakage assays. Cytotoxicity was preceded by reduced glutathione (GSH) depletion at 3 h without glutathione disulfide formation. Exposure to 30 mM Met resulted in increased cytotoxicity and GSH depletion. It is interesting to note that female hepatocytes were resistant to Met-induced cytotoxicity at these concentrations and showed increased cellular GSH levels compared with hepatocytes exposed to medium alone. The effects of amino-oxyacetic acid (AOAA), an inhibitor of Met transamination, and 3-deazaadenosine (3-DA), an inhibitor of the Met transmethylation pathway enzyme S-adenosylhomocysteine hydrolase, on Met toxicity in male hepatocytes were then examined. Addition of 0.2 mM AOAA partially blocked Met-induced GSH depletion and cytotoxicity, whereas 0.1 mM 3-DA potentiated Met-induced toxicity. Exposure of male hepatocytes to 0.3 mM 3-methylthiopropionic acid (3-MTP), a known Met transamination metabolite, resulted in cytotoxicity and cellular GSH depletion similar to that observed with 30 mM Met, whereas incubations with D-methionine resulted in no toxicity. Female hepatocytes were less sensitive to 3-MTP toxicity than males, which may partially explain their resistance to Met toxicity. Taken together, these results suggest that Met transamination and not transmethylation plays a major role in Met toxicity in male mouse hepatocytes.

  17. Hepatocyte differentiation.

    PubMed

    Olsavsky Goyak, Katy M; Laurenzana, Elizabeth M; Omiecinski, Curtis J

    2010-01-01

    Increasingly, research suggests that for certain systems, animal models are insufficient for human toxicology testing. The development of robust, in vitro models of human toxicity is required to decrease our dependence on potentially misleading in vivo animal studies. A critical development in human toxicology testing is the use of human primary hepatocytes to model processes that occur in the intact liver. However, in order to serve as an appropriate model, primary hepatocytes must be maintained in such a way that they persist in their differentiated state. While many hepatocyte culture methods exist, the two-dimensional collagen "sandwich" system combined with a serum-free medium, supplemented with physiological glucocorticoid concentrations, appears to robustly maintain hepatocyte character. Studies in rat and human hepatocytes have shown that when cultured under these conditions, hepatocytes maintain many markers of differentiation including morphology, expression of plasma proteins, hepatic nuclear factors, phase I and II metabolic enzymes. Functionally, these culture conditions also preserve hepatic stress response pathways, such as the SAPK and MAPK pathways, as well as prototypical xenobiotic induction responses. This chapter will briefly review culture methodologies but will primarily focus on hallmark hepatocyte structural, expression and functional markers that characterize the differentiation status of the hepatocyte.

  18. 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

  19. Inactivation of hepatocyte nuclear factor-4α mediates alcohol-induced downregulation of intestinal tight junction proteins

    PubMed Central

    Zhong, Wei; Zhao, Yantao; McClain, Craig J.; Kang, Y. James

    2010-01-01

    Chronic alcohol exposure has been shown to increase the gut permeability in the distal intestine, in part, through induction of zinc deficiency. The present study evaluated the molecular mechanisms whereby zinc deficiency mediates alcohol-induced intestinal barrier dysfunction. Examination of zinc finger transcription factors in the gastrointestinal tract of mice revealed a prominent distribution of hepatocyte nuclear factor-4α (HNF-4α). HNF-4α exclusively localizes in the epithelial nuclei and exhibited an increased abundance in mRNA and protein levels in the distal intestine. Chronic alcohol exposure to mice repressed the HNF-4α gene expression in the ileum and reduced the protein level and DNA binding activity of HNF-4α in all of the intestinal segments with the most remarkable changes in the ileum. Chronic alcohol exposure also decreased the mRNA levels of tight junction proteins, particularly in the ileum. Caco-2 cell culture studies were conducted to determine the role of HNF-4α in regulation of the epithelial tight junction and barrier function. Knockdown of HNF-4α in Caco-2 cells decreased the mRNA and protein levels of tight junction proteins in association with disruption of the epithelial barrier. Alcohol treatment inactivated HNF-4α, which was prevented by N-acetyl-cysteine or zinc. The link between zinc and HNF-4α function was confirmed by zinc deprivation, which inhibited HNF-4α DNA binding activity. These results indicate that inactivation of HNF-4α due to oxidative stress and zinc deficiency is likely a novel mechanism contributing to the deleterious effects of alcohol on the tight junctions and the intestinal barrier function. PMID:20576917

  20. Induction of apoptosis and autophagy via mitochondria- and PI3K/Akt/mTOR-mediated pathways by E. adenophorum in hepatocytes of saanen goat

    PubMed Central

    Luo, Biao; Qiao, Yan; Xu, Ruiguang; Zuo, Zhicai; Deng, Junliang; Nong, Xiang; Peng, Guangneng; He, Wei; Wei, Yahui; Hu, Yanchun

    2016-01-01

    E. adenophorum has reported to cause hepatotoxicity. But, the precise effects of E. adenophorum on hepatocytes is unclear. Saanen goats were fed on E. adenophorum to detect the cytotoxicity effects of E. adenophorum on hepatocytes. Our study has shown that the typical apoptotic features, the increasing apoptotic hepatocytes and activated caspase-9, −3 and the subsequent cleavage of PARP indicated the potent pro-apoptotic effects of E. adenophorum. Moreover, the translocation of Bax and Cyt c between mitochondria and cytosol triggering the forming of apoptosome proved that the mitochondria-mediated apoptosis was triggered by E. adenophorum. Furthermore, E. adenophorum increased the MDC-positive autophagic vacuoles and the subcellular localization of punctate LC3, the ratio of LC3-II/LC3-I and the protein levels of Beclin 1, but decreased that of P62, indicating the potent pro-autophagic effects of E. adenophorum. In addition, E. adenophorum significantly inhibited the protein leves of p-PI3K, p-Akt and p-mTORC1, but increased PTEN and p-AMPK. Also, the p-mTORC2 and p-Akt Ser473 were inhibited, indicating that the supression of mTORC2/Akt pathway could induce the autophagy of hepatocytes. The autophagy-realted results indicated that the inhibition of PI3K/Akt/mTORC1- and mTORC2/Akt-mediated pathways contributed to the pro-autophagic activity of E. adenophorum. These findings provide new insights to understand the mechanisms involved in E. adenophorum-caused hepatotoxicity of Saanen goat. PMID:27391155

  1. Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas.

    PubMed Central

    Olivero, M.; Rizzo, M.; Madeddu, R.; Casadio, C.; Pennacchietti, S.; Nicotra, M. R.; Prat, M.; Maggi, G.; Arena, N.; Natali, P. G.; Comoglio, P. M.; Di Renzo, M. F.

    1996-01-01

    Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the invasive growth of epithelial cells via the c-MET oncogene-encoded receptor. In normal lung, both the receptor and the ligand are detected, and the latter is known to be a mitogenic and a motogenic factor for both cultured bronchial epithelial cells and non-small-cell carcinoma lines. Here, ligand and receptor expression was examined in 42 samples of primary human non-small-cell lung carcinoma of different histotype. Each carcinoma sample was compared with adjacent normal lung tissue. The Met/HGF receptor was found to be 2 to 10-fold increased in 25% of carcinoma samples (P = 0.0113). The ligand, HGF/SF, was found to be 10 to 100-fold overexpressed in carcinoma samples (P < 0.0001). Notably, while HGF/SF was occasionally detectable and found exclusively as a single-chain inactive precursor in normal tissues, it was constantly in the biologically-active heterodimeric form in carcinomas. Immunohistochemical staining showed homogeneous expression of both the receptor and the ligand in carcinoma samples, whereas staining was barely detectable in their normal counterparts. These data show that HGF/SF is overexpressed and consistently activated in non-small-cell lung carcinomas and may contribute to the invasive growth of lung cancer. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8980383

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

    PubMed

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

    2013-01-01

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

  3. Therapeutic effect of hepatocyte growth factor-secreting mesenchymal stem cells in a rat model of liver fibrosis

    PubMed Central

    Kim, Myung-Deok; Kim, Sung-Soo; Cha, Hyun-Young; Jang, Seung-Hun; Chang, Da-Young; Kim, Wookhwan; Suh-Kim, Haeyoung; Lee, Jae-Ho

    2014-01-01

    Bone marrow-derived mesenchymal stromal cells (MSCs) have been reported to be beneficial for the treatment of liver fibrosis. Here, we investigated the use of genetically engineered MSCs that overexpress hepatocyte growth factor (HGF) as a means to improve their therapeutic effect in liver fibrosis. Liver fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. HGF-secreting MSCs (MSCs/HGF) were prepared by transducing MSCs with an adenovirus carrying HGF-encoding cDNA. MSCs or MSCs/HGF were injected directly into the spleen of fibrotic rats. Tissue fibrosis was assessed by histological analysis 12 days after stem cell injection. Although treatment with MSCs reduced fibrosis, treatment with MSCs/HGF produced a more significant reduction and was associated with elevated HGF levels in the portal vein. Collagen levels in the liver extract were decreased after MSC/HGF therapy, suggesting recovery from fibrosis. Furthermore, liver function was improved in animals receiving MSCs/HGF, indicating that MSC/HGF therapy resulted not only in reduction of liver fibrosis but also in improvement of hepatocyte function. Assessment of cell and biochemical parameters revealed that mRNA levels of the fibrogenic cytokines PDGF-bb and TGF-β1 were significantly decreased after MSC/HGF therapy. Subsequent to the decrease in collagen, expression of matrix metalloprotease-9 (MMP-9), MMP-13, MMP-14 and urokinase-type plasminogen activator was augmented following MSC/HGF, whereas tissue inhibitor of metalloprotease-1 (TIMP-1) expression was reduced. In conclusion, therapy with MSCs/HGF resulted in an improved therapeutic effect compared with MSCs alone, probably because of the anti-fibrotic activity of HGF. Thus, MSC/HGF represents a promising approach toward a cell therapy for liver fibrosis. PMID:25145391

  4. 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

  5. Pigment epithelium-derived factor (PEDF) suppresses IL-1β-mediated c-Jun N-terminal kinase (JNK) activation to improve hepatocyte insulin signaling.

    PubMed

    Gattu, Arijeet K; Birkenfeld, Andreas L; Iwakiri, Yasuko; Jay, Steven; Saltzman, Mark; Doll, Jennifer; Protiva, Petr; Samuel, Varman T; Crawford, Susan E; Chung, Chuhan

    2014-04-01

    Pigment epithelium-derived factor (PEDF) is an antiinflammatory protein that circulates at high levels in the metabolic syndrome. Metabolic studies of PEDF knockout (KO) mice were conducted to investigate the relationship between PEDF, inflammatory markers, and metabolic homeostasis. Male PEDF KO mice demonstrated a phenotype consisting of increased adiposity, glucose intolerance, and elevated serum levels of metabolites associated with the metabolic syndrome. Genome expression analysis revealed an increase in IL-1β signaling in the livers of PEDF KO mice that was accompanied by impaired IRS and Akt signaling. In human hepatocytes, PEDF blocked the effects of an IL-1β challenge by suppressing activation of the inflammatory mediator c-Jun N-terminal kinase while restoring Akt signaling. RNA interference of PEDF in human hepatocytes was permissive for c-Jun N-terminal kinase activation and decreased Akt signaling. A metabolomics profile identified elevated circulating levels of tricarboxyclic acid cycle intermediates including succinate, an inducer of IL-1β, in PEDF KO mice. Succinate-dependent IL-1β expression was blocked by PEDF in PEDF KO, but not wild-type hepatocytes. In vivo, PEDF restoration reduced hyperglycemia and improved hepatic insulin signaling in PEDF KO mice. These findings identify elevated PEDF as a homeostatic mechanism in the human metabolic syndrome.

  6. 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

  7. Transforming Growth Factor β Controls the Directional Migration of Hepatocyte Cohorts by Modulating Their Adhesion to Fibronectin

    PubMed Central

    Binamé, Fabien; Lassus, Patrice

    2008-01-01

    Transforming growth factor β (TGF-β) has a strong impact on liver development and physiopathology, exercised through its pleiotropic effects on growth, differentiation, survival, and migration. When exposed to TGF-β, the mhAT3F cells, immortalized, highly differentiated hepatocytes, maintained their epithelial morphology and underwent dramatic alterations of adhesion, leading to partial or complete detachment from a culture plate, followed by readhesion and spreading. These alterations of adhesive behavior were caused by sequential changes in expression of the α5β1 integrin and of its ligand, the fibronectin. The altered specificity of anchorage to the extracellular matrix gave rise to changes in cells' collective motility: cohorts adhering to fibronectin maintained a persistent, directional motility, with ezrin-rich pathfinder cells protruding from the tips of the cohorts. The absence of adhesion to fibronectin prevented the appearance of polarized pathfinders and lead to random, oscillatory motility. Our data suggest a novel role for TGF-β in the control of collective migration of epithelial cohorts. PMID:18094041

  8. 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

  9. Ganglioside GD3 induces convergence and synergism of adhesion and hepatocyte growth factor/Met signals in melanomas.

    PubMed

    Furukawa, Keiko; Kambe, Mariko; Miyata, Maiko; Ohkawa, Yuki; Tajima, Orie; Furukawa, Koichi

    2014-01-01

    Ganglioside GD3 is highly expressed in human melanomas and enhances malignant properties of melanomas, such as cell proliferation and invasion activity. In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL-I). Although stimulation of melanoma N1 cells (GD3+ and GD3-) with either HGF or adhesion to CL-I did not show marked differences in the phosphorylation levels of Akt at Ser473 and Thr308 between two types of cells, simultaneous treatment resulted in definite and markedly increased activation of Akt in GD3+ cells. Similar increases were also shown in Erk1/2 phosphorylation levels with the costimulation in GD3+ cells. When resistance to induced apoptosis by H2O2 was examined, only GD3+ cells treated with both HGF and adhesion to CL-I showed clearly low percentages of dead cells compared with GD3- cells or GD3+ cells treated with either one of the stimulants. Cell growth measured by 5-ethynyl-2' deoxyuridine uptake also showed synergistic effects in GD3+ cells. These results suggested that GD3 plays a crucial role in the convergence of multiple signals, leading to the synergistic effects of those signals on malignant properties of melanomas. © 2013 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  10. Ganglioside GD3 induces convergence and synergism of adhesion and hepatocyte growth factor/Met signals in melanomas

    PubMed Central

    Furukawa, Keiko; Kambe, Mariko; Miyata, Maiko; Ohkawa, Yuki; Tajima, Orie; Furukawa, Koichi

    2014-01-01

    Ganglioside GD3 is highly expressed in human melanomas and enhances malignant properties of melanomas, such as cell proliferation and invasion activity. In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL-I). Although stimulation of melanoma N1 cells (GD3+ and GD3−) with either HGF or adhesion to CL-I did not show marked differences in the phosphorylation levels of Akt at Ser473 and Thr308 between two types of cells, simultaneous treatment resulted in definite and markedly increased activation of Akt in GD3+ cells. Similar increases were also shown in Erk1/2 phosphorylation levels with the costimulation in GD3+ cells. When resistance to induced apoptosis by H2O2 was examined, only GD3+ cells treated with both HGF and adhesion to CL-I showed clearly low percentages of dead cells compared with GD3− cells or GD3+ cells treated with either one of the stimulants. Cell growth measured by 5-ethynyl-2‘ deoxyuridine uptake also showed synergistic effects in GD3+ cells. These results suggested that GD3 plays a crucial role in the convergence of multiple signals, leading to the synergistic effects of those signals on malignant properties of melanomas. PMID:24372645

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

    SciTech Connect

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

    2015-12-25

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

  12. Expression of CD31, Met/hepatocyte growth factor receptor and bone morphogenetic protein in bone metastasis of osteosarcoma.

    PubMed

    Arihiro, K; Inai, K

    2001-02-01

    The mechanism of metastasis of osteosarcoma cells to other bones has not yet fully been clarified. The purpose of the present study was to examine whether various factors involve the formation of osteosarcoma metastatic foci in other bones. Immunohistochemically, CD31 expression in osteosarcoma with no bone metastasis and osteosarcoma with bone metastasis was noted in 10 and 75% of cases, respectively. Met/hepatocyte growth factor (HGF) receptor expression in osteosarcoma with no bone metastasis and osteosarcoma with bone metastasis was noted in 90 and 25% of cases, respectively. Bone morphogenetic protein (BMP) expression in osteosarcoma with no bone metastasis and osteosarcoma with bone metastasis was noted in 20 and 75% of cases, respectively. Metastasis of osteosarcoma cells to other bones was significantly correlated with expression of BMP and CD31 and with no expression of Met/HGF receptor protein in osteosarcoma cells. In contrast, expression of insulin-like growth factor receptor in osteosarcoma cells did not correlate significantly with bone metastasis. These results suggest that formation of metastatic foci of osteosarcoma cells in other bones is regulated by CD31, which is associated with migration between endothelial cells, by BMP, which can induce and activate various mesenchymal cells affecting bone formation, and by escape of effect by HGF, which promotes differentiation of osteosarcoma cells.

  13. Hepatocyte growth factor and alternative splice variants - expression, regulation and implications in osteogenesis and bone health and repair.

    PubMed

    Frisch, Rachel N; Curtis, Kevin M; Aenlle, Kristina K; Howard, Guy A

    2016-09-01

    Bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into multiple cell types, including osteoblasts, chondrocytes, and adipocytes. These pluripotent cells secrete hepatocyte growth factor (HGF), which regulates cell growth, survival, motility, migration, mitogenesis and is important for tissue development/regeneration. HGF has four splice variants, NK1, NK2, NK3, and NK4 which have varying functions and affinities for the HGF receptor, cMET. HGF promotes osteoblastic differentiation of MSCs into bone forming cells, playing a role in bone development, health and repair. This review will focus on the effects of HGF in osteogenesis, bone repair and bone health, including structural and functional insights into the role of HGF in the body. Approximately 6.2 million Americans experience a fracture annually, with 5-10% being mal- or non-union fractures. HGF is important in priming MSCs for osteogenic differentiation in vitro and is currently being studied to assess its role during bone repair in vivo. Due to the high turnover rate of systemic HGF, non-classic modes of HGF-treatment, including naked-plasmid HGF delivery and the use of HGF splice variants (NK1 & NK2) are being studied to find safe and efficacious treatments for bone disorders, such as mal- or non-union fractures.

  14. 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.

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

    PubMed

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

    2016-06-01

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

  16. 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.

  17. 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.

  18. Recombinant human hepatocyte growth factor (HGF), but not rat HGF, elicits glomerular injury and albuminuria in normal rats via an immune complex-dependent mechanism.

    PubMed

    Mizuno, Shinya; Ikebuchi, Fumie; Fukuta, Kazuhiro; Kato, Takashi; Matsumoto, Kunio; Adachi, Kiichi; Abe, Tetsushi; Nakamura, Toshikazu

    2011-03-01

    1. Hepatocyte growth factor (HGF) has the therapeutic potential to improve renal fibrosis and proteinuria in rodents with chronic kidney disease. In contrast, long-term administration of human HGF to normal rats reportedly elicits proteinuria. Thus, the role of HGF during proteinuria remains contentious. The aim of the present study was to demonstrate that human HGF is antigenic to rodents and that immune complex formation causes proteinuria. 2. We administered either human or rat HGF to normal rats for 28 days. Albuminuria was evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The renal phenotypes of the two HGF treatments were examined using histological techniques. 3. Administration of human HGF (1 mg/kg per day, i.v.) to rats led to severe albuminuria and glomerular hypertrophy in association with increased blood levels of anti-human HGF IgG and IgG deposition in mesangial areas. Furthermore, an immune complex between human HGF and anti-human HGF IgG stimulated the production of proteinuric cytokines (including transforming growth factor-β) in rat cultured mesangial cells. In contrast, treatment of healthy rats with rat HGF for 4 weeks caused neither mesangial IgG deposition nor elevated anti-HGF IgG in the blood. Overall, rat HGF did not provoke albuminuria. 4. We conclude that human HGF produces pseudotoxic effects in normal rat kidneys via an immune complex-mediated pathway, whereas syngenic HGF is safe due to less deposition of glomerular IgG. Our results affirm the safety of the repeated use of syngenic HGF for the treatment of chronic organ diseases, such as renal fibrosis and liver cirrhosis.

  19. A TLR and non-TLR mediated innate response to lentiviruses restricts hepatocyte entry and can be ameliorated by pharmacological blockade.

    PubMed

    Agudo, Judith; Ruzo, Albert; Kitur, Kipyegon; Sachidanandam, Ravi; Blander, J Magarian; Brown, Brian D

    2012-12-01

    Lentiviral vector (LV)-mediated gene transfer is a promising method of gene therapy. We previously reported that systemic injection of HIV-based LV triggers a transient inflammatory response. Here, we carried out studies to better characterize this response, and to develop a strategy to overcome the adverse effects of interferon (IFN) on LV-mediated gene transfer. We profiled gene expression in the liver after LV administration using deep-sequencing (RNA-seq), and identified several innate response pathways. We examined the response to LV in MyD88-TRIF knockout mice, which are incapable of toll-like receptor (TLR) signaling. Unexpectedly, the IFN response to LV was not reduced in the liver indicating that a non-TLR pathway can recognize LV in this organ. Indeed, blocking reverse transcription with azidothymidine (AZT) reduced the IFN response only in the liver, suggesting that proviral DNA can be a trigger. To block the inflammatory response, we pretreated mice with a short course of dexamethasone (Dex). At 4 hours post-treatment, all the IFN-induced genes were normalized. By blocking the inflammatory response, hepatocyte transduction was dramatically increased, which in turn doubled the level of human factor IX (FIX) produced by a hepatocyte-specific LV. Our studies uncover new insights into LV-induced immune responses in the liver, and provide a means to increase the safety and efficiency of LV-mediated gene transfer.

  20. Independent repression of bile acid synthesis and activation of c-Jun N-terminal kinase (JNK) by activated hepatocyte fibroblast growth factor receptor 4 (FGFR4) and bile acids.

    PubMed

    Yu, Chundong; Wang, Fen; Jin, Chengliu; Huang, Xinqiang; McKeehan, Wallace L

    2005-05-06

    The fibroblast growth factor (FGF) receptor complex is a regulator of adult organ homeostasis in addition to its central role in embryonic development and wound healing. FGF receptor 4 (FGFR4) is the sole FGFR receptor kinase that is significantly expressed in mature hepatocytes. Previously, we showed that mice lacking mouse FGFR4 (mR4(-/-)) exhibited elevated fecal bile acids, bile acid pool size, and expression of liver cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for canonical neutral bile acid synthesis. To prove that hepatocyte FGFR4 was a negative regulator of cholesterol metabolism and bile acid synthesis independent of background, we generated transgenic mice overexpressing a constitutively active human FGFR4 (CahR4) in hepatocytes and crossed them with the FGFR4-deficient mice to generate CahR4/mR4(-/-) mice. In mice expressing active FGFR4 in liver, fecal bile acid excretion was 64%, bile acid pool size was 47%, and Cyp7a1 expression was 10-30% of wild-type mice. The repressed level of Cyp7a1 expression was resistant to induction by a high cholesterol diet relative to wild-type mice. Expression of CahR4 in mR4(-/-) mouse livers depressed bile acid synthesis below wild-type levels from the elevated levels observed in mR4(-/-). Levels of phosphorylated c-Jun N-terminal kinase (JNK), which is part of a pathway implicated in bile acid-mediated repression of synthesis, was 30% of wild-type levels in mR4(-/-) livers, whereas CahR4 livers exhibited an average 2-fold increase. However, cholate still strongly induced phospho-JNK in mR4(-/-) livers. These results confirm that hepatocyte FGFR4 regulates bile acid synthesis by repression of Cyp7a1 expression. Hepatocyte FGFR4 may contribute to the repression of bile acid synthesis through JNK signaling but is not required for activation of JNK signaling by bile acids.

  1. 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'...

  2. 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'...

  3. 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. Copyright 2010 Elsevier B.V. All rights reserved.

  4. 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.

  5. Association between Hepatocyte Growth Factor (HGF) Gene Polymorphisms and Serum HGF Levels in Patients with Rheumatoid Arthritis.

    PubMed

    Kara, Fatih; Yildirim, Abdulkadir; Gumusdere, Musa; Karatay, Saliha; Yildirim, Kadir; Bakan, Ebubekir

    2014-10-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by proliferation and insufficient apoptosis of synovial cell, inflammatory cell infiltration, angiogenesis, and destruction of joints. Hepatocyte growth factor (HGF) has many functions, such as regulation of inflammation, angiogenesis, and inhibition of apoptosis. The purpose of this study was to investigate the association between intron 13 C/A and intron 14 T/C HGF gene polymorphisms and serum HGF levels in patients with RA. 100 patients with RA and 123 healthy controls were included in this study. Serum HGF concentrations were measured using ELISA kit. Gene polymorphisms were determined by allelic discrimination analysis using the real-time PCR method. HGF levels, frequency of AA genotype and A allele for intron 13 C/A polymorphism and frequency of CC genotype and C allele for intron 14 T/C polymorphism were increased in patients with RA compared to healthy controls. There was no overall associations between genotypes and serum HGF concentrations in both patient and control groups. Our results indicate that HGF protein and gene may play an important role in the etiopathogenesis of RA. However, further studies are required for a better understanding of mechanisms related to the disease process.

  6. Proinflammatory Cytokines (IL-1α, IL-6) and Hepatocyte Growth Factor in Patients with Alcoholic Liver Cirrhosis

    PubMed Central

    Prystupa, Andrzej; Kiciński, Paweł; Sak, Jarosław; Boguszewska-Czubara, Anna; Toruń-Jurkowska, Anna; Załuska, Wojciech

    2015-01-01

    Background. The aim of the study was to assess the activity of interleukin-1α, interleukin-6, and hepatocyte growth factor protein (HGF) in serum of patients with alcoholic liver cirrhosis. Materials and Methods. Sixty patients with alcoholic liver cirrhosis treated in various hospitals were randomly enrolled. The stage of cirrhosis was assessed according to the Child-Turcotte-Pugh scoring system. The control group consisted of ten healthy persons without liver disease, who did not drink alcohol. Additionally, the group of alcoholics without liver cirrhosis was included in the study. The activity of interleukin-1α, interleukin-6, and HGF in blood plasma of patients and controls was measured using the sandwich enzyme immunoassay technique with commercially available quantitative ELISA test kits. Results. Higher concentrations of HGF protein were demonstrated in patients with Child class B and Child class C liver cirrhosis, compared to controls and alcoholics without liver cirrhosis. Moreover, significantly higher concentrations of HGF protein were found in patients with Child class C liver cirrhosis compared to patients with Child class A liver cirrhosis (p < 0.05). The concentrations of interleukin-1α in patients with Child class B and Child class C liver cirrhosis were significantly higher in comparison with controls. Significantly higher concentrations of interleukin-6 were demonstrated in Child class C, compared to Child class A. PMID:26448742

  7. Expression of hypoxia-inducible factor-1α and hepatocyte growth factor in development of fibrosis in the transplanted kidney.

    PubMed

    Kellenberger, Terese; Marcussen, Niels; Nyengaard, Jens R; Wogensen, Lise; Jespersen, Bente

    2015-02-01

    Late renal graft loss is associated with interstitial fibrosis. Hypoxia-inducible factor-1α (HIF-1α) is thought to facilitate fibrosis through interaction with TGF-β1, while hepatocyte growth factor (HGF) may act antifibrotic in the kidney allograft. The aim of this study was to investigate the expression of HIF-1α and HGF in protocol biopsies as possible prognostic biomarkers for renal fibrosis. Thirty-nine renal transplant recipients were included in the study. Protocol biopsies performed 1 and 2 years after transplantation were used for immunohistochemistry analysis. The correlation between HIF-1α/HGF and the Banff score was analysed. In addition, progression in renal fibrosis and graft survival among recipients with high or low expression of HIF-1α/HGF after transplantation was compared. There was no significant correlation between fibrosis and the HIF-1α expression 1 and 2 years after transplantation, but an inverse significant correlation between the HGF expression and the fibrosis score 1 year after transplantation was shown. Even when adjusting for human leucocyte antigen mismatches, there was a significant relationship between fibrosis and HGF expression. Graft survival was not significantly correlated to HIF-1α or HGF at 1 year, although the trend was towards better graft survival with high HGF. HGF may have antifibrotic effects in human renal transplants. (Central.Denmark.Region.Committee number: 1-10-72-318-13).

  8. Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues.

    PubMed Central

    Tashiro, K; Hagiya, M; Nishizawa, T; Seki, T; Shimonishi, M; Shimizu, S; Nakamura, T

    1990-01-01

    The primary structure of rat hepatocyte growth factor (HGF) was elucidated by determining the base sequence of the complementary DNA (cDNA) of HGF. The cDNA for rat HGF was isolated by screening a liver cDNA library with oligonucleotides based on the partial N-terminal amino acid sequence of the beta subunit of purified rat HGF. HGF is encoded in an mRNA of about 6 kilobases. Both alpha and beta subunits of HGF are specified in a single open reading frame for a 728-amino acid protein with a calculated molecular weight of 82,904. The N-terminal part of HGF has a signal sequence and a prosequence with 30 and 25 amino acid residues, respectively. The mature heterodimer structure is derived proteolytically from this single pre-pro precursor polypeptide. The calculated molecular weights of the alpha and beta subunits are 50,664 and 25,883, respectively, and each subunit has two potential N-linked glycosylation sites. The amino acid sequence of HGF is 38% identical with that of plasminogen. The alpha subunit of HGF contains four "kringle" structures, and the beta subunit has 37% amino acid identity with the serine protease domain of plasmin. Northern blot analysis revealed that HGF mRNA was expressed in rat various tissues, including the liver, kidney, lung, and brain. Images PMID:2139229

  9. Hepatocyte growth factor/scatter factor enhances the invasion of mesothelioma cell lines and the expression of matrix metalloproteinases

    PubMed Central

    Harvey, P; Clark, I M; Jaurand, M-C; Warn, R M; Edwards, D R

    2000-01-01

    Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional factor involved both in development and tissue repair, as well as pathological processes such as cancer and metastasis. It has been identified in vivo in many types of tumours together with its tyrosine kinase receptor, Met. We show here that exogenous HGF/SF acts as a strong chemoattractant for human mesothelioma cell lines. The factor also enhanced cell adhesion to and invasion into Matrigel. The mesothelioma cell lines synthesized a panel of matrix metalloproteinases critical for tumour progression such as MMP-1, 2, 3, 9 and membrane-bound MT1-MMP. HGF/SF stimulated the expression of MMP-1, 9 and MT1-MMP and had a slight effect on expression of the MMP inhibitor TIMP-1 but not TIMP-2. However, there was no simple correlation between the levels of MMPs and TIMPs of the cell lines and their different invasion properties or between HGF/SF stimulatory effects on MMP expression and invasion. In addition, effects of protease inhibitors on invasion suggested that serine proteases were also expressed in human mesothelioma cell lines and were involved in HGF/SF-induced invasion. The results show a predominant role for HGF/SF in mesothelioma cell invasion, stimulating simultaneously adhesion, motility, invasion and regulation of MMP and TIMP levels. © 2000 Cancer Research Campaign PMID:11027427

  10. Hepatocyte growth factor secreted by ovarian cancer cells stimulates peritoneal implantation via the mesothelial-mesenchymal transition of the peritoneum.

    PubMed

    Nakamura, Michihiko; Ono, Yoshihiro J; Kanemura, Masanori; Tanaka, Tomohito; Hayashi, Masami; Terai, Yoshito; Ohmichi, Masahide

    2015-11-01

    A current working model for the metastatic process of ovarian carcinoma suggests that cancer cells are shed from the ovarian tumor into the peritoneal cavity and attach to the layer of mesothelial cells that line the inner surface of the peritoneum, and several studies suggest that hepatocyte growth factor (HGF) plays an important role in the dissemination of ovarian cancer. Our objectives were to evaluate the HGF expression of ovarian cancer using clinical data and assess the effect of HGF secreted from human ovarian cancer cells to human mesothelial cells. HGF expression was immunohistochemically evaluated in 165 epithelial ovarian cancer patients arranged as tissue microarrays. HGF expression in four ovarian cancer cell lines was evaluated by using semi-quantitative polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay. The effect of ovarian cancer cell derived HGF to the human mesothelial cells was assessed by using morphologic analysis, Western blotting and cell invasion assay. The effect of HGF on ovarian cancer metastasis was assessed by using in vivo experimental model. The clinical data showed a significantly high correlation between the HGF expression and the cancer stage. The in vivo and in vitro experimental models revealed that HGF secreted by ovarian cancer cells induces the mesothelial-to-mesenchymal transition and stimulates the invasion of mesothelial cells. Furthermore, manipulating the HGF activity affected the degree of dissemination and ascite formation. We demonstrated that HGF secreted by ovarian cancer cells plays an important role in cancer peritoneal implantation. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Decreased Hepatocyte Growth Factor (HGF) and Gamma Aminobutyric Acid (GABA) in Individuals with Obsessive-Compulsive Disorder (OCD).

    PubMed

    Russo, Anthony J; Pietsch, Stefanie C

    2013-01-01

    There is support for the role of gamma aminobutyric acid (GABA) in the etiology of mood disorders. 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 and GABA as well as symptom severity in individuals with obsessive-compulsive disorder (OCD). Plasma from 15 individuals with OCD (9 males, 6 females;, mean age 38.7 years) and 17 neurotypical controls (10 males, 7 females; mean age 35.2 years) was assessed for HGF, GABA, urokinase plasminogen activator (uPA), and urokinase plasminogen activator receptor (uPAR) concentration using enzyme-linked immunosorbest assays ELISAs. Symptom severity was assessed in these OCD individuals and compared with HGF and GABA concentrations. In this preliminary study, individuals with OCD had significantly decreased HGF levels, decreased plasma levels of GABA and decreased uPA. We found that both uPA and uPAR levels correlate with HGF. Both low uPA and low uPAR levels correlate with high symptom severity in individuals with OCD. Low GABA levels in OCD individuals also correlate with high symptom severity. These results demonstrate a preliminary association between HGF, GABA, uPA levels, and OCD and suggest that plasma GABA and uPA levels are related to symptom severity in individuals with OCD.

  12. Repeated intravenous injection of recombinant human hepatocyte growth factor ameliorates liver cirrhosis but causes albuminuria in rats.

    PubMed

    Kusumoto, Kazunori; Ido, Akio; Moriuchi, Akihiro; Katsura, Toshiya; Kim, Ildeok; Takahama, Yuka; Numata, Masatsugu; Kodama, Mayumi; Hasuike, Satoru; Nagata, Kenji; Uto, Hirofumi; Inui, Ken-Ichi; Tsubouchi, Hirohito

    2006-03-01

    Hepatocyte growth factor (HGF) is a promising agent for the treatment of liver cirrhosis because of its mitogenic and anti-fibrotic effects. We investigated the effect of recombinant human HGF (rh-HGF) on cirrhosis development; its pharmacokinetics and nephrotoxicity in rats with liver cirrhosis induced by 4-week treatment with dimethylnitrosamine (DMN). rh-HGF (0.3 mg/kg) was intravenously administered to rats once a day for 4 weeks in parallel with DMN treatment or twice a day for the last 2 weeks of DMN treatment. Repeated doses of rh-HGF increased the liver weight and serum albumin, and reduced serum ALT. The development of hepatic fibrosis was inhibited more efficiently by extended low-dose treatment with rh-HGF. In cirrhotic rats, serum levels of rh-HGF increased and clearance was decreased, leading to an increase in the area under the plasma-concentration time curve and a decrease in the steady-state volume of distribution. Repeated doses of rh-HGF led to increased urinary albumin excretion, but no rh-HGF-treated animals developed increased serum creatinine levels. Urinary albumin excretion returned to baseline after the cessation of rh-HGF. These results suggest that extended treatment with rh-HGF is required for the attenuation of cirrhosis, and repeated doses of rh-HGF cause adverse effects in extra-hepatic organs. These issues must be resolved before the widespread application of rh-HGF in the treatment of liver cirrhosis.

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

    PubMed Central

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

    2014-01-01

    Previous reports described 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. PMID:25101856

  14. 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

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

    PubMed

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

    2016-07-01

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

  16. Inhibition by interleukin-1 beta and tumor necrosis factor-alpha of the insulin-like growth factor I messenger ribonucleic acid response to growth hormone in rat hepatocyte primary culture.

    PubMed

    Thissen, J P; Verniers, J

    1997-03-01

    The cytokines are the putative mediators of the catabolic reaction that accompanies infection and trauma. Evidence suggests that their catabolic actions are indirect and potentially mediated through changes in hormonal axis such as the hypothalamo-pituitary-adrenal axis. Insulin-like growth factor I (IGF-I) is a GH-dependent growth factor that regulates the protein metabolism. To determine whether cytokines can directly inhibit the production of IGF-I by the liver, we investigated the regulation of IGF-I gene expression by interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha (10 ng/ml) in a model of rat primary cultured hepatocytes. Hepatocytes were isolated by liver collagenase perfusion and cultured on Matrigel 48 h before experiments. Each experiment was performed in at least three different animals. In the absence of GH, IL-1 beta and TNF-alpha did not affect the IGF-I messenger RNA (mRNA) basal levels, whereas IL-6 increased it by a factor of 2.5 after 24 h (P < 0.05). GH (500 ng/ml) alone stimulated the IGF-I gene expression markedly (5-to 10-fold increase) after 24 h (P < 0.001). IL-1 beta, and TNF-alpha to a lesser extent, dramatically inhibited the IGF-I mRNA response to GH (IL-1 beta: -82%, P < 0.001 and TNF-alpha: -47%, P < 0.01). The half-maximal inhibition of the IGF-I mRNA response to GH was observed for a concentration of IL-1 beta between 0.1 and 1 ng/ml. Moreover, IL-1 beta abolished the IL-6-induced IGF-I mRNA response. In contrast, IL-6 did not impair the IGF-I mRNA response to GH. To determine the potential role of the GH receptor (GHR) and the GH-binding protein (GHBP) in this GH resistance, we assessed the GHR and GHBP mRNAs response to these cytokines. GH alone did not affect the GHR/GHBP mRNA levels. IL-1 beta markedly decreased the GHR and GHBP mRNA levels (respectively, -68% and -60%, P < 0.05). Neither TNF-alpha nor IL-6 affected the GHR/GHBP gene expression. In conclusion, our results show that IL-1 beta, and TNF-alpha to

  17. Evaluation of the effect of static magnetic fields combined with human hepatocyte growth factor on human satellite cell cultures.

    PubMed

    Birk, Richard; Sommer, Ulrich; Faber, Anne; Aderhold, Christoph; Schulz, Johannes D; Hörmann, Karl; Goessler, Ulrich Reinhart; Stern-Straeter, Jens

    2014-06-01

    Tissue engineering is a promising research field, which aims to create new functional muscle tissue in vitro, by utilizing the myogenic differentiation potential of human stem cells. The objective of the present study was to determine the effect of static magnetic fields (SMF), combined with the use of the myogenic differentiation enhancing hepatocyte growth factor (HGF), on human satellite cell cultures, which are one of the preferred stem cell sources in skeletal muscle tissue engineering. We performed almarBlue® proliferation assays and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) for the following myogenic markers: desmin (DES), myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), myosin heavy chain (MYH) and α1 actin (ACTA1) to detect the effects on myogenic maturation. Additionally, immunohistochemical staining (ICC) and fusion index (FI) determination as independent markers of differentiation were performed on satellite cell cultures stimulated with HGF and HGF + SMF with an intensity of 80 mT. ICC verified the muscle phenotype at all time points. SMF enhanced the proliferation of satellite cell cultures treated with HGF. RT-PCR analysis, ICC and FI calculation revealed the effects of HGF/SMF on the investigated differentiation markers and stimulation with HGF and SMF verified the continuing maturation, however no significant increase in analysed markers could be detected when compared with control cultures treated with serum cessation. In conclusion, HGF or HGF + SMF stimulation of human satellite cell cultures did not lead to the desired enhancement of myogenic maturation of human satellite cell cultures compared with cell cultures stimulated with growth factor reduction.

  18. Arecoline-induced growth arrest and p21WAF1 expression are dependent on p53 in rat hepatocytes.

    PubMed

    Chou, Wen-Wen; Guh, Jinn-Yuh; Tsai, Jung-Fa; Hwang, Chi-Ching; Chen, Hung-Chun; Huang, Jau-Shyang; Yang, Yu-Lin; Hung, Wen-Chun; Chuang, Lea-Yea

    2008-01-14

    Betel-quid use is associated with the risk of liver cirrhosis and hepatocellular carcinoma and arecoline, the major alkaloid of betel-quid, is hepatotoxic in mice. Therefore, we studied the cytotoxic and genotoxic effects of arecoline in normal rat hepatocytes (Clone-9 cells). Arecoline dose-dependently (0.1-1mM) decreased cell cycle-dependent proliferation while inducing DNA damage at 24h. Moreover, arecoline (1mM)-induced apoptosis and necrosis at 24h. Arecoline dose-dependently (0.1-0.5mM) increased transforming growth factor-beta (TGF-beta) mRNA, gene transcription and bioactivity and neutralizing TGF-beta antibody attenuated arecoline (0.5mM)-inhibited cell proliferation at 24h. Arecoline (0.5mM) also increased p21(WAF1) protein expression and p21(WAF1) gene transcription. Moreover, arecoline (0.5mM) time-dependently (8-24h) increased p53 serine 15 phosphorylation. Pifithrin-alpha (p53 inhibitor) and the loss of the two p53-binding elements in the p21(WAF1) gene promoter attenuated arecoline-induced p21(WAF1) gene transcription at 24h. Pifithrin-alpha also attenuated arecoline (0.5mM)-inhibited cell proliferation at 24h. We concluded that arecoline induces cytotoxicity, DNA damage, G(0)/G(1) cell cycle arrest, TGF-beta1, p21(WAF1) and activates p53 in Clone-9 cells. Moreover, arecoline-induced p21(WAF1) is dependent on p53 while arecoline-inhibited growth is dependent on both TGF-beta and p53.

  19. 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

  20. Hepatocyte Growth Factor and Interleukin-6 in Prostate Cancer Bone Metastasis

    DTIC Science & Technology

    2006-06-01

    LHRH agonist on the bone marrow environment. Another strategy of reducing androgen levels is by the downregulation of GnRH receptors in the...Interleukin-6 in Prostate Cancer Bone Metastasis PRINCIPAL INVESTIGATOR: Beatrice S. Knudsen, M.D., Ph.D. CONTRACTING ORGANIZATION...Growth Factor and Interleukin-6 in Prostate Cancer Bone Metastasis 5a. CONTRACT NUMBER 5b. GRANT NUMBER DAMD17-02-1-0159 5c. PROGRAM ELEMENT

  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. Madecassoside ameliorates bleomycin‐induced pulmonary fibrosis in mice through promoting the generation of hepatocyte growth factor via PPAR‐γ in colon

    PubMed Central

    Xia, Ying; Xia, Yu‐Feng; Lv, Qi; Yue, Meng‐Fan; Qiao, Si‐Miao; Yang, Yan

    2016-01-01

    Background and Purpose Madecassoside has potent anti‐pulmonary fibrosis (PF) effects when administered p.o., despite having extremely low oral bioavailability. Herein, we explored the mechanism of this anti‐PF effect with regard to gut hormones. Experimental Approach A PF model was established in mice by intratracheal instillation of bleomycin. Haematoxylin and eosin stain and Masson's trichrome stain were used to assess histological changes in the lung. Quantitative‐PCR and Western blot detected mRNA and protein levels, respectively, and cytokines were measured by ELISA. Small interfering RNA was used for gene‐silencing. EMSA was applied to detect DNA‐binding activity. Key Results Administration of madecassoside, p.o., but not its main metabolite madecassic acid, exhibited a direct anti‐PF effect in mice. However, i.p. madecassoside had no anti‐PF effect. Madecassoside increased the expression of hepatocyte growth factor (HGF) in colon tissues, and HGF receptor antagonists attenuated its anti‐PF effect. Madecassoside facilitated the secretion of HGF from colonic epithelial cells by activating the PPAR‐γ pathway, as shown by an up‐regulation of PPAR‐γ mRNA expression, nuclear translocation and DNA‐binding activity both in vitro and in vivo. Also GW9662, a selective PPAR‐γ antagonist, almost completely prevented the madecassoside‐induced increased expression of HGF and amelioration of PF. Conclusions and Implications The potent anti‐PF effects induced by p.o. madecassoside in mice are not mediated by its metabolites or itself after absorption into blood. Instead, madecassoside increases the activity of PPAR‐γ, which subsequently increases HGF expression in colonic epithelial cells. HGF then enters into the circulation and lung tissue to exert an anti‐PF effect. PMID:26750154

  3. M10, a Caspase Cleavage Product of the Hepatocyte Growth Factor Receptor, Interacts with Smad2 and Demonstrates Anti-Fibrotic Properties in Vitro and in Vivo

    PubMed Central

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

    2016-01-01

    Hepatocyte growth factor receptor, also known as cellular mesenchymal-epithelial transition factor (c-MET, MET), is an important antifibrotic molecule that protects various tissues, including lung, from injury and fibrosis. The intracellular cytoplasmic tail of MET contains a caspase-3 recognition motif “DEVD-T” that upon cleavage by caspase-3 generates a 10 amino acid peptide, TRPASFWETS, designated as “M10”. M10 contains at its N-terminus the uncharged amino acid proline (P) directly after a cationic amino acid arginine (R) which favors the transport of the peptide through membranes. M10, when added to cell culture medium, remains in the cytoplasm and nuclei of cells for up to 24 hours. M10 effectively decreases collagen in both scleroderma and TGFβ-stimulated normal lung and skin fibroblasts. M10 interacts with the MH2 domain of Smad2 and inhibits TGFβ-induced Smad2 phosphorylation, suggesting that the antifibrotic effects of M10 are mediated in part by counteracting Smad-dependent fibrogenic pathways. In the bleomycin murine model of pulmonary fibrosis, M10 noticeably reduced lung inflammation and fibrosis. Ashcroft fibrosis scores and lung collagen content were significantly lower in bleomycin-treated mice receiving M10 as compared with bleomycin-treated mice receiving scrambled peptide. We conclude that M10 peptide interacts with Smad2 and demonstrates strong antifibrotic effects in vitro and in vivo in an animal model of lung fibrosis and should be considered as a potential therapeutic agent for systemic sclerosis and other fibrosing diseases. PMID:26772959

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

    PubMed

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

    2016-04-01

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

  5. Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM.

    PubMed

    Rohrbeck, Leona; Gong, Jia-Nan; Lee, Erinna F; Kueh, Andrew J; Behren, Andreas; Tai, Lin; Lessene, Guillaume; Huang, David C S; Fairlie, Walter D; Strasser, Andreas; Herold, Marco J

    2016-12-01

    A large proportion of melanomas harbour the activating BRAF(V600E) mutation that renders these cells dependent on MAPK signalling for their survival. Although the highly specific and clinically approved BRAF(V600E) kinase inhibitor, PLX4032, induces apoptosis of melanoma cells bearing this mutation, the underlying molecular mechanisms are not fully understood. Here, we reveal that PLX4032-induced apoptosis depends on the induction of the pro-apoptotic BH3-only protein PUMA with a minor contribution of its relative BIM. Apoptosis could be significantly augmented when PLX4032 was combined with an inhibitor of the pro-survival protein BCL-XL, whereas neutralization of the pro-survival family member BCL-2 caused no additional cell death. Although the initial response to PLX4032 in melanoma patients is very potent, resistance to the drug eventually develops and relapse occurs. Several factors can cause melanoma cells to develop resistance to PLX4032; one of them is the activation of the receptor tyrosine kinase cMET on melanoma cells by its ligand, hepatocyte growth factor (HGF), provided by the tumour microenvironment or the cancer cells themselves. We found that HGF mediates resistance of cMET-expressing BRAF mutant melanoma cells to PLX4032-induced apoptosis through downregulation of PUMA and BIM rather than by increasing the expression of pro-survival BCL-2-like proteins. These results suggest that resistance to PLX4032 may be overcome by specifically increasing the levels of PUMA and BIM in melanoma cells through alternative signalling cascades or by blocking pro-survival BCL-2 family members with suitable BH3 mimetic compounds.

  6. 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

    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; Sladek, Frances M

    2015-10-01

    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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Madecassoside ameliorates bleomycin-induced pulmonary fibrosis in mice through promoting the generation of hepatocyte growth factor via PPAR-γ in colon.

    PubMed

    Xia, Ying; Xia, Yu-Feng; Lv, Qi; Yue, Meng-Fan; Qiao, Si-Miao; Yang, Yan; Wei, Zhi-Feng; Dai, Yue

    2016-04-01

    Madecassoside has potent anti-pulmonary fibrosis (PF) effects when administered p.o., despite having extremely low oral bioavailability. Herein, we explored the mechanism of this anti-PF effect with regard to gut hormones. A PF model was established in mice by intratracheal instillation of bleomycin. Haematoxylin and eosin stain and Masson's trichrome stain were used to assess histological changes in the lung. Quantitative-PCR and Western blot detected mRNA and protein levels, respectively, and cytokines were measured by ELISA. Small interfering RNA was used for gene-silencing. EMSA was applied to detect DNA-binding activity. Administration of madecassoside, p.o., but not its main metabolite madecassic acid, exhibited a direct anti-PF effect in mice. However, i.p. madecassoside had no anti-PF effect. Madecassoside increased the expression of hepatocyte growth factor (HGF) in colon tissues, and HGF receptor antagonists attenuated its anti-PF effect. Madecassoside facilitated the secretion of HGF from colonic epithelial cells by activating the PPAR-γ pathway, as shown by an up-regulation of PPAR-γ mRNA expression, nuclear translocation and DNA-binding activity both in vitro and in vivo. Also GW9662, a selective PPAR-γ antagonist, almost completely prevented the madecassoside-induced increased expression of HGF and amelioration of PF. The potent anti-PF effects induced by p.o. madecassoside in mice are not mediated by its metabolites or itself after absorption into blood. Instead, madecassoside increases the activity of PPAR-γ, which subsequently increases HGF expression in colonic epithelial cells. HGF then enters into the circulation and lung tissue to exert an anti-PF effect. © 2016 The British Pharmacological Society.

  8. 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

  9. Celastrol attenuates mitochondrial dysfunction and inflammation in palmitate-mediated insulin resistance in C3A hepatocytes.

    PubMed

    Abu Bakar, Mohamad Hafizi; Sarmidi, Mohamad Roji; Tan, Joo Shun; Mohamad Rosdi, Mohamad Norisham

    2017-03-15

    Accumulating evidence indicates that mitochondrial dysfunction-induced inflammation is among the convergence points for the greatest hallmarks of hepatic insulin resistance. Celastrol, an anti-inflammatory compound from the root of Tripterygium Wilfordii has been reported to mitigate insulin resistance and inflammation in animal disease models. Nevertheless, the specific mechanistic actions of celastrol in modulating such improvements at the cellular level remain obscure. The present study sought to explore the mechanistic roles of celastrol upon insulin resistance induced by palmitate in C3A human hepatocytes. The hepatocytes exposed to palmitate (0.75mM) for 48h exhibited reduced both basal and insulin-stimulated glucose uptake, mitochondrial dysfunction, leading to increased mitochondrial oxidative stress with diminished fatty acid oxidation. Elevated expressions of nuclear factor-kappa B p65 (NF-κB p65), c-Jun NH(2)-terminal kinase (JNK) signaling pathways and the amplified release of pro-inflammatory cytokines including IL-8, IL-6, TNF-α and CRP were observed following palmitate treatment. Consistently, palmitate reduced and augmented phosphorylated Tyrosine-612 and Serine-307 of insulin receptor substrate-1 (IRS-1) proteins, respectively in hepatocytes. However, celastrol at the optimum concentration of 30nM was able to reverse these deleterious occasions and protected the cells from mitochondrial dysfunction and insulin resistance. Importantly, we presented evidence for the first time that celastrol efficiently prevented palmitate-induced insulin resistance in hepatocytes at least, via improved mitochondrial functions and insulin signaling pathways. In summary, the present investigation underlines a conceivable mechanism to elucidate the cytoprotective potential of celastrol in attenuating mitochondrial dysfunction and inflammation against the development of hepatic insulin resistance. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Co-culture of hepatoma cells with hepatocytic precursor (stem-like) cells inhibits tumor cell growth and invasion by downregulating Akt/NF-κB expression

    PubMed Central

    Sui, Cheng-Jun; Xu, Miao; Li, Wei-Qing; Yang, Jia-Mei; Yan, Hong-Zhu; Liu, Hui-Min; Xia, Chun-Yan; Yu, Hong-Yu

    2016-01-01

    Hepatocytic stem cells (HSCs) have inhibitory effects on hepatocarcinoma cells. The present study investigated the effects of HSC activity in hepatocarcinoma cells in vitro. A Transwell co-culture system of hepatocytic precursor (stem-like) WB-F344 cells and hepatoma CBRH-7919 cells was used to assess HSC activity in metastasized hepatoma cells in vitro. Nude mouse xenografts were used to assess HSC activity in vivo. Co-culture of hepatoma CBRH-7919 cells with WB-F344 cells suppressed the growth and colony formation, tumor cell migration and invasion capacity of CBRH-7919 cells. The nude mouse xenograft assay demonstrated that the xenograft size of CBRH-7919 cells following co-culture with WB-F344 cells was significantly smaller compared with that of control cells. Furthermore, the expression levels of the epithelial markers E-cadherin and β-catenin were downregulated, while the mesenchymal markers α-SMA and vimentin were upregulated. Co-culture of CBRH-7919 cells with WB-F344 cells downregulated NF-κB and phospho-Akt expression. In conclusion, hepatocytic precursor (stem-like) WB-F344 cells inhibited the growth, colony formation and invasion capacity of metastasized hepatoma CBRH-7919 cells in vitro and in vivo by downregulating Akt/NF-κB signaling. PMID:27895771

  11. 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.

  12. Downregulating SOCS3 with siRNA ameliorates insulin signaling and glucose metabolism in hepatocytes of IUGR rats with catch-up growth.

    PubMed

    Ye, Juan; Zheng, Ruidan; Wang, Qun; Liao, Lihong; Ying, Yanqin; Lu, Huiling; Cianflone, Katherine; Ning, Qin; Luo, Xiaoping

    2012-12-01

    Individuals with intrauterine growth retardation (IUGR) who demonstrate a catch-up in body weight are prone to insulin resistance. High expressions of suppressor of cytokine signaling 3 (SOCS3) are thought to aggravate insulin resistance. We hypothesized that downregulating SOCS3 expression via small interfering RNA (siRNA) might have beneficial effects on insulin-resistant hepatocytes of catch-up growth IUGR rats (CG-IUGRs). An IUGR rat model was employed via maternal nutritional restriction. After evaluating metabolic states of CG-IUGR offspring, effective SOCS3-specific siRNA (siSOCS3) was transfected into cultured hepatocytes using liposomes. mRNA levels of SOCS3, insulin receptor substrates (IRSs), phosphatidylinositol 3-kinase (PI3K), and Akt2, key gluconeogenesis genes, were assessed via real-time PCR. Protein expression and phosphorylation changes were evaluated via western blot. CG-IUGR hepatocytes showed increases in SOCS3 and gluconeogenic gene expressions, and decreases in IRS1 and PI3K expressions as compared with controls. After transfecting CG-IUGR hepatocytes with siSOCS3, protein levels of IRS1, PI3K, and phosphorylated Akt2 were higher as compared with those of untransfected CG-IUGR cells. Transcriptional suppression effects of gluconeogenesis genes were more obvious in siSOCS3-treated cells after insulin stimulation. Additional insights were provided to understand mechanisms of insulin resistance in CG-IUGR rats. Downregulating SOCS3 might improve insulin signaling transduction and ameliorate hepatic glucose metabolism in insulin-resistant CG-IUGR rats in vitro.

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

    PubMed Central

    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. PMID:7822413

  14. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2016-04-28

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

  16. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. 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.

  18. Gene transfer of hepatocyte growth factor gene improves learning and memory in the chronic stage of cerebral infarction.

    PubMed

    Shimamura, Munehisa; Sato, Naoyuki; Waguri, Satoshi; Uchiyama, Yasuo; Hayashi, Takuya; Iida, Hidehiro; Nakamura, Toshikazu; Ogihara, Toshio; Kaneda, Yasufumi; Morishita, Ryuichi

    2006-04-01

    There is no specific treatment to improve the functional recovery in the chronic stage of ischemic stroke. To provide the new therapeutic options, we examined the effect of overexpression of hepatocyte growth factor (HGF) in the chronic stage of cerebral infarction by transferring the HGF gene into the brain using hemagglutinating virus of Japan envelope vector. Sixty rats were exposed to permanent middle cerebral artery occlusion (day 1). Based on the sensorimotor deficits at day 7, the rats were divided equally into control vector or HGF-treated rats. At day 56, rats transfected with the HGF gene showed a significant recovery of learning and memory in Morris water maze tests (control vector 50+/-4 s; HGF 33+/-5 s; P<0.05) and passive avoidance task (control vector 132.4+/-37.5 s; HGF 214.8+/-26.5 s; P<0.05). Although the total volume of cerebral infarction was not related to the outcome, immunohistochemical analysis for Cdc42 and synaptophysin in the peri-infarct region revealed that HGF enhanced the neurite extension and increased synapses. Immunohistochemistry for glial fibriary acidic protein revealed that the formation of glial scar was also prevented by HGF gene treatment. Additionally, the number of the arteries was increased in the HGF group at day 56. These data demonstrated that HGF has a pivotal role for the functional recovery after cerebral infarction through neuritogenesis, improved microcirculation, and the prevention of gliosis. Our results also provide evidence for the feasibility of gene therapy in the chronic stage of cerebral infarction.

  19. Hepatocyte growth factor/scatter factor induces a variety of tissue- specific morphogenic programs in epithelial cells

    PubMed Central

    1995-01-01

    Hepatocyte growth factor/scatter factor (HGF/SF) is the mesenchymal ligand of the epithelial tyrosine kinase receptor c-Met. In vitro, HGF/SF has morphogenic properties, e.g., induces kidney epithelial cells to form branching ducts in collagen gels. Mutation of the HGF/SF gene in mice results in embryonic lethality due to severe liver and placenta defects. Here, we have evaluated the morphogenic activity of HGF/SF with a large variety of epithelial cells grown in three- dimensional collagen matrices. We found that HGF/SF induces SW 1222 colon carcinoma cells to form crypt-like structures. In these organoids, cells exhibit apical/basolateral polarity and build a well- developed brush border towards the lumen. Capan 2 pancreas carcinoma cells, upon addition of HGF/SF, develop large hollow spheroids lined with a tight layer of polarized cells. Collagen inside the cysts is digested and the cells show features of pancreatic ducts. HGF/SF induces EpH4 mammary epithelial cells to form long branches with end- buds that resemble developing mammary ducts. pRNS-1-1 prostate epithelial cells in the presence of HGF/SF develop long ducts with distal branching as found in the prostate. Finally, HGF/SF simulates alveolar differentiation in LX-1 lung carcinoma cells. Expression of transfected HGF/SF cDNA in LX-1 lung carcinoma and EpH4 mammary epithelial cells induce morphogenesis in an autocrine manner. In the cell lines tested, HGF/SF activated the Met receptor by phosphorylation of tyrosine residues. These data show that HGF/SF induces intrinsic, tissue-specific morphogenic activities in a wide variety of epithelial cells. Apparently, HGF/SF triggers respective endogenous programs and is thus an inductive, not an instructive, mesenchymal effector for epithelial morphogenesis. PMID:8522613

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

    PubMed

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

    2015-01-01

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

  1. Early treatment with hepatocyte growth factor improves cardiac function in experimental heart failure induced by myocardial infarction.

    PubMed

    Jin, Hongkui; Yang, Renhui; Li, Wei; Ogasawara, Annie K; Schwall, Ralph; Eberhard, David A; Zheng, Zhong; Kahn, David; Paoni, Nicholas F

    2003-02-01

    Plasma levels of hepatocyte growth factor (HGF) are increased within hours of cardiac ischemia/reperfusion in rats, and HGF has been shown to be cardioprotective toward acute ischemic injury. Myocardial levels of HGF mRNA and protein are increased for several days after myocardial infarction (MI), however, indicating a possible additional protective effect of HGF toward the progression of MI to heart failure. The purpose of this study was to determine whether HGF administration during the time course of endogenous cardiac HGF induction would lead to long-term improvement in cardiac function in rats with MI. MI was induced by 2-h occlusion of the left coronary artery, followed by reperfusion. HGF was given by intravenous infusion at 0.45 mg/kg/day for 6 days beginning on the day after surgery. Cardiac function and hemodynamic parameters were measured by using indwelling catheters and perivascular flow probes in conscious animals 8 weeks post-MI. Myocardial infarcts were approximately 30% of the left ventricle, and there was no difference in infarct size between the vehicle-treated and HGF-treated groups. Compared with untreated sham-operated rats, vehicle-treated MI animals had significantly lower cardiac index and stroke volume index and higher systemic vascular resistance, indicating heart failure developed. Treatment with HGF caused a significant increase in cardiac index and stroke volume index and a reduction in systemic vascular resistance in rats with MI, restoring these parameters close to those observed in sham-operated control animals. These results provide direct evidence that HGF may be of benefit to cardiovascular function in ischemic cardiomyopathy.

  2. 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.

  3. 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

  4. 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.

  5. Caffeic acid attenuated acetaminophen-induced hepatotoxicity by inhibiting ERK1/2-mediated early growth response-1 transcriptional activation.

    PubMed

    Pang, Chun; Shi, Liang; Sheng, Yuchen; Zheng, Zhiyong; Wei, Hai; Wang, Zhengtao; Ji, Lili

    2016-12-25

    Caffeic acid (CA) is a natural compound abundant in fruits, coffee and plants. This study aims to investigate the involved mechanism of the therapeutic detoxification of CA against acetaminophen (APAP)-induced hepatotoxicity. CA (10, 30 mg/kg) was orally given to mice at 1 h after mice were pre-administrated with APAP (300 mg/kg). The therapeutic detoxification of CA against APAP-induced hepatotoxicity was observed by detecting serum aminotransferases, liver malondialdehyde (MDA) amount and liver histological evaluation in vivo. CA reduced APAP-induced increase in the mRNA expression of early growth response 1 (Egr1) in hepatocytes, and inhibited APAP-induced Egr1 transcriptional activation in vitro and in vivo. CA reduced the increased expression of growth arrest and DNA-damage-inducible protein (Gadd45)α induced by APAP in hepatocytes. Moreover, Egr1 siRNA reduced Gadd45α expression and reversed APAP-induced cytotoxicity in hepatocytes. Further results showed that CA blocked APAP-induced activation of extracellular-regulated protein kinase (ERK1/2) signaling cascade in vivo and in vitro. In addition, the application of ERK1/2 inhibitors (PD98059 and U0126) abrogated the nuclear translocation of Egr1 induced by APAP in hepatocytes. In conclusion, this study demonstrated the therapeutic detoxification of CA against APAP-induced liver injury, and the inhibition of CA on ERK1/2-mediated Egr1 transcriptional activation was involved in this process. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. HIF-1α and HIF-2α are critically involved in hypoxia-induced lipid accumulation in hepatocytes through reducing PGC-1α-mediated fatty acid β-oxidation.

    PubMed

    Liu, Yanlong; Ma, Zhenhua; Zhao, Cuiqing; Wang, Yuhua; Wu, Guicheng; Xiao, Jian; McClain, Craig J; Li, Xiaokun; Feng, Wenke

    2014-04-21

    During periods of cellular hypoxia, hepatocytes adapt to consume less oxygen by shifting energy production from mitochondrial fatty acid β-oxidation to glycolysis. One of the earliest responses to pathologic hypoxia is the activation of the hypoxia-inducible factor (HIF). In the present study, we examined whether HIF-1 and HIF-2 were involved in the regulation of fatty acid synthesis and β-oxidation. We showed that hypoxia induced fat accumulation in the livers of mice and in HepG2 cells. These hypoxia-induced changes in fatty acid metabolism were mediated by suppressing fatty acid β-oxidation, without significantly influencing fatty acid synthesis. Exposing hepatocytes to 1% O2 reduced the mRNA expression of carnitine palmitoyltransferase 1 (CPT-1), which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for β-oxidation. Moreover, hypoxia exposure reduced proliferator-activated receptor-γ coactivator-1α (PGC-1α) protein levels, which plays an important role in regulation of β-oxidation. Exposure of HIF-1α or HIF-2α deficient hepatocytes to hypoxia abrogated the reduction in PGC-1α and CPT-1 expression and cellular lipid accumulation observed in normal hepatocytes exposed to hypoxia. These results suggest that both HIF-1α and HIF-2α are involved in hypoxia-induced lipid accumulation in hepatocytes via reducing PGC-1α mediated fatty acid β-oxidation.

  7. Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

    PubMed Central

    Trebicka, Jonel; Schievenbusch, Stephanie; Strack, Ingo; Molnar, Levente; von Brandenstein, Melanie; Töx, Ulrich; Nischt, Roswitha; Coutelle, Oliver; Dienes, Hans Peter; Odenthal, Margarete

    2011-01-01

    Background In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC. Methodology HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3′-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting. Principal Findings The 3′-UTR of the collagen-1 and −4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis. Conclusions Upregulation of miRNA-29 by HGF

  8. Effect of intramuscular injection of hepatocyte growth factor plasmid DNA with electroporation on bleomycin-induced lung fibrosis in rats.

    PubMed

    Long, Xiang; Xiong, Sheng-dao; Xiong, Wei-ning; Xu, Yong-jian

    2007-08-20

    So far, there is no efficient treatment for pulmonary fibrosis. The objective of this study was to determine whether intramuscular injection of the hepatocyte growth factor (HGF) plasmid DNA by in vivo electroporation could prevent bleomycin-induced pulmonary fibrosis in rats, and to investigate the possible mechanisms. Twenty male Wistar rats were randomly divided into four groups: control group (group C), model group (group M), early intervention group (group I) and late intervention group (group II). Groups M, I and II were intratracheally infused with bleomycin, then injected the plasmid pcDNA3.1-hHGF to group I on day 7, 14 and 21. Group II received the same treatment like Group I on day 14 and 21. All the rats were killed on day 28 after bleomycin injection. We detected Homo HGF expression in the rats with ELISA method and estimated the pathological fibrosis score of lung tissue using hematoxylin eosin (HE) and Massion staining. The mRNA expression of transforming growth factor-beta1 (TGF-beta1), cycloxygenase-2 (COX-2), and rat HGF in rat pulmonary parenchyma were evaluated by RT-PCR. Immunohistochemistry and Western blotting were performed to determine the protein expression of transforming TGF-beta1 and COX-2 in lung parenchyma. The plasmid pcDNA3.1-hHGF could express hHGF in NIH3T3 cells and the hHGF protein is secreted into the culture medium. The expression of hHGF protein could be monitored in quadriceps muscle, plasma and lung in Groups I and II. Pulmonary fibrosis levels of Groups I and II were obviously lower than that of group M (P < 0.05). Expression of TGF-beta1 protein and mRNA in lung tissue was markedly decreased in Groups I and II compared with Group M (P < 0.05). The level of expression of HGF and COX-2 mRNA was higher in Groups I and II than in Group M (P < 0.05). Injection of the plasmid pcDNA3.1-hHGF into skeletal muscle with electroporation has a potential role in the treatment of bleomycin-induced lung fibrosis. Exogenous HGF may

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

    SciTech Connect

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

    2010-07-20

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

  10. Mediators in cell growth and differentiation

    SciTech Connect

    Ford, R.J.; Maizel, A.L.

    1985-01-01

    This book contains papers divided among seven sections. The section headings are: Cell Cycle and Control of Cell Growth, Growth Factors for Nonlymphoid Cells, Colony-Stimulating Factors, Stem Cells and Hematopoiesis, Lymphoid Growth Factors, Growth Factors in Neoplasia, Interferon, and Differentiation in Normal and Neoplastic Cells.

  11. Severity of Plasma Leakage Is Associated With High Levels of Interferon γ-Inducible Protein 10, Hepatocyte Growth Factor, Matrix Metalloproteinase 2 (MMP-2), and MMP-9 During Dengue Virus Infection.

    PubMed

    Her, Zhisheng; Kam, Yiu-Wing; Gan, Victor C; Lee, Bernett; Thein, Tun-Linn; Tan, Jeslin J L; Lee, Linda K; Fink, Katja; Lye, David C; Rénia, Laurent; Leo, Yee-Sin; Ng, Lisa F P

    2017-01-01

     Dengue virus infection typically causes mild dengue fever, but, in severe cases, life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) occur. The pathophysiological hallmark of DHF and DSS is plasma leakage that leads to enhanced vascular permeability, likely due to a cytokine storm.  Ninety patients with dengue during 2010-2012 in Singapore were prospectively recruited and stratified according to their disease phase, primary and secondary infection status, and disease severity, measured by plasma leakage. Clinical parameters were recorded throughout the disease progression. The levels of various immune mediators were quantified using comprehensive multiplex microbead-based immunoassays for 46 immune mediators.  Associations between clinical parameters and immune mediators were analyzed using various statistical methods. Potential immune markers, including interleukin 1 receptor antagonist, interferon γ-inducible protein 10, hepatocyte growth factor, soluble p75 tumor necrosis factor α receptor, vascular cell adhesion molecule 1, and matrix metalloproteinase 2, were significantly associated with significant plasma leakage. Secondary dengue virus infections were also shown to influence disease outcome in terms of disease severity.  This study identified several key markers for exacerbated dengue pathogenesis, notably plasma leakage. This will allow a better understanding of the molecular mechanisms of DHF and DSS in patients with dengue. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  12. Effectiveness of Intracavernous Delivery of Recombinant Human Hepatocyte Growth Factor on Erectile Function in the Streptozotocin-Induced Diabetic Mouse.

    PubMed

    Das, Nando Dulal; Yin, Guo Nan; Choi, Min Ji; Song, Kang-Moon; Park, Jin-Mi; Limanjaya, Anita; Ghatak, Kalyan; Minh, Nguyen Nhat; Ock, Jiyeon; Park, Soo-Hwan; Kim, Ho Min; Ryu, Ji-Kan; Suh, Jun-Kyu

    2016-11-01

    Diabetic erectile dysfunction is a disease mostly of vascular origin and men with diabetic erectile dysfunction respond poorly to oral phosphodiesterase-5 inhibitors. Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an essential role in the regulation of cell proliferation, survival, and angiogenesis. To determine the effectiveness of recombinant human (rh)-HGF in restoring erectile function in diabetic mice. Four groups of mice were used: control non-diabetic mice and streptozotocin-induced diabetic mice receiving two successive intracavernous injections of phosphate buffered saline (days -3 and 0), a single intracavernous injection of rh-HGF (day 0), or two successive intracavernous injections of rh-HGF (days -3 and 0). We also examined the effect of rh-HGF in primary cultured mouse cavernous endothelial cells and in major pelvic ganglion culture in vitro, which was incubated under a normal-glucose (5 mmol/L) or a high-glucose (30 mmol/L) condition. Two weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve and the penis was harvested for histologic studies. Repeated intracavernous injections of rh-HGF protein induced significant restoration of erectile function in diabetic mice (89-100% of control values), whereas a single intracavernous injection of rh-HGF protein elicited modest improvement. Rh-HGF significantly induced phosphorylation of its receptor c-Met, increased the content of endothelial cells and smooth muscle cells, and decreased the generation of reactive oxygen species (superoxide anion and peroxynitrite) and extravasation of oxidized low-density lipoprotein in diabetic mice. Under the high-glucose condition, rh-HGF protein also promoted tube formation in mouse cavernous endothelial cells and enhanced neurite sprouting in major pelvic ganglion culture in vitro. The dual angiogenic and neurotrophic effects of HGF could open a new avenue through which diabetic erectile dysfunction

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

    PubMed Central

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

    2016-01-01

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

  14. Neurohumoral mediation of exercise-induced growth.

    PubMed

    Borer, K T

    1994-06-01

    To assess exercise effects on growth, other variables modulating growth need to be taken into account. Endogenous control of growth proceeds from local actions of growth factors and dependence on nutrition abundance through guidance by growth hormone (GH) and other anabolic hormones to neuroendocrine suppression of growth. Nutrient abundance controls the reparative growth of lean body mass in adulthood by coupling it to anabolic endocrine reflexes. Growth is blocked when catabolic endocrine reflexes govern energy expenditure. The relationship between exercise intensity and growth is nonlinear. Growth is an intermittent process. Its expression and stimulation are dependent on ultradian and circadian rhythms of energy metabolism and neurohumoral release. High-resistance exercise selectively stimulates growth of the musculoskeletal system through expression of growth factor genes in the challenged tissues and without the GH guidance or abundant nutritional support. Habitual endurance exercise stimulates reparative growth of lean body mass through the neuroendocrine adaptations including increased pulsatile GH secretion. These also facilitate oxidative utilization of storage lipids thereby contributing to the regulation of body composition in adulthood. In the absence of sufficient high-resistance and endurance exercise regulation of adult body mass is impaired: excess LBM is lost during energy deficit, and excess fat accumulates during energy surplus.

  15. [Influence of hepatocyte growth factor on iNOS, NO and IL-1β in the cerebrum during cerebral ischemia/reperfusion in rats].

    PubMed

    He, Fang; Ye, Bei; Chen, Jianzhen; Sun, Xiaoyan; Li, Chang

    2014-01-01

    To explore the effect of hepatocyte growth factor (HGF) on inducible nitric oxide synthase (iNOS), NO and interleukin-1β (IL-1β) in the cerebrum of rats subjected to cerebral ischemia/reperfusion (I/R). Sprague-Dawley rats were randomly divided into 5 groups: a sham group, an I/R group,an HGF1 group, an HGF2 group, and an HGF3 group. The latter 3 groups were respectively injected 15, 30 and 60 μg/kg HGF. The focal cerebral I/R model was established by sutureoccluded method. After 1.5 h ischemia followed by 24 h reperfusion, the iNOS activity and NO content in the ischemic cerebral tissue were assessed. The expression of iNOS mRNA and IL-1β mRNA was detected. The level of iNOS protein and IL-1β content were determined. In addition, cultured cerebral cortical neurons in vitro were exposed to I/R. Then the expression of iNOS and IL-1β protein in the neurons was detected, and NO content was assessed. The iNOS activity and NO content in the ischemic cerebral tissue were increased. The expression of iNOS mRNA and IL-1β mRNA was upregulated. The level of iNOS protein and IL- 1β content were increased. Administration of HGF decreased the iNOS activity and NO content, and downregulated the expression of iNOS mRNA, IL-1β mRNA, iNOS protein and IL-1β content in the ischemic cerebral tissue. HGF decreased the expression of IL-1β, iNOS protein and NO content in the cortical neurons exposed to I/R in vitro. HGF can inhibit the expression of IL-1β and decrease the expression of iNOS and content of NO, which is probably one of the mechanisms mediating the protection of HGF against cerebral ischemia injury.

  16. The activation and differential signalling of the growth hormone receptor induced by pGH or anti-idiotypic monoclonal antibodies in primary rat hepatocytes.

    PubMed

    Li, Wei; Lan, Hainan; Liu, Huimin; Fu, Zhiling; Yang, Yanhong; Han, Weiwei; Guo, Feng; Liu, Yu; Zhang, Hui; Liu, Jingsheng; Zheng, Xin

    2013-08-25

    In this report, we have developed a panel of monoclonal anti-idiotypic antibodies to pGH by immunising BALB/c mice with a purified monoclonal anti-pGH antibody (1A3), among which one mAb, termed CG-8F, was selected for further characterisation. We found that CG-8F behaved as a typical Ab2β, not only conformationally competing with pGH for 1A3 but also exhibiting recognition for GHR in a rat hepatocyte model. We next examined the resulting signal transduction pathways triggered by this antibody in rat hepatocytes and found that both pGH and CG-8F could trigger the JAK2-STAT1/3/5-mediated signal transduction pathway. Furthermore, the phosphorylation kinetics of pSTAT1/3/5 induced by either pGH or CG-8F were remarkably similar in the dose-response and time course rat hepatocyte experiments. In contrast, only pGH, but not CG-8F, was capable of inducing ERK phosphorylation. Further experimental studies indicated that the two functional binding sites on CG-8F are required for GHR activation. This study partially reveals the mechanism of action of GH anti-idiotypic antibodies and also indicates that monoclonal anti-idiotypic antibodies represent an effective way to produce GH mimics, suggesting that it is possible to produce signal-specific cytokine agonists using an anti-idiotypic antibody approach. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  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

    2016-02-02

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2014-04-01

    Hawthorn (Crataegus oxyacantha L.), a plant used in traditional medicine, is a rich source of procyanidins which have been reported to exhibit antioxidant and anti-carcinogenic activity. In this study, we assessed the effect of hawthorn bark extract (HBE) on Nrf2 pathway activation in THLE-2 and HepG2 cells. Treatment with 1.1 µg/mL, 5.5 µg/mL and 11 µg/mL of HBE resulted in the translocation of Nrf2 from the cytosol to the nucleus in both cell lines; however, the accumulation of phosphorylated Nrf2 was observed only in THLE-2. Accordingly, treatment of cells with HBE was associated with an increase in the mRNA and protein level of such Nrf2-dependent genes as glutathione S-transferases (GSTA, GSTP, GSTM, GSTT), NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) (0.2-1.1-fold change, p < 0.05), however, only in normal THLE-2 hepatocytes. The induction of NQO1 correlated with an increased level of p53 (0.21-0.42-fold change, p < 0.05). These effects may be related to induction of phosphorylation of upstream ERK and JNK kinases. Collectively, the results suggest that the Nrf2/ARE pathway may play an important role in the regulation of procyanidin-mediated antioxidant/detoxifying effects in hepatocytes, and this may explain the hepatoprotective and chemopreventive properties of these phytochemicals. Copyright © 2013 John Wiley & Sons, Ltd.

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

    PubMed

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

    2014-12-01

    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. Copyright © 2014 the American Physiological Society.

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

    PubMed Central

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

    2014-01-01

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

  2. Insulin- and leptin-mediated control of aquaglyceroporins in human adipocytes and hepatocytes is mediated via the PI3K/Akt/mTOR signaling cascade.

    PubMed

    Rodríguez, Amaia; Catalán, Victoria; Gómez-Ambrosi, Javier; García-Navarro, Socorro; Rotellar, Fernando; Valentí, Víctor; Silva, Camilo; Gil, María J; Salvador, Javier; Burrell, María A; Calamita, Giuseppe; Malagón, María M; Frühbeck, Gema

    2011-04-01

    Glycerol constitutes an important metabolite for the control of lipid accumulation and glucose homeostasis. The impact of obesity and obesity-associated type 2 diabetes as well as the potential regulatory role of insulin and leptin on aquaglyceroporins (AQP) 3, 7, and 9 were analyzed. The tissue distribution and expression of AQP in biopsies of omental and sc adipose tissue as well as liver were analyzed in lean and obese Caucasian volunteers (n = 63). The effect of insulin (1, 10, and 100 nmol/liter) and leptin (0.1, 1, and 10 nmol/liter) on the expression of the glycerol channels was determined in vitro in human omental adipocytes and HepG2 hepatocytes. The translocation of AQP in response to insulin and isoproterenol was analyzed by immunocytochemistry. In addition to the well-known expression of AQP7 in adipose tissue, AQP3 and AQP9 were also expressed in both omental and sc adipose tissue. Obese type 2 diabetes patients showed higher expression of AQP in visceral adipose tissue and lower expression of AQP7 in sc adipose tissue and hepatic AQP9. The staining of AQP9 in the plasma membrane of adipocytes was reinforced by insulin, whereas isoproterenol induced the translocation of AQP3 and AQP7 from the lipid droplets to the plasma membrane. Insulin up-regulated all AQP, whereas leptin up-regulated AQP3 and down-regulated AQP7 and AQP9 in adipocytes and hepatocytes. These effects were abrogated by both the phosphatidylinositol 3-kinase inhibitor wortmannin and the mammalian target of rapamycin inhibitor rapamycin. Our findings show, for the first time, that insulin and leptin regulate the AQP through the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway in human visceral adipocytes and hepatocytes. AQP3 and AQP7 may facilitate glycerol efflux from adipose tissue while reducing the glycerol influx into hepatocytes via AQP9 to prevent the excessive lipid accumulation and the subsequent aggravation of hyperglycemia in human obesity.

  3. Pro-osteogenic effects of fibrin glue in treatment of avascular necrosis of the femoral head in vivo by hepatocyte growth factor-transgenic mesenchymal stem cells

    PubMed Central

    2014-01-01

    Background Autologous transplantation of modified mesenchymal stem cells (MSCs) is a promising candidate for the treatment of the refractory clinical disease, avascular necrosis of the femoral head (ANFH). Our previous attempts by compounding MSCs with medical fibrin glue to treat ANFH in animal model have achieved excellent effects. However, the underlying molecular mechanism is unclear, especially on the transgenic gene expression. Methods Rabbit MSCs were isolated and compounded with fibrin glue. Following degrading of fibrin glue, proliferation, viability, expression of transgenic hepatocyte growth factor gene as well as osteogenic differentiation of MSCs were evaluated together with that of uncompounded MSCs. Fibrin glue-compounded MSCs were transplanted into the lesion of ANFH model, and the therapeutic efficacy was compared with uncompounded MSCs. One-Way ANOVA was used to determine the statistical significance among treatment groups. Results Fibrin glue compounding will not affect molecular activities of MSCs, including hepatocyte growth factor (HGF) secretion, cell proliferation and viability, and osteogenic differentiation in vitro. When applying fibrin glue-compounded MSCs for the therapy of ANFH in vivo, fibrin glue functioned as a drug delivery system and provided a sustaining microenvironment for MSCs which helped the relatively long-term secretion of HGF in the femoral head lesion and resulted in improved therapeutic efficacy when compared with uncompounded MSCs as indicated by hematoxylin-eosin staining and immunohistochemistry of osteocalcin, CD105 and HGF. Conclusion Transplantation of fibrin glue-compounding MSCs is a promising novel method for ANFH therapy. PMID:24885252

  4. Carnosic acid protects normal mouse hepatocytes against H2 O2 -induced cytotoxicity via sirtuin 1-mediated signaling.

    PubMed

    Wang, Ting; Takikawa, Yasuhiro

    2016-02-01

    Carnosic acid (CA) is well known for its antioxidant properties. The aim of this study was to examine the effects of CA on cytotoxicity under oxidative stress. Primary hepatocytes and AML12 cells were treated with: (i) 0.1 μM, 1 μM and 10 μM CA; (ii) 3 mM H2 O2 with or without 1 μM CA; or (iii) 3 mM H2 O2 with 1 μM CA and 0.04 μM sirtuin 1 (SIRT1) inhibitor EX527 or 10 μM mitogen-activated protein kinase (MAPK) inhibitor U0126. Cell viability, intracellular reactive oxygen species (ROS) and lactate dehydrogenase (LDH) leakage were determined. In addition, total protein levels of cleaved caspase 3, SIRT1, phosphorylated Nrf2, 5'-adenosine monophosphate-activated protein kinase (AMPK) and MAPKs were evaluated by western blot analysis and suspension array system. First, although 10 μM CA produced cytotoxicity, CA at concentrations at or below 1 μM did not inhibit cell viability. Second, H2 O2 increased total cellular ROS and LDH leakage and decreased cell viability, whereas co-treatment with H2 O2 and 1 μM CA significantly inhibited these effects of H2 O2 . Third, CA at 1 μM increased protein levels of SIRT1. Pretreatment with EX527 or transfection of siRNA-targeting SIRT1 weakened the protective effects of CA against H2 O2 -induced cell death. Fourth, H2 O2 induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes. U0126 inhibited oxidative damage induced by H2 O2 . Co-treatment with CA inhibited ERK1/2 activation induced by H2 O2 . Our data indicate that CA protects against oxidative stress-induced cytotoxicity via SIRT1 by regulating subsequent downstream factors such as ERK1/2. © 2015 The Japan Society of Hepatology.

  5. Growth hormone regulates the expression of hepatocyte nuclear factor-3 gamma and other liver-enriched transcription factors in the bovine liver.

    PubMed

    Eleswarapu, S; Jiang, H

    2005-01-01

    Growth hormone (GH) regulates the expression of many genes in the liver, and for some genes this regulation may be mediated through liver-enriched transcription factors (LETFs). As part of the long-term goal to investigate the role of LETFs in GH regulation of gene expression in the liver, in this study we determined the effect of GH administration on the expression of 10 LETFs, including hepatocyte nuclear factor (HNF)-1alpha, HNF-1beta, HNF-3alpha, HNF-3beta, HNF-3gamma, HNF-4alpha, HNF-6, CCAAT/enhancer-binding protein (C/EBP) alpha, C/EBPbeta, and albumin D-element binding protein (DBP) in the bovine liver. Eighteen non-lactating and non-pregnant Angus cows were assigned randomly to three groups (n=6 per group) and each cow received a single intramuscular injection of 500 mg slow-release recombinant bovine GH. Liver biopsy samples were taken from group 1 cows 6 h after GH administration, from group 2 cows 24 h after GH administration, and from group 3 cows 1 week after GH administration. Liver biopsies were also collected from group 3 cows 1 day before GH administration, serving as pre-GH controls. The LETF mRNAs in these liver samples were quantified using ribonuclease protection assays with probes generated from bovine LETF cDNAs cloned by standard reverse transcription-polymerase chain reaction. The levels of HNF-3gamma and HNF-6 mRNAs were higher (P< 0.05) in the cows 24 h and 1 week after GH administration than in the untreated cows or the cows 6 h after GH administration. The levels of HNF-4alpha mRNA were higher (P< 0.05) in the cows 1 week after GH administration than in the other three groups of cows. The levels of C/EBPalpha mRNA were higher (P< 0.05) in the cows 24 h after GH administration than in the untreated cows or the cows 6 h after GH administration. The levels of HNF-3alpha mRNA were higher (P< 0.05) in the cows 6 h after GH administration but were lower (P< 0.05) in the cows 24 h or 1 week after GH administration compared with those in the

  6. Patient-specific hepatocyte-like cells derived from induced pluripotent stem cells model pazopanib-mediated hepatotoxicity

    PubMed Central

    Choudhury, Yukti; Toh, Yi Chin; Xing, Jiangwa; Qu, Yinghua; Poh, Jonathan; Huan, Li; Tan, Hui Shan; Kanesvaran, Ravindran; Yu, Hanry; Tan, Min-Han

    2017-01-01

    Idiosyncratic drug-induced hepatotoxicity is a major cause of liver damage and drug pipeline failure, and is difficult to study as patient-specific features are not readily incorporated in traditional hepatotoxicity testing approaches using population pooled cell sources. Here we demonstrate the use of patient-specific hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells for modeling idiosyncratic hepatotoxicity to pazopanib (PZ), a tyrosine kinase inhibitor drug associated with significant hepatotoxicity of unknown mechanistic basis. In vitro cytotoxicity assays confirmed that HLCs from patients with clinically identified hepatotoxicity were more sensitive to PZ-induced toxicity than other individuals, while a prototype hepatotoxin acetaminophen was similarly toxic to all HLCs studied. Transcriptional analyses showed that PZ induces oxidative stress (OS) in HLCs in general, but in HLCs from susceptible individuals, PZ causes relative disruption of iron metabolism and higher burden of OS. Our study establishes the first patient-specific HLC-based platform for idiosyncratic hepatotoxicity testing, incorporating multiple potential causative factors and permitting the correlation of transcriptomic and cellular responses to clinical phenotypes. Establishment of patient-specific HLCs with clinical phenotypes representing population variations will be valuable for pharmaceutical drug testing. PMID:28120901

  7. Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells

    PubMed Central

    Lee, Patrick C; Truong, Brian; Vega-Crespo, Agustin; Gilmore, W Blake; Hermann, Kip; Angarita, Stephanie AK; Tang, Jonathan K; Chang, Katherine M; Wininger, Austin E; Lam, Alex K; Schoenberg, Benjamen E; Cederbaum, Stephen D; Pyle, April D; Byrne, James A; Lipshutz, Gerald S

    2016-01-01

    Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism. PMID:27898091

  8. 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

  9. (Na,K)-ATPase-mediated cation pumping in cultured rat hepatocytes. Rapid modulation by alanine and taurocholate transport and characterization of its relationship to intracellular sodium concentration.

    PubMed

    Van Dyke, R W; Scharschmidt, B F

    1983-11-10

    (Na,K)-ATPase is thought to maintain the transmembrane electrochemical sodium gradient which powers secondary active sodium-coupled transport of a variety of solutes including amino acids and bile acids. However, little is known regarding the effect of sodium-coupled solute transport on intracellular sodium concentration ( [Na]ic) and on (Na,K)-ATPase-mediated cation pumping in the intact cell. In order to address this question, we have measured 22Na uptake rate, steady state 22Na content, and ouabain-suppressible 86Rb uptake rate in primary cultures of adult rat hepatocytes under a variety of conditions. Compared with control conditions (sodium uptake rate = 6.00 +/- 0.40 nmol X min-1 X mg-1; [Na]ic = 11.96 +/- 0.54 mM; cation pumping = 2.53 +/- 0.18 nmol X min-1 X mg-1), cation pumping was increased by taurocholate (less than or equal to 158%), alanine (less than or equal to 246%), monensin (less than or equal to 400%), and cold exposure (less than or equal to 525%), and this increase was accompanied by increases in Na uptake and [Na]ic. In contrast, preincubation in low sodium medium decreased all three variables. These changes in cation pumping were blocked in the absence of extracellular sodium and were not accompanied by changes in ouabain-suppressible ATP hydrolysis measured in cell homogenate. An overall plot of cation pumping versus [Na]ic yielded a sigmoid-shaped curve. Values for KNa (17.8 +/- 1.4 mM) and Vmax (8.98 +/- 0.62 nmol X min-1 X mg-1) for cation pumping were estimated assuming three sodium sites per pump unit. These findings indicate that: 1) uptake of alanine and taurocholate is associated with a rapid increase in (Na,K)-ATPase cation pumping; 2) this increase probably results from an increase in pumping per pump unit rather than an increase in the total number of pump units, and it appears to be mediated via an increase in sodium influx and [Na]ic; 3) [Na]ic under control conditions is close to the apparent KNa of cation pumping, implying

  10. Hepatocyte proliferation induced by a single dose of a peroxisome proliferator.

    PubMed Central

    Ohmura, T.; Ledda-Columbano, G. M.; Piga, R.; Columbano, A.; Glemba, J.; Katyal, S. L.; Locker, J.; Shinozuka, H.

    1996-01-01

    In compensatory hyperplasia after partial hepatectomy or liver cell injury, hepatocyte proliferation is triggered by coordinated actions of growth factor such as hepatocyte growth factor and transforming growth factor-alpha and -beta. Initiation of hepatocyte DNA synthesis is preceded by the activation of the set of early growth response genes mediated by enhanced nuclear factor-kappa B binding to DNA. Using an experimental model to induce hepatocyte DNA synthesis in vivo by a single dose of a peroxisome proliferator, which does not induce liver cell necrosis (direct hyperplasia), we investigated whether peroxisome proliferator-induced hepatocyte proliferation involved an induction of known growth factors, an activation of early growth response genes, and nuclear factor-kappa B. A single intragastric administration of 250 mg/kg BR931 (4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio-(N-beta-hydroxyethyl) acetamide) to male wistar rats induced a wave of hepatocyte DNA synthesis starting after 12 hours and peaking at approximately 24 to 36 hours. The response was dose dependent. The treatment also induced the expression of the mRNA for the peroxisomal bifunctional enzyme, one of the peroxisome-related fatty acid beta-oxidation enzymes. Pretreatment of rats with dexamethasone (2 mg/kg) inhibited both hepatocyte DNA synthesis and the induction of the peroxisomal bifunctional enzyme gene. Northern blot analyses of liver RNA during a period preceding the onset of DNA synthesis revealed no induction of hepatocyte growth factor, transforming growth factor-alpha, or tumor necrosis factor-alpha mRNAs. No induction of early growth response genes, liver regeneration factor-1, or c-myc was detected. Furthermore, gel mobility shift assays showed no enhanced nuclear factor-kappa B binding to its DNA consensus sequence after BR931 treatment, whereas control studies demonstrated a distinct increase in binding after partial hepatectomy or lead nitrate treatment. The results suggest that

  11. 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

  12. Control of glycolysis in cultured chick embryo hepatocytes. Fructose 2,6-bisphosphate content and phosphofructokinase-1 activity are stimulated by insulin and epidermal growth factor.

    PubMed Central

    Hamer, M J; Dickson, A J

    1990-01-01

    Chick embryo hepatocytes were maintained in monolayer culture in a serum-free chemically defined medium for periods of up to 2 days. Over this time period, insulin provoked selective increases (up to 5-fold) in factors relevant to the control of glycolysis: the activities of phosphofructokinase-1 (PFK-1), phosphofructokinase-2 (PFK-2) and hexokinase isoenzymes and the content of fructose 2,6-bisphosphate (F26BP). Half-maximal effects of insulin on pFK-1 activity were in the physiological range (0.1 nM). Changes in enzyme activities and F26BP content in response to insulin were correlated with stimulation of glycolytic flux as estimated by radioisotopic flux. These data are discussed in relation to known changes which occur in hepatic glycolytic activity and PFK-1 activity in the intact chick around hatching. The effects of insulin on F26BP content, PFK-1 activity and glycolytic flux were mimicked by epidermal growth factor (EGF). In contrast, phorbol esters produced minimal actions on any of the above parameters. Our data indicate that protein kinase C is not involved in the actions of insulin or EGF in control of F26BP content or PFK-1 activity. This work indicates that the related tyrosyl kinase receptors of insulin and EGF may provoke identical responses within hepatocytes, but through the utilization of different transduction systems which merge to common control points. Images Fig. 1 PMID:2143894

  13. Electrochemical sensing of hepatocyte viability.

    PubMed

    Tsai, Hweiyan; Tsai, Shang-heng; Ting, Wei-Jen; Hu, Chao-Chin; Fuh, C Bor

    2014-05-21

    We investigated the use of amperometric and chronoamperometric methods with a double mediator system and screen-printed electrodes (SPEs) for the electrochemical sensing of hepatocyte viability. Cell counts were determined based on measuring cellular respiration via interaction of electroactive redox mediators. The oxidation currents of chronoamperometric measurement were proportional to the concentrations of ferrocyanide which was produced via interaction of cellular respiration, succinate and ferricyanide. The integrated oxidation charges increased linearly with the density of the cultured primary rat hepatocytes over a range of 1 × 10(5) to 5 × 10(5) cells per well (slope = 1.98 (±0.08) μC per 10(5) cells; R(2) = 0.9969), and the detection limit was 7600 (±300) cells per well based on S/N = 3. Each density of cells was cultured in triple replicates and individual cell samples were evaluated. The results of the cytotoxic effect of the chronoamperometric method are comparable to those of the tetrazolium-based colorimetric assay. The chronoamperometric method with ferricyanide and succinate mediators is an efficient, alternative method for assessing the viability of primary hepatocytes which can be completed in 20 min. Succinate did not provide an efficient electron shuttle between cytosolic respiratory redox activity of cancer cells and extracellular ferricyanide, an effect that may be useful for distinguishing hepatocarcinoma cells from healthy hepatocytes.

  14. Adipocyte exosomes induce transforming growth factor beta pathway dysregulation in hepatocytes: a novel paradigm for obesity-related liver disease.

    PubMed

    Koeck, Emily S; Iordanskaia, Tatiana; Sevilla, Samantha; Ferrante, Sarah C; Hubal, Monica J; Freishtat, Robert J; Nadler, Evan P

    2014-12-01

    The pathogenesis of nonalcoholic fatty liver disease (NAFLD) has been attributed to increased systemic inflammation and insulin resistance mediated by visceral adipose tissue (VAT), although the exact mechanisms are undefined. Exosomes are membrane-derived vesicles containing messenger RNA, microRNA, and proteins, which have been implicated in cancer, neurodegenerative, and autoimmune diseases, which we postulated may be involved in obesity-related diseases. We isolated exosomes from VAT, characterized their content, and identified their potential targets. Targets included the transforming growth factor beta (TGF-β) pathway, which has been linked to NAFLD. We hypothesized that adipocyte exosomes would integrate into HepG2 and hepatic stellate cell lines and cause dysregulation of the TGF-β pathway. Exosomes from VAT from obese and lean patients were isolated and fluorescently labeled, then applied to cultured hepatic cell lines. After incubation, culture slides were imaged to detect exosome uptake. In separate experiments, exosomes were applied to cultured cells and incubated 48-h. Gene expression of TGF-β pathway mediators was analyzed by polymerase chain reaction, and compared with cells, which were not exposed to exosomes. Fluorescent-labeled exosomes integrated into both cell types and deposited in a perinuclear distribution. Exosome exposure caused increased tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and integrin ανβ-5 expression and decreased matrix metalloproteinase-7 and plasminogen activator inhibitor-1 expression in to HepG2 cells and increased expression of TIMP-1, TIMP-4, Smad-3, integrins ανβ-5 and ανβ-8, and matrix metalloproteinase-9 in hepatic stellate cells. Exosomes from VAT integrate into liver cells and induce dysregulation of TGF-β pathway members in vitro and offers an intriguing possibility for the pathogenesis of NAFLD. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2015-03-01

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

  16. Hepatocyte differentiation of WIF-B cells includes a high capacity of interleukin-6-mediated induction of alpha 1-acid glycoprotein and alpha 2-macroglobulin.

    PubMed

    Guillonneau, F; Drechou, A; Poüs, C; Chevalier, S; Lardeux, B; Cassio, D; Durand, G

    1999-01-11

    Responsiveness to cytokine-mediated acute inflammatory stimuli of the highly differentiated and polarized WIF-B hybrid cell line was studied by measuring the induction of alpha 1-acid glycoprotein and alpha 2-macroglobulin mRNAs after interleukin-1, interleukin-6 and tumor necrosis factor-alpha treatments in the presence of dexamethasone. Compared with their Fao parent, WIF-B cells were 10 times more responsive to 24-h interleukin-6 induction regarding alpha 2-macroglobulin induction. At variance from the response measured in Fao cells, the late effects of interleukin-6 treatment confirmed the higher sensitivity of WIF-B cells to this cytokine as a 72-h treatment as 10 times more effective than a 24-h treatment at inducting alpha 1-acid glycoprotein mRNA. These findings highlight the hepatocyte differentiation of WIF-B cells compared with other hepatoma cell lines, with respect to the regulation of acute-phase protein gene expression. They also make WIF-B cells a convenient model to study the molecular effects of interleukin-6 in terms of transduction and/or transcription, and the many cross-talks that occur during the regulation of acute-phase protein gene expression.

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

    SciTech Connect

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

    1987-05-01

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

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

    PubMed

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

    2016-05-01

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

  19. In vivo transfer of hepatocyte growth factor gene accelerates proliferation of hepatic oval cells in a 2-acetylaminofluorene/partial hepatectomy model in rats.

    PubMed

    Shiota, G; Kunisada, T; Oyama, K; Udagawa, A; Nomi, T; Tanaka, K; Tsutsumi, A; Isono, M; Nakamura, T; Hamada, H; Sakatani, T; Sell, S; Sato, K; Ito, H; Kawasaki, H

    2000-03-31

    To clarify the effect of hepatocyte growth factor (HGF) on proliferation of hepatic oval cells, we transferred HGF gene into liver of the Solt-Farber rat model. Male Fisher 344 rats were infected with a recombinant adenovirus carrying the cDNA for HGF (pAxCAHGF) from tail vein. HGF mRNA showed its peak at 4 days, and diminished thereafter. The total and proliferating cell nuclear antigen-positive hepatic oval cells were significantly elevated in HGF-transferred rats, in which stem cell factor and c-kit mRNA increased at each time point. Our results suggest that in vivo transfer of the HGF gene into liver accelerates proliferation of hepatic oval cells in the Solt-Farber model in rats.

  20. Development of a rapid semi-quantitative immunochromatographic assay for serum hepatocyte growth factor and its usefulness in acute liver failure.

    PubMed

    Uto, Hirofumi; Ido, Akio; Kusumoto, Kazunori; Hasuike, Satoru; Nagata, Kenji; Hayashi, Katsuhiro; Yamagishi, Toshiya; Gohda, Eiichi; Tsubouchi, Hirohito

    2005-12-01

    Measurement of serum human hepatocyte growth factor (HGF) by enzyme-linked immunosorbent assay (ELISA) is useful for the early diagnosis and prediction of prognosis of patients with acute liver failure (ALF). This ELISA methodology, however, is neither rapid nor convenient for use at the bedside. In this study, we have developed a rapid semi-quantitative immunochromatographic (IC) assay and evaluated its usefulness in assessing patients with acute hepatic injury. Only 100mul of serum is required; the assay can be easily completed in 20min. The values obtained using this novel assay correlated well with the values obtained using the standard ELISA protocol. In addition, the values obtained in the IC assay correlated with clinical course; increased serum HGF levels were associated with an increased frequency of ALF and death. These results indicate that this rapid semi-quantitative IC assay for HGF is useful for the early diagnosis of ALF and prediction of clinical outcome in acute hepatic injury.

  1. 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.

  2. In vivo cell lineage analysis during chemical hepatocarcinogenesis in rats using retroviral-mediated gene transfer: evidence for dedifferentiation of mature hepatocytes.

    PubMed

    Gournay, Jérôme; Auvigne, Isabelle; Pichard, Virginie; Ligeza, Catherine; Bralet, Marie-Pierre; Ferry, Nicolas

    2002-06-01

    Feeding adult rats with a diet containing 2-acetylaminofluorene (2-AAF) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Although oval cells may be facultative liver stem cells, the actual relationship between oval cells and liver cancer has not been clearly established in vivo. Our goal was to label hepatic cells in vivo using retroviral vectors and follow their fate during the early steps of chemically induced hepatocarcinogenesis. Oval cell proliferation was induced by continuous feeding with a carcinogenic diet containing 2-AAF. We used two different strategies to genetically label hepatic cells: (a) labeling of proliferating cells in rats fed 2-AAF by injecting recombinant retroviral vectors containing the beta-galactosidase gene either in a peripheral vein or in the common bile duct at the peak of oval cell proliferation and (b) prelabeling of hepatocytes by intravenously injecting recombinant vectors 1 day after partial hepatectomy and 1 week before subsequent administration of 2-AAF. Using the first strategy, transgene expression occurred in both oval cells and hepatocytes. Using the second strategy, we could selectively label, and hence study the fate of, differentiated hepatocytes. In the latter case, we observed clusters of beta-galactosidase-positive hepatocytes, some of them also expressing preneoplastic markers such as gamma-glutamyl transpeptidase as well as the placental form of glutathione-S-transferase. These results demonstrate that preneoplastic foci can originate from mature hepatocytes and are consistent with the hypothesis that dedifferentiation of mature hepatocytes may occur during the course of carcinogenic regimen.

  3. 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.

  4. Regulation of growth hormone (GH) receptor (GHR1 and GHR2) mRNA level by GH and metabolic hormones in primary cultured tilapia hepatocytes.

    PubMed

    Pierce, A L; Breves, J P; Moriyama, S; Uchida, K; Grau, E G

    2012-10-01

    Growth hormone (GH) regulates essential physiological functions in teleost fishes, including growth, metabolism, and osmoregulation. Recent studies have identified two clades of putative receptors for GH (GHR1 clade and GHR2 clade) in fishes, both of which are highly expressed in the liver. Moreover, the liver is an important target for the anabolic effects of GH via endocrine IGFs, and liver sensitivity to GH is modulated by metabolic hormones. We investigated the effects of GH, insulin, glucagon, cortisol and triiodothyronine on GHR1 and GHR2 mRNA levels in primary cultured tilapia hepatocytes. Physiological concentrations of GH strongly stimulated GHR2 mRNA level (0.5-50×10(-9) M), but did not affect GHR1 mRNA level. Insulin suppressed stimulation of GHR2 mRNA level by GH (10(-8)-10(-6) M). Insulin increased basal GHR1 mRNA level (10(-8)-10(-6) M). Cortisol increased basal GHR2 mRNA level (10(-7)-10(-6) M), but did not consistently affect GH-stimulated GHR2 mRNA level. Cortisol increased basal GHR1 mRNA level (10(-9)-10(-6) M). Glucagon suppressed GH-stimulated GHR2 mRNA level and increased basal GHR1 mRNA level at a supraphysiological concentration (10(-6) M). A single injection of GH (5 μg/g) increased liver GHR2 mRNA level, and insulin injection (5 μg/g) decreased both basal and GH-stimulated GHR2 mRNA levels after 6 h. In contrast, insulin and GH injection had little effect on liver GHR1 mRNA level. This study shows that GHR1 and GHR2 gene expression are differentially regulated by physiological levels of GH and insulin in tilapia primary hepatocytes. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Insulin-like growth factor 1: common mediator of multiple enterotrophic hormones and growth factors

    PubMed Central

    Bortvedt, Sarah F.; Lund, P. Kay

    2013-01-01

    Purpose of review To summarize recent evidence that IGF1 mediates growth effects of multiple trophic factors and discuss clinical relevance. Recent findings Recent reviews and original reports indicate benefits of growth hormone (GH) and long-acting glucagon-like peptide 2 (GLP2) analogues in short bowel syndrome and Crohn’s disease. This review highlights evidence that biomarkers of sustained small intestinal growth or mucosal healing and evaluation of intestinal epithelial stem cell biomarkers may improve clinical measures of intestinal growth or response to trophic hormones. Compelling evidence that IGF1 mediates growth effects of GH and GLP2 on intestine or linear growth in preclinical models of resection or Crohn’s disease is presented, along with a concept that these hormones or IGF1 may enhance sustained growth if given early after bowel resection. Evidence that SOCS protein induction by GH or GLP2 in normal or inflamed intestine, may limit IGF1-induced growth, but protect against risk of dysplasia or fibrosis is reviewed. Whether IGF1 receptor mediates IGF1 action and potential roles of insulin receptors are addressed. Summary IGF1 has a central role in mediating trophic hormone action in small intestine. Better understanding of benefits and risks of IGF1, receptors that mediate IGF1 action, and factors that limit undesirable growth are needed. PMID:22241077

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

    PubMed Central

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

    2015-01-01

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

  7. 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.

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

    PubMed

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

    2015-01-01

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

  9. 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

  10. 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

  11. D-Glucose uptake in fish hepatocytes: mediated by transporter in rainbow trout (Oncorhynchus mykiss), but only by diffusion in prespawning lamprey (Lampetra fluviatilis) and in RTH-149 cell line.

    PubMed

    Mannerström, Marika; Tähti, Hanna; Tiihonen, Kirsti; Salama, Annika

    2003-11-01

    The transport of D-glucose into rainbow trout (Oncorhynchus mykiss) and river lamprey (Lampetra fluviatilis) hepatocytes, as well as into rainbow trout hepatoblastoma cell line RTH-149 was studied using tracer methods. The half-time for D-glucose equilibration was 15 s for rainbow trout. The half-times for the non-metabolizable D-glucose analog, 3-O-methyl-D-glucose equilibration were 8 s, 37 s and 38 s for rainbow trout, lamprey and RTH-149 cells, respectively. The 3-O-methyl-D-glucose was taken up by rainbow trout hepatocytes by facilitated diffusion in addition to simple diffusion. The uptake showed saturation kinetics with the K(m) of 37 mM and V(max) of 62 mmol kg(-1) cells min(-1). The uptake was sensitive to phloretin and cytochalasin B, but not affected by ouabain. The 3-O-methyl-D-glucose uptake by lamprey hepatocytes and RTH-149 cells showed no indication of saturation up to 160 mM, and was not affected by phloretin, cytochalasin B or ouabain, which suggests the mode of transport to be by passive diffusion. However, immunocytochemical stainings revealed the existence of mammalian type GLUT1 and GLUT2 transporters in all cells studied. The lack of a functioning carrier-mediated glucose uptake in lamprey hepatocytes might be due to its physiological state (prespawning starvation). The minor 3-O-methyl-D-glucose uptake into RTH-149 cells compared to freshly isolated rainbow trout hepatocytes might reflect low metabolic activity of the cell lines. Under the conditions applied the RTH-149 cell line is no suitable in vitro model for glucose transport in fish cells.

  12. 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.

  13. 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

  14. 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…

  15. 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…

  16. Common carp (Cyprinus carpio) insulin-like growth factor binding protein-2 (IGFBP-2): molecular cloning, expression profiles, and hormonal regulation in hepatocytes.

    PubMed

    Chen, Wenbo; Li, Wensheng; Lin, Haoran

    2009-05-01

    In the present study, we cloned IGFBP-2 cDNA from common carp (Cyprinus carpio) liver. The 1879 bp full-length cDNA encodes 274 amino acid residues containing a putative signal peptide of 22 residues. Two IGFBP-2 transcripts with estimated sizes of 2.2 and 1.5 kb have been detected with Northern blot analysis in liver. Relatively high levels of IGFBP-2 mRNA were observed in all regions of brain, liver, pituitary, ovary and testis. Intermediate levels were observed in white muscle, thymus gland and head kidney, while in retina, heart and other tissues IGFBP-2 mRNA levels were very low. A significant level of IGFBP-2 mRNA was firstly detected at lens formation stage, and it continued to increase to the highest level at blood cycling stage, and fell to a relatively high level until hatching. The expression pattern of IGFBP-2 mRNA was similar during different stages of testis and ovary. At recrudescing stage the expression level was extremely low, but it sharply increased to a high level at matured stage, and finally brought back to the very low level at regressed stage. Hepatocytes IGFBP-2 mRNA was greatly reduced by growth hormone but increased by insulin. PD-98059 and LY-294002, the specific inhibitor of MEK and PI3K, increased IGFBP-2 mRNA expression level and completely blocked the inhibitory effect of GH. It is suggested that the MAPK and PI3 kinase-signaling pathways were involved in the decrease of IGFBP-2 mRNA expression induced by GH in primary cultured hepatocytes.

  17. Early exposure to ethanol or red wine and long-lasting effects in aged mice. A study on nerve growth factor, brain-derived neurotrophic factor, hepatocyte growth factor, and vascular endothelial growth factor.

    PubMed

    Ceccanti, Mauro; Mancinelli, Rosanna; Tirassa, Paola; Laviola, Giovanni; Rossi, Simona; Romeo, Marina; Fiore, Marco

    2012-02-01

    Prenatal ethanol exposure produces severe changes in brain, liver, and kidney through mechanisms involving growth factors. These molecules regulate survival, differentiation, maintenance, and connectivity of brain, liver, and kidney cells. Despite the abundant available data on the short and mid-lasting effects of ethanol intoxication, only few data show the long-lasting damage induced by early ethanol administration. The aim of this study was to investigate changes in nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) in brain areas, liver, and kidney of 18-mo-old male mice exposed perinatally to ethanol at 11% vol or to red wine at the same ethanol concentration. The authors found that ethanol per se elevated NGF, BDNF, HGF, and VEGF measured by ELISA in brain limbic system areas. In the liver, early exposure to ethanol solution and red wine depleted BDNF and VEGF concentrations. In the kidney, red wine exposure only decreased VEGF. In conclusion, the present study shows that, in aged mice, early administration of ethanol solution induced long-lasting damage at growth factor levels in frontal cortex, hippocampus, and liver but not in kidney. Otherwise, in mice exposed to red wine, significant changes were observed in the liver and kidney but not in the hippocampus and frontal cortex. The brain differences in ethanol-induced toxicity when ethanol is administered alone or in red wine may be related to compounds with antioxidant properties present in the red wine. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  18. Clinical significance of hepatocyte growth factor, platelet-derived growth factor-AB, and transforming growth factor-alpha in bone marrow and peripheral blood of patients with multiple myeloma.

    PubMed

    Kara, Ismail Oguz; Sahin, Berksoy; Gunesacar, Ramazan; Unsal, Cagatay

    2006-01-01

    Angiogenesis is a process that plays an important role in the growth and progression of cancer; growing evidence suggests that neovascularization is important in hematologic malignancies. Increased angiogenic potential has been identified in multiple myeloma (MM). In this study, investigators simultaneously measured the levels of hepatocyte growth factor (HGF), platelet-derived growth factor-AB (PDGFAB), and transforming growth factor-alpha (TGF-alpha) through enzyme-linked immunosorbent assay in the bone marrow (BM) and peripheral blood (PB) of 30 patients with MM and 10 healthy controls. Differences in HGF values in BM sera were significant (P=.001) between patients and controls. In detailed analyses of HGF, PDGF-AB, and TGF-alpha, according to disease stage, a significant correlation was found between disease stage and BM HGF (P=.047), BM TGF-alpha (P=.021), and PB PDGF-AB (P=.006), respectively. When correlations between all other parameters were analyzed, significance was noted between PB TGF-alpha and lactate dehydrogenase (P=.02), PB TGF-alpha and PB HGF (P=.002), BM TGF-alpha and CD38 (P=.046), BM TGF-alpha and BM HGF (P=.000), BM TGF-alpha and BM PDGF-AB (P=.048), BM HGF and PB HGF (P=.044), and BM PDGF-AB and PB PDGF-AB (P=.000). BM HGF levels had a significant effect on overall survival, with disease severity assessed in terms of disease stage (P=.0018, log-rank test). These data show that in patients with MM, high levels of BM HGF, BM TGF-alpha, and PB PDGF-AB were associated with advanced disease stage; in addition, HGF played a significant role in disease processing and was related to disease severity. These findings have also led to the concept of a symbiotic relationship between the growth of myeloma cells and HGF, TGF-alpha, and PDGFAB in BM.

  19. 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

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

    PubMed

    Doricakova, Aneta; Vrzal, Radim

    2015-11-04

    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.

  1. 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

  2. Fibroblast growth factor 21 mediates specific glucagon actions.

    PubMed

    Habegger, Kirk M; Stemmer, Kerstin; Cheng, Christine; Müller, Timo D; Heppner, Kristy M; Ottaway, Nickki; Holland, Jenna; Hembree, Jazzminn L; Smiley, David; Gelfanov, Vasily; Krishna, Radha; Arafat, Ayman M; Konkar, Anish; Belli, Sara; Kapps, Martin; Woods, Stephen C; Hofmann, Susanna M; D'Alessio, David; Pfluger, Paul T; Perez-Tilve, Diego; Seeley, Randy J; Konishi, Morichika; Itoh, Nobuyujki; Kharitonenkov, Alexei; Spranger, Joachim; DiMarchi, Richard D; Tschöp, Matthias H

    2013-05-01

    Glucagon, an essential regulator of glucose homeostasis, also modulates lipid metabolism and promotes weight loss, as reflected by the wasting observed in glucagonoma patients. Recently, coagonist peptides that include glucagon agonism have emerged as promising therapeutic candidates for the treatment of obesity and diabetes. We developed a novel stable and soluble glucagon receptor (GcgR) agonist, which allowed for in vivo dissection of glucagon action. As expected, chronic GcgR agonism in mice resulted in hyperglycemia and lower body fat and plasma cholesterol. Notably, GcgR activation also raised hepatic expression and circulating levels of fibroblast growth factor 21 (FGF21). This effect was retained in isolated primary hepatocytes from wild-type (WT) mice, but not GcgR knockout mice. We confirmed this link in healthy human volunteers, where injection of natural glucagon increased plasma FGF21 within hours. Functional relevance was evidenced in mice with genetic deletion of FGF21, where GcgR activation failed to induce the body weight loss and lipid metabolism changes observed in WT mice. Taken together, these data reveal for the first time that glucagon controls glucose, energy, and lipid metabolism at least in part via FGF21-dependent pathways.

  3. Molecular link between auxin and ROS-mediated polar growth.

    PubMed

    Mangano, Silvina; Denita-Juarez, Silvina Paola; Choi, Hee-Seung; Marzol, Eliana; Hwang, Youra; Ranocha, Philippe; Velasquez, Silvia Melina; Borassi, Cecilia; Barberini, María Laura; Aptekmann, Ariel Alejandro; Muschietti, Jorge Prometeo; Nadra, Alejandro Daniel; Dunand, Christophe; Cho, Hyung-Taeg; Estevez, José Manuel

    2017-05-16

    Root hair polar growth is endogenously controlled by auxin and sustained by oscillating levels of reactive oxygen species (ROS). These cells extend several hundred-fold their original size toward signals important for plant survival. Although their final cell size is of fundamental importance, the molecular mechanisms that control it remain largely unknown. Here we show that ROS production is controlled by the transcription factor RSL4, which in turn is transcriptionally regulated by auxin through several auxin response factors (ARFs). In this manner, auxin controls ROS-mediated polar growth by activating RSL4, which then up-regulates the expression of genes encoding NADPH oxidases (also known as RESPIRATORY BURST OXIDASE HOMOLOG proteins) and class III peroxidases, which catalyze ROS production. Chemical or genetic interference with ROS balance or peroxidase activity affects root hair final cell size. Overall, our findings establish a molecular link between auxin and ROS-mediated polar root hair growth.

  4. Actin growth profile in clathrin-mediated endocytosis

    NASA Astrophysics Data System (ADS)

    Tweten, D. J.; Bayly, P. V.; Carlsson, A. E.

    2017-05-01

    Clathrin-mediated endocytosis in yeast is driven by a protein patch containing close to 100 different types of proteins. Among the proteins are 5000 -10 000 copies of polymerized actin, and successful endocytosis requires growth of the actin network. Since it is not known exactly how actin network growth drives endocytosis, we calculate the spatial distribution of actin growth required to generate the force that drives the process. First, we establish the force distribution that must be supplied by actin growth, by combining membrane-bending profiles obtained via electron microscopy with established theories of membrane mechanics. Next, we determine the profile of actin growth, using a continuum mechanics approach and an iterative procedure starting with an actin growth profile obtained from a linear analysis. The profile has fairly constant growth outside a central hole of radius 45-50 nm, but very little growth in this hole. This growth profile can reproduce the required forces if the actin shear modulus exceeds 80 kPa, and the growing filaments can exert very large polymerization forces. The growth profile prediction could be tested via electron-microscopy or super-resolution experiments in which the turgor pressure is suddenly turned off.

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed

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

    2015-08-19

    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 (Met(fl/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, Met(fl/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 Met(fl/fl); Wnt1Cre+ mice. Finally, Met(fl/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. 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 understood. We show that

  8. Evidence for an LKB1/AMPK/eNOS Cascade Regulated by HGF, S-Adenosylmethionine and NO in Hepatocyte Proliferation

    PubMed Central

    Vázquez, Mercedes; Ariz, Usue; Varela-Rey, Marta; Embade, Nieves; Martínez, Nuria; Fernández, David; Gómez, Laura; Lamas, Santiago; Lu, Shelly C; Martínez-Chantar, M Luz; Mato, José M

    2008-01-01

    S-Adenosylmethionine (SAMe) is involved in numerous complex hepatic processes such as hepatocyte proliferation, death, inflammatory responses, and anti-oxidant defense. One of the most relevant actions of SAMe is the inhibition of hepatocyte proliferation during liver regeneration. In hepatocytes, SAMe regulates the levels of cytoplasmic HuR, an RNA-binding protein that increases the half-life of target mRNA such as cyclin D1 and A2, via inhibition of HGF-mediated AMP-activated protein kinase (AMPK) phosphorylation. Because AMPK is activated by the tumor suppressor kinase LKB1, and AMPK activates endothelial nitric oxide (NO) synthase (eNOS), and NO synthesis is of great importance for hepatocyte proliferation, we hypothesized that in hepatocytes HGF may induce the phosphorylation of LKB1, AMPK and eNOS through a process regulated by SAMe, and that this cascade might be crucial for hepatocyte growth. Here we demonstrate that the proliferative response of hepatocytes involves eNOS phosphorylation via HGF-mediated LKB1 and AMPK phosphorylation, and that this process is regulated by SAMe and NO. We also show that knockdown of LKB1, AMPK, or eNOS with specific iRNA inhibits HGF-mediated hepatocyte proliferation. Finally, we found that the LKB1/AMPK/eNOS cascade is activated during liver regeneration after partial hepatectomy and that this process is impaired in mice treated with SAMe before hepatectomy, in knockout mice deficient in hepatic SAMe, and in eNOS knockout mice. Conclusion We have identified an LKB1/AMPK/eNOS cascade regulated by HGF, SAMe and NO that functions as a critical determinant of hepatocyte proliferation during liver regeneration after partial hepatectomy. PMID:19177591

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

    PubMed

    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

    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. 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. Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion.

  10. 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

  11. Endothelial Signals Modulate Hepatocyte Apicobasal Polarization in Zebrafish

    PubMed Central

    Sakaguchi, Takuya F.; Sadler, Kirsten C.; Crosnier, Cecile; Stainier, Didier Y.R.

    2009-01-01

    Summary Emerging evidence indicates that paracrine signals from endothelial cells play a role in tissue differentiation and organ formation [1–3]. Here, we identify a novel role for endothelial cells in modulating hepatocyte polarization during liver organogenesis. We find that in zebrafish, the apical domain of the hepatocytes predicts the location of the intrahepatic biliary network. The refinement of hepatocyte polarization coincides with the invasion of endothelial cells into the liver, and these endothelial cells migrate along the maturing basal surface of the hepatocytes. Using genetic, pharmacological, and transplantation experiments, we provide evidence that endothelial cells influence the polarization of the adjacent hepatocytes. This influence of endothelial cells on hepatocytes is mediated at least in part by the cell-surface protein Heart of glass and contributes to the establishment of coordinately aligned hepatocyte apical membranes and evenly spaced intrahepatic conduits. PMID:18951027

  12. 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.

  13. Hepatocyte growth factor fusion protein having collagen-binding activity (CBD-HGF) accelerates re-endothelialization and intimal hyperplasia in balloon-injured rat carotid artery.

    PubMed

    Ohkawara, Nana; Ueda, Hiroki; Shinozaki, Shohei; Kitajima, Takashi; Ito, Yoshihiro; Asaoka, Hiroshi; Kawakami, Akio; Kaneko, Eiji; Shimokado, Kentaro

    2007-08-01

    Hepatocyte growth factor (HGF) is known to stimulate endothelial cell proliferation. However, re-endothelialization is not enhanced when the native protein is administered to the injured artery, probably due to the short half-life of HGF at the site of injury. Therefore, the effects of an HGF fusion protein having collagen-binding activity (CBD-HGF) on re-endothelialization and neointimal formation was studied in the balloon-injured rat carotid artery. The left common carotid artery of male Sprague-Dawley rats was injured with an inflated balloon catheter, and then treated with CBD-HGF 10 microg/mL), HGF (10 micro g/mL) or saline (control) for 15 min. After 14 days, the rats were injected with Evans blue and sacrificed. The re-endothelialized area was significantly greater in the CBD-HGF- treated rats than in the control or HGF -treated rats. Neointimal formation was significantly more pronounced in the CBD-HGF treated rats than in other rat groups. Both HGF and CBD-HGF stimulated proliferation of vascular smooth muscle cells as well as endothelial cells in vitro. Consistent with this, cultured smooth muscle cells were shown to express the HGF receptor (c-Met). CBD-HGF accelerates re-endothelialization and neointimal formation in vivo. CBD fusion protein is a useful vehicle to deliver vascular growth factors to injured arteries.

  14. Hepatic stellate cell-targeted delivery of hepatocyte growth factor transgene via bile duct infusion enhances its expression at fibrotic foci to regress dimethylnitrosamine-induced liver fibrosis.

    PubMed

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

    2013-05-01

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

  15. Hepatocyte growth factor promoting the proliferation of human eccrine sweat gland epithelial cells is relative to AKT signal channel and β-catenin.

    PubMed

    Lei, Xia; Wu, Jinjin; Liu, Bo; Lu, Yuangang

    2012-01-01

    Hepatocyte growth factor (HGF) is a multi-effective molecule, playing important roles in organ growth, tumorigenesis and trauma healing. This experiment aims at studying the promoting function of HGF on the proliferation of human eccrine sweat gland epithelial cells (hESGc) and its relative signal channels. After HGF at different concentrations were added into cells, MTT was adopted to detect the cell proliferations, Annexin-V/PI the cell apoptosis, and Westernblot the expressions of p-AKT, AKT, p-ERK, p-GSK3β, p-IKBα, and β-catenin in hESGc. After adding siRNA c-Met to block HGF or LY294002 to inhibit p-AKT, we used MTT to detect the proliferation of hESGc and Westernblot to detect the expression of β-catenin. As a result, 20-40 ng/mL HGF could promote the proliferation of hESGc and inhibit its apoptosis. HGF could promote the expressions of p-AKT1/2/3, p-ERK, p-GSK3β, p-IKBα, and β-catenin. The additions of siRNA c-Met to block HGF or LY294002 to inhibit p-AKT could downregulate β-catenin and inhibit the proliferation promotion caused by HGF. Consequently, we concluded HGF can promote the proliferation of human eccrine sweat gland epithelial cells, which is relative to AKT signal channel and β-catenin.

  16. Prostaglandin E2 promotes hepatic bile acid synthesis by an E prostanoid receptor 3-mediated hepatocyte nuclear receptor 4α/cholesterol 7α-hydroxylase pathway in mice.

    PubMed

    Yan, Shuai; Tang, Juan; Zhang, Yuyao; Wang, Yuanyang; Zuo, Shengkai; Shen, Yujun; Zhang, Qianqian; Chen, Di; Yu, Yu; Wang, Kai; Duan, Sheng-Zhong; Yu, Ying

    2017-03-01

    Prostaglandin E2 (PGE2 ) is an important lipid mediator of inflammation. However, whether and how PGE2 regulates hepatic cholesterol metabolism remains unknown. We found that expression of the PGE2 receptor, E prostanoid receptor 3 (EP3) expression is remarkably increased in hepatocytes in response to hyperlipidemic stress. Hepatocyte-specific deletion of EP3 receptor (EP3(hep-/-) ) results in hypercholesterolemia and augments diet-induced atherosclerosis in low-density lipoprotein receptor knockout (Ldlr(-/-) ) mice. Cholesterol 7α-hydroxylase (CYP7A1) is down-regulated in livers of EP3(hep-/-) Ldlr(-/-) mice, leading to suppressed hepatic bile acid (BA) biosynthesis. Mechanistically, hepatic-EP3 deficiency suppresses CYP7A1 expression by elevating protein kinase A (PKA)-dependent Ser143 phosphorylation of hepatocyte nuclear receptor 4α (HNF4α). Disruption of the PKA-HNF4α interaction and BA sequestration rescue impaired BA excretion and ameliorated atherosclerosis in EP3(hep-/-) Ldlr(-/-) mice.

  17. 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

  18. 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

  19. TEAD mediates YAP-dependent gene induction and growth control.

    PubMed

    Zhao, Bin; Ye, Xin; Yu, Jindan; Li, Li; Li, Weiquan; Li, Siming; Yu, Jianjun; Lin, Jiandie D; Wang, Cun-Yu; Chinnaiyan, Arul M; Lai, Zhi-Chun; Guan, Kun-Liang

    2008-07-15

    The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. Here we demonstrate that the TEAD family transcription factors are essential in mediating YAP-dependent gene expression. TEAD is also required for YAP-induced cell growth, oncogenic transformation, and epithelial-mesenchymal transition. CTGF is identified as a direct YAP target gene important for cell growth. Moreover, the functional relationship between YAP and TEAD is conserved in Drosophila Yki (the YAP homolog) and Scalloped (the TEAD homolog). Our study reveals TEAD as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP.

  20. TEAD mediates YAP-dependent gene induction and growth control

    PubMed Central

    Zhao, Bin; Ye, Xin; Yu, Jindan; Li, Li; Li, Weiquan; Li, Siming; Yu, Jianjun; Lin, Jiandie D.; Wang, Cun-Yu; Chinnaiyan, Arul M.; Lai, Zhi-Chun; Guan, Kun-Liang

    2008-01-01

    The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. Here we demonstrate that the TEAD family transcription factors are essential in mediating YAP-dependent gene expression. TEAD is also required for YAP-induced cell growth, oncogenic transformation, and epithelial–mesenchymal transition. CTGF is identified as a direct YAP target gene important for cell growth. Moreover, the functional relationship between YAP and TEAD is conserved in Drosophila Yki (the YAP homolog) and Scalloped (the TEAD homolog). Our study reveals TEAD as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP. PMID:18579750

  1. Growth factors as mediators of exercise actions on the brain.

    PubMed

    Llorens-Martín, M; Torres-Alemán, I; Trejo, José L

    2008-01-01

    Physical exercise has long been recognized as highly beneficial for brain and body health. The molecular mechanisms responsible for translation of exercise stimuli in the brain have claimed attention due to mounting evidence for the neuroprotective actions of the exercise and its positive effects in preventing both ageing and neurodegenerative disease. These molecular mediators are currently under investigation with new tools able to yield deep insights into the neurobiology of exercise. In the present work we focus on the evidence pertaining to the mediation of exercise effects by insulin-like growth factor 1 (IGF1), as recent reports suggest that this growth factor shows brain area-specific, temporal rank-sensitive, and behavioural task-dependent features in response to exercise.

  2. Ah receptor- and TCDD-mediated liver tumor promotion: clonal selection and expansion of cells evading growth arrest and apoptosis.

    PubMed

    Bock, Karl Walter; Köhle, Christoph

    2005-05-15

    The Ah receptor (AhR) has been characterized as a ligand-activated transcription factor which belongs to the bHLH/PAS (basic helix-loop-helix/Per-Arnt-Sim) family of chemosensors. Transgenic mouse models revealed adaptive and developmental functions of the AhR in the absence of exogenous ligands. Use of persistent agonists such as dioxins including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds demonstrated that the AhR mediates a plethora of species- and tissue-dependent toxicities, including chloracne, wasting, teratogenicity, immunotoxicity, liver tumor promotion and carcinogenicity. However, molecular mechanisms underlying most aspects of these toxic responses as well as biological functions of the AhR are currently unknown. Previous studies of liver tumor promotion in the two-stage hepatocarcinogenesis model indicated that TCDD mediates clonal expansion of 'initiated' preneoplastic hepatocytes, identified as enzyme-altered foci (EAF) by inhibiting apoptosis and bypassing AhR-mediated growth arrest. In contrast, the Ah receptor has been shown in cell models to stimulate growth arrest and apoptosis. Possible underlying mechanisms of these AhR responses are discussed, including enhanced metabolism of retinoic acid which attenuates TGFbeta-mediated apoptosis and interaction of the Ah receptor with the hypophosphorylated retinoblastoma tumor suppressor protein. The discrepancy between in vivo findings in EAF and AhR functions may be solved by hypothesizing that sustained activation of the Ah receptor generates a strong selective pressure in liver treated with genotoxic carcinogens leading to selection and expansion of clones evading growth arrest and apoptosis. Models are discussed which may facilitate verification of this hypothesis.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

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

    PubMed Central

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

    2015-01-01

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

  10. Hepatocyte growth factor induces glucose uptake in 3T3-L1 adipocytes through A Gab1/phosphatidylinositol 3-kinase/Glut4 pathway.

    PubMed

    Bertola, Adeline; Bonnafous, Stéphanie; Cormont, Mireille; Anty, Rodolphe; Tanti, Jean-François; Tran, Albert; Le Marchand-Brustel, Yannick; Gual, Philippe

    2007-04-06

    Adipose tissue is a source of hepatocyte growth factor (HGF), and circulating HGF levels have been associated with elevated body mass index in human. However, the effects of HGF on adipocyte functions have not yet been investigated. We show here that in 3T3-L1 adipocytes HGF stimulates the phosphatidylinositol (PI) 3-kinase-dependent protein kinase B (PKB) activity, AS160 phosphorylation, Glut4 translocation, and consequently, glucose uptake. The initial steps involved in HGF- and insulin-induced glucose uptake are different. HGF enhanced the tyrosine phosphorylation of Gab1, leading to the recruitment of the p85-regulated subunit of PI 3-kinase, whereas p85 was exclusively recruited by IRS1 in response to insulin. In adipocytes rendered insulin-resistant by a long-lasting tumor necrosis factor alpha treatment, the protein level of Gab1 was strongly decreased, and HGF-stimulated PKB activation and glucose uptake were also altered. Moreover, treatment of 3T3-L1 adipocytes with thiazolidinedione, an anti-diabetic drug, enhanced the expression of both HGF and its receptor. These data provide the first evidence that in vitro HGF promotes glucose uptake through a Gab1/PI 3-kinase/PKB/AS160 pathway which was altered in tumor necrosis factor alpha-treated adipocytes.

  11. 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.

  12. Modulation of hepatocyte growth factor plasma levels in relation to the dose of exogenous heparin administered: an experimental study in rats.

    PubMed

    Moreno, E; Meneu, J C; Calvo, J; Pérez, B; Sesma, A G; Manrique, A; Vegh, I; Aragón, A M; Grau, M; Gimeno, A; Jiménez, C; Gómez, R; Moreno, A; Abradelo, M; García, I; de la Calle, A

    2005-11-01

    Partial liver transplantation has been consolidated to be a valid treatment option. We sought to understand the factors that modulate and may be harnessed to accelerate hepatocyte regeneration. We sought to determine the impact of heparin on m-hepatocyte growth factor (HGF) plasma concentrations. Sixteen rats were assigned to four groups of four animals each: group A, without heparin; group B, 600 IU/kg; group C, 1000 IU/kg, group D, 1400 IU/kg. Blood samples (0.5 mL) were obtained from each rat at baseline and at 30, 60, 120, and 240 minutes. After the samples were centrifuged to separate supernates from the cell phase they were stored at -20 degrees C in the m-HGF reagent and subsequently tested using enzyme-linked immunosorbent assay. Results were analyzed using SPSS 11.5 statistical software. Among the 16 rats, one died at 110 minutes, just prior to the last extraction. The remaining rats were sacrificed. Mean weight was: 466 +/- 64.24 g with no intergroup differences (P = .149). The comparative results (using Student t test) were: baseline A(1-4) versus A(1-4) 30 minutes: P < .05; baseline A(1-4) versus A(1-4) 60 minutes: P < .05; baseline A(1-4) versus A(1-4) 120 minutes: P = .10 (NS); baseline A(1-4) versus A(1-4) 240 minutes: P = .15 (NS). No significant differences were fou