Migration Of Hepatocytes Research Articles

Magnetotactic hepatocytes promote liver repopulation after transplantation

Clinical application of hepatocyte transplantation has been halted due to the low engraftment efficiency and limited proliferation capacity of donor hepatocytes in the host liver. Here, we developed a magneto-plasmonic nanohybrid to spatiotemporally drive the primary hepatocytes into the host liver under the external magnetic field that significantly improves the engraftment efficiency of donor hepatocytes. A magnetic lipid-coated-gold-nanorods/iron-oxide nanocluster (GNR-IO) (LGI) was designed for efficient magneto-actuation and photoacoustic imaging-guided cell tracking during the hepatocyte transplantation. By optimizing the concentration of LGI (100 μg /mL) and the configuration of a magnetic field (260 mT), we were able to increase the engraftment efficiency of primary hepatocytes into the host livers of Fah-/- mice, a model with the genetic deficit of fumarylacetoacetate hydrolase (FAH) resulting in liver injuries, for 3.89-fold of the control by one week of the transplantation. Further assessment of repopulation of hepatocytes in the host liver indicated the LGI (100 μg /mL) + magnetic field (260 mT) group had up to 15 % repopulation of the host liver by three-weeks-post-transplantation and reached full repopulation by 6-weeks-post-transplantation. This was 3 ∼ 5 times more rapid than the Fah-/- mice transplanted with hepatocytes without the LGI and the magnetic field. RNA-sequencing of the hepatocytes incubated with LGI indicated that the loading of LGIs enhanced the migration, survival, and engraftment of hepatocytes by regulating the tumor necrosis factor (TNF)-signaling pathway and reducing the macrophage in host liver. Furthermore, LGI-labeled hepatocytes could be tracked in host livers with photoacoustic imaging for at least one week post transplantation. Most of the LGI were cleared from the host systems via the kidneys by six weeks. The novel strategy established in this study, which utilized nanoparticles combined with the magnetic field to improve the efficiency of primary hepatocyte engraftment and repopulation, can provide a feasible scheme for the clinical application of hepatocyte transplantation in the treatment of liver failure.

Effect of curcumin on malignant hepatocytes and mitochondria studied using atomic force microscopy

Mitochondria are emerging as potential targets for the cancer treatment. In this study, the effects of curcumin on the activity, migration, and mitochondrial membrane potential (MMP) of malignant hepatocytes (SMMC-7721 cells) were determined using cell viability, migration, and MMP assays. Changes in the morphology and biomechanics of SMMC-7721 cells and their mitochondria were studied using both optical microscopy and atomic force microscopy (AFM). The cell survival rate, migration and MMP depended on the concentration of curcumin. Optical microscopy studies showed that curcumin altered the cell morphology. AFM studies showed that the changes in the morphology and nanomechanics of SMMC-7721 cells and their mitochondria, were induced by curcumin. As the concentration of curcumin increased, the cell length, width, and adhesion decreased, but the height, roughness and Young's modulus increased. In contrast, the mitochondrial length, width, height and roughness increased, but the adhesion and Young's modulus decreased. There was a close relationship between mitochondria and cells in terms of function, morphology and biomechanics. This study shows the effects of curcumin on SMMC-7721 cells and their mitochondria from biology and biophysics perspectives. The findings aid in comprehensively understanding the interactions between mitochondria and malignant hepatocytes.

LncRNA-Gm9866 promotes liver fibrosis by activating TGFβ/Smad signaling via targeting Fam98b

ObjectiveThe exact mechanism and target molecules of liver fibrosis have remained largely elusive. Here, we investigated the role of long noncoding RNA Gm9866(lncRNA-Gm9866) on liver fibrosis.MethodsThe transcription of lncRNA-Gm9866 in activated cells and mouse fibrotic livers was determined by quantitative polymerase chain reaction (qRT-PCR). The effects of lentivirus-mediated knockdown or overexpression of lncRNA-Gm9866 in liver fibrosis were examined in vitro and in vivo. Furthermore, bioinformatics analysis, cell samples validation, fluorescence in situ hybridization (FISH) co-localization, RNA binding protein immunoprecipitation (RIP), actinomycin D test and Western blot (WB) were carried out to explore the potential mechanism of lncRNA-Gm9866.ResultsThe expression of α-smooth muscle actin (α-SMA), Collagen I (COL-1) and lncRNA-Gm9866 were significantly increased in tissues and cells. Overexpressing lncRNA-Gm9866 promoted the activation of hepatic stellate cells (HSCs). Silencing lncRNA-Gm9866 inhibited the activation of HSCs and transforming growth factor-β1 (TGFβ1) induced fibrosis. Overexpressing lncRNA-Gm9866 promoted hepatocytes (HCs) apoptosis and the expression of pro-fibrogenic genes, inhibited the proliferation and migration of HCs. Knockdown of lncRNA-Gm9866 inhibited the apoptosis of HCs, the expression of pro-fibrogenic genes, TGFβ1 induced fibrosis and the occurrence of carbon tetrachloride (CCl4)-induced liver fibrosis, and promoted the proliferation and migration of HCs. Mechanistically, lncRNA-Gm9866 may directly bine with Fam98b. Silencing Fam98b in stably overexpressing lncRNA-Gm9866 cell lines reversed the increase of pro-fibrogenic genes and pro-apoptotic genes, fibrosis related pathway protein TGFβ1, Smad2/3, p-Smad2/3 and Notch3 induced by overexpressing lncRNA-Gm9866.ConclusionsLncRNA-Gm9866 may regulate TGFβ/Smad and Notch pathways by targeting Fam98b to regulate liver fibrosis. LncRNA-Gm9866 may be a new target for diagnosis and treatment of liver fibrosis.

Hepatitis C Virus Core Protein Promotes the Metastasis of Human Hepatocytes by Activating the MAPK/ERK/PEA3-SRF/c-Fos/MMPs Axis

Previous studies have shown that the hepatitis C virus (HCV) core protein plays an important role in the metastasis of hepatocellular carcinoma (HCC) cells. This study aimed to identify the potential mechanism of HCV core protein in HCC. A transcription factor microarray analysis was performed to identify the factors regulated by the HCV core protein. A comprehensive bioinformatics analysis approach was utilized to predict the functions, regulatory signaling pathways and downstream target genes of the differentially regulated transcription factors. Dual-luciferase assays, qPCR, Western blotting, ERK pathway inhibition experiments and siRNA knockdown experiments were performed to verify the effects of the HCV core protein on PEA3, SRF and c-Fos, as well asthe underlying mechanism. The migration/invasion assay and scratch assay served to confirm the metastasis-promoting mechanism of the HCV core protein. The results demonstrated that altered expression of PEA3, SRF and c-Fos mediated by the HCV core protein were associated with the MAPK/ERK pathway. c-Fos was a downstream target protein of PEA3 and SRF. Knockdown of PEA3-SRF/c-Fos expression and ERK pathway components suppressed the migration and invasion activity of hepatocytes by affecting MMP2 and MMP9 expression. We provided preliminary evidence that the role of the HCV core protein in promoting metastasis is at least partially dependent on the activation of the MAPK/ERK/PEA3-SRF/c-Fos/MMP2/MMP9 axis. These findings reveal a novel mechanism by which the HCV core protein promotes HCC metastasis and may provide new therapeutic targets for patients with metastatic HCC.

Murine Falcor/LL35 lncRNA Contributes to Glucose and Lipid Metabolism In Vitro and In Vivo

Glucose and lipid metabolism are crucial functional systems in eukaryotes. A large number of experimental studies both in animal models and humans have shown that long non-coding RNAs (lncRNAs) play an important role in glucose and lipid metabolism. Previously, human lncRNA DEANR1/linc00261 was described as a tumor suppressor that regulates a variety of biological processes such as cell proliferation, apoptosis, glucose metabolism and tumorigenesis. Here we report that murine lncRNA Falcor/LL35, a proposed functional analog of human DEANR1/linc00261, is predominantly expressed in murine normal hepatocytes and downregulated in HCC and after partial hepatectomy. The application of high-throughput approaches such as RNA-seq, LC-MS proteomics, lipidomics and metabolomics analysis allowed changes to be found in the transcriptome, proteome, lipidome and metabolome of hepatocytes after LL35 depletion. We revealed that LL35 is involved in the regulation of glycolysis and lipid biosynthesis in vitro and in vivo. Moreover, LL35 affects Notch and NF-κB signaling pathways in normal hepatocytes. All observed changes result in the decrease in the proliferation and migration of hepatocytes. We demonstrated similar phenotype changes between murine LL35 and human linc00261 depletion in vitro and in vivo that opens the opportunity to translate results for LL35 from a liver murine model to possible functions of human lncRNA linc00261.

Increased Phosphatase of Regenerating Liver-1 by Placental Stem Cells Promotes Hepatic Regeneration in a Bile-Duct-Ligated Rat Model.

Phosphatase of regenerating liver-1 (PRL-1) controls various cellular processes and liver regeneration. However, the roles of PRL-1 in liver regeneration induced by chorionic-plate-derived mesenchymal stem cells (CP-MSCs) transplantation remain unknown. Here, we found that increased PRL-1 expression by CP-MSC transplantation enhanced liver regeneration in a bile duct ligation (BDL) rat model by promoting the migration and proliferation of hepatocytes. Engrafted CP-MSCs promoted liver function via enhanced hepatocyte proliferation through increased PRL-1 expression in vivo and in vitro. Moreover, higher increased expression of PRL-1 regulated CP-MSC migration into BDL-injured rat liver through enhancement of migration-related signals by increasing Rho family proteins. The dual effects of PRL-1 on proliferation of hepatocytes and migration of CP-MSCs were substantially reduced when PRL-1 was silenced with siRNA-PRL-1 treatment. These findings suggest that PRL-1 may serve as a multifunctional enhancer for therapeutic applications of CP-MSC transplantation.

Plasmodium sporozoites on the move: Switching from cell traversal to productive invasion of hepatocytes.

Parasites of the genus Plasmodium, the etiological agent of malaria, are transmitted through the bite of anopheline mosquitoes, which deposit sporozoites into the host skin. Sporozoites migrate through the dermis, enter the bloodstream, and rapidly traffic to the liver. They cross the liver sinusoidal barrier and traverse several hepatocytes before switching to productive invasion of a final one for replication inside a parasitophorous vacuole. Cell traversal and productive invasion are functionally independent processes that require proteins secreted from specialized secretory organelles known as micronemes. In this review, we summarize the current understanding of how sporozoites traverse through cells and productively invade hepatocytes, and discuss the role of environmental sensing in switching from a migratory to an invasive state. We propose that timely controlled secretion of distinct microneme subsets could play a key role in successful migration and infection of hepatocytes. A better understanding of these essential biological features of the Plasmodium sporozoite may contribute to the development of new strategies to fight against the very first and asymptomatic stage of malaria.

Decellularized hepatic extracellular matrix hydrogel attenuates hepatic stellate cell activation and liver fibrosis.

Liver fibrosis results from excessive accumulation of extracellular matrix (ECM) proteins that distort the hepatic architecture. Progression of liver fibrosis results in cirrhosis and liver failure, and often, liver transplantation is required. The decellularized liver tissue contains different components that mimic the natural hepatic environment. We hypothesized that a decellularized liver hydrogel can be used to replace the necrotic hepatocytes and damaged ECM. Therefore, our aim in this study is to develop a therapy for treating liver fibrosis. Mice livers were decellularized and processed to form a hepatic hydrogel. We evaluated the biocompatibility and bioactivity of the hydrogel. The ability of the hydrogel to enhance the migration of hepatocytes and endothelial cells was investigated. Human hepatic stellate cell line (LX-2) activated by transforming growth factor-β1 (TGF-β1) was used as in vitro model for fibrogenesis. Then, the hydrogel was injected into the liver parenchyma of mice after the induction of liver fibrosis using thioacetamide. The resulting hydrogel maintained a complex composition, which included glycosaminoglycans, collagen, elastin, and growth factors. Hepatocytes and endothelial cells were shown to migrate toward the hydrogel in vitro. Liver hydrogel improved TGF-β1-induced LX-2 cells activation via blocking the TGF-β1/Smad pathway. The matrix was delivered successfully in vivo and enhanced the reduction of fibrosis and recovery to a nearly normal structure. In conclusion, we have demonstrated that the liver hydrogel can be utilized as an injectable biomaterial for liver tissue engineering in order to reduce the degree of fibrosis.

Fe (III)@TA@IGF-2 microspheres loaded hydrogel for liver injury treatment

As one of the most commonly used materials in liver tissue engineering, hydrogel has received much attention in recent years. In this work, we prepared a gelatin methacrylate (GelMA)/oxidized hyaluronic acid (OHA)/galactosylated chitosan (Gal-CS)/Fe (III)@TA@IGF-2 200 (TA200) hydrogel loaded with insulin-like growth factor 2 (IGF-2) for regeneration of damaged hepatocytes. Fe (III)@TA microspheres served as carrier to achieve sustained release of IGF-2 to promote hepatocytes regeneration. Galactose ligands could bind to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes. Galactosylated chitosan could significantly increase the specific function of hepatocytes. The hydrogel we prepared had a storage modulus of 1100 Pa and was suitable for migration of hepatocytes. The release ratio of IGF-2 could reach up to 90% within 14 days. For carbon tetrachloride (CCl4) induced human hepatic stellate cell line LX2 damage, GelMA/OHA/Gal-CS/TA200 hydrogel could significantly improve the survival of LX2 cells. The expression of HNF-4α and transferrin was detected in LX2 cells treated with hydrogel, indicating that the specific function of the liver was also restored. In summary, the GelMA/OHA/Gal-CS/TA200 hydrogels could be used as new tissue engineering scaffolds for the construction of artificial livers.

Upregulation of RUNX1 Suppresses Proliferation and Migration through Repressing VEGFA Expression in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, and occurs in people with chronic liver diseases. Current treatment methods include surgery, transplant, and chemotherapy. Our study demonstrates runt-related transcription factor 1 (RUNX1) as a novel molecule in the initiation and development of HCC, and the role of its interaction with vascular endothelial growth factor A (VEGFA) in HCC. We showed the suppressive role of RUNX1 in the proliferation and migration of hepatocytes. In addition, the repressor RUNX1 functioned as a transcription factor on the promoter of VEGFA to inhibit the expression of VEGFA. Study in the HCC cells demonstrated that the suppression of HCC proliferation and migration was masked in the presence of overexpressed VEGFA. Introduction of RUNX1 into HCC mice model significantly limited the tumor growth. In summary, our study demonstrated that RUNX1 functions as a repressor in the HCC and this suppressive function was dependent on its effect on VEGFA.

Retraction: Role of Hepatitis C Virus Induced Osteopontin in Epithelial to Mesenchymal Transition, Migration and Invasion of Hepatocytes.

Retraction: Role of Hepatitis C Virus Induced Osteopontin in Epithelial to Mesenchymal Transition, Migration and Invasion of Hepatocytes.

Osteopontin Regulates Hepatitis C Virus (HCV) Replication and Assembly by Interacting with HCV Proteins and Lipid Droplets and by Binding to Receptors αVβ3 and CD44

Hepatitis C virus (HCV) replication and assembly occur at the specialized site of endoplasmic reticulum (ER) membranes and lipid droplets (LDs), respectively. Recently, several host proteins have been shown to be involved in HCV replication and assembly. In the present study, we demonstrated the important relationship among osteopontin (OPN), the ER, and LDs. OPN is a secreted phosphoprotein, and overexpression of OPN in hepatocellular carcinoma (HCC) tissue can lead to invasion and metastasis. OPN expression is also enhanced in HCV-associated HCC. Our recent studies have demonstrated the induction, proteolytic cleavage, and secretion of OPN in response to HCV infection. We also defined the critical role of secreted OPN in human hepatoma cell migration and invasion through binding to receptors integrin αVβ3 and CD44. However, the role of HCV-induced OPN in the HCV life cycle has not been elucidated. In this study, we showed a significant reduction in HCV replication, assembly, and infectivity in HCV-infected cells transfected with small interfering RNA (siRNA) against OPN, αVβ3, and CD44. We also observed the association of endogenous OPN with HCV proteins (NS3, NS5A, NS4A/B, NS5B, and core). Confocal microscopy revealed the colocalization of OPN with HCV NS5A and core in the ER and LDs, indicating a possible role for OPN in HCV replication and assembly. Interestingly, the secreted OPN activated HCV replication, infectivity, and assembly through binding to αVβ3 and CD44. Collectively, these observations provide evidence that HCV-induced OPN is critical for HCV replication and assembly.IMPORTANCE Recently, our studies uncovered the critical role of HCV-induced endogenous and secreted OPN in migration and invasion of hepatocytes. However, the role of OPN in the HCV life cycle has not been elucidated. In this study, we investigated the importance of OPN in HCV replication and assembly. We demonstrated that endogenous OPN associates with HCV NS3, NS5A, NS5B, and core proteins, which are in close proximity to the ER and LDs. Moreover, we showed that the interactions of secreted OPN with cell surface receptors αVβ3 and CD44 are critical for HCV replication and assembly. These observations provide evidence that HCV-induced endogenous and secreted OPN play pivotal roles in HCV replication and assembly in HCV-infected cells. Taken together, our findings clearly demonstrate that targeting OPN may provide opportunities for therapeutic intervention of HCV pathogenesis.

Function and therapeutic advances of chemokine and its receptor in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of hepatic pathology, ranging from simple accumulation of fat in its most benign form, steatohepatitis, to cirrhosis in its most advanced form. The prevalence of NAFLD is 20–30% in adults, and 10–20% of patients with NAFLD progress to nonalcoholic steatohepatitis (NASH) which is predicted to be the leading cause of liver transplantation over the next 10 years. Therefore, it is essential to explore effective diagnostic and treatment strategies for NAFLD patients. Chemokines are a family of small and highly conserved proteins (molecular weight ranging from 8 to 12 kDa) involved in regulating the migration and activities of hepatocytes, Kupffer cells (KCs), hepatic stellate cells (HSCs), endothelial cells and circulating immune cells. Accumulating data show that chemokine and its receptor act vital roles in the pathogenesis of NAFLD. Herein, we summarize the involvement of the chemokine and its receptor in the pathogenesis of NAFLD and explore the novel pharmacotherapeutic avenues for patients with NAFLD.

Spontaneous hepatocyte migration towards an endothelial cell tube network.

A crucial part of the engineering liver tissue is contribution of nonparenchymal cells and maintenance of a complex three-dimensional (3D) structure in vitro for their normal physiology and function. We generated 3D hepatic tissue using primary isolated rat hepatocytes and an endothelial cell tube network from human endothelial vein epithelial cells (HUVECs). To create the 3D hepatic tissue, coculture of primary hepatocytes and tube-structured HUVECs was performed on a Matrigel®. After the HUVECs formed the tube structures, primary isolated rat hepatocytes were inoculated onto the HUVEC tube-structured layer and cultured for 24hr. We investigated the cell migration, cellular interaction, and distributions of HUVEC tube structures and hepatocytes using multi cell-imaging incubator, confocal microscopy, and electron microscopy analyses. During the culture time, time-lapse imaging showed spontaneous migration of the hepatocytes in the gel, and after the 24-hr culture period, the vast majority of the hepatocytes had moved and adhered to the surface of the HUVEC tube structures. A confocal microscopy assay confirmed this unique 3D cellular interaction between hepatocytes and HUVEC tube structures. The hepatocytes were able to maintain their spherical shape, as well as HUVECs (tube-like form with tubular cavity). We speculate that coculturing of hepatocytes and endothelial cells replicates part of their normal physiology and may help induce migration in vitro and the growth of complex biological tissue structures.

Role of Glypican-3 in the growth, migration and invasion of primary hepatocytes isolated from patients with hepatocellular carcinoma.

Recently, Glypican-3 (GPC3) has been identified as a potential hepatocellular carcinoma (HCC) diagnostic and/or therapeutic target. GPC3 has been found to be up-regulated in HCC and to be absent in normal and cirrhotic liver. As yet, however, the molecular characteristics of GPC3 and its role in HCC cell physiology and development are still undefined. Human hepatocyte cultures were established from 10 HCC patients. Additional liver samples were obtained from 5 patients without cirrhosis and/or HCC. Soft agar colony formation, (co-)immunofluorescence and Western blot assays were used to characterize the hapatocyte cultures. The expression of GPC3 in the hepatocytes was silenced using siRNA, after which, apoptosis, scratch wound migration and transwell invasion assays were performed. We found that in HCC precursor hepatocytes GPC3 is increasingly expressed in different forms and at different locations, i.e., a non-cleaved form (70kDa) was found to be localized in the cytoplasm while a N-terminal cleaved form (N-GPC3: 40kDa) was fond to be localized in the cytoplasm and at the extracellular side of hepatocyte membranes. In addition, we found that the non-cleaved form of GPC3 co-localizes with Furin-Convertase in the Golgi apparatus. We also found that, similar to GPC3, Furin-Convertase is expressed in HCC precursor cells, suggesting a role in GPC3 processing. Subsequent siRNA-mediated GPC3 silencing resulted in a temporary inhibition of cell proliferation, migration and ivasion, while inducing apoptosis in transformed hepatocytes. Our data reveal new aspects of the role of GPC3 in early hepatocyte transformation. In addition we conclude that GPC3 may serve as a new HCC immune-therapeutic target.

Controlling the selective and directional migration of hepatocytes by a complementary density gradient of glycosylated hyperbranched polymers and poly(ethylene glycol) molecules.

Repair and regeneration of defected tissues and organs depends strongly on the directional migration of targeted cells, for example, the enhancement of directional migration of hepatocytes could be helpful in liver regeneration and transplantation. Herein a complementary gradient of galactose-modified hyperbranched polymers (LA-HPMA) and poly(ethylene glycol) (PEG) molecules was designed and prepared on a same substrate. Characterizations of X-ray photoelectron spectrometry and quartz crystal microbalance with dissipation (QCM-d) demonstrated the unidirectional change in grafting density of LA-HPMA and PEG molecules, respectively. On the LA-HPMA/PEG complementary gradient surface, the human hepatoma (HepG2) cells showed preferential orientation and enhanced directional migration toward the region of lower PEG density and higher LA-HPMA density. By contrast, the mouse embryonic fibroblasts (NIH3T3) showed random migration irrelevant to the gradient. The success of the complementary gradient relies on the specific interaction between galactose and asialoglycoprotein receptor (ASGPR) expressed on HepG2 cells. STATEMENT OF SIGNIFICANCE.

RhoC overexpression-induced migration and invasion of human normal hepatocytes

Objective To investigate the role of Ras-homologous GTPase C (RhoC) overexpres-sion in inducing migration and invasion for malignant transformation in normal human hepatocytes.Methods pcDNA3-RhoC vector was transfected into human normal liver cell HL7702.Scratch-healing test combined with Transwell was used to examine cell migration and invasion,and matrix metalloproteinases (MMPs)gelatinase activities were assayed through zymography.Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the migration and invasion associated genes.RhoC transfected HL7702 cells were inoculated into the nude mice to test tumor formation.Results Cells transfected with pcDNA3-RhoC acquired migration capacity,and the migration distance was notably increased as compared with parental cells and negative control groups [(63.33 ± 10.07)％ vs.(28.67 ± 7.02)％ and (28.33 ±6.66)％,P ＜0.01].The number of pcDNA3-RhoC cells through the Transwell was increased significantly (65.33 ± 6.80 vs.24.33 ± 5.79 and 23.33 ± 5.73,P ＜ 0.01),and the invasion capacity was significantly enhanced in pcDNA3-RhoC-transfected group as compared with control groups (43.67 ±7.59 vs.13.33 ± 3.40 and 15.33 ± 4.11,P ＜ 0.01).MMPs gelatinase activities were increased,the expression of migration and invasion associated genes and proteins such as MMP-2,MMP-9,vascular endothelial growth factor (VEGF),P27RF-Rho and Survivin was significantly up-regulated,and the expression of tissue inhibitor of tissue inhibitorof metalloproteinase 1 (TIMP1),TIMP2,p53 and phosphatase and tensin homolog deleted on chromosome ten (PTEN) was down-regulated in pcDNA3-RhoC-transfected group as compared with control group.The tumor formation ratio in nude mice was 40％.Conclusion RhoC overexpression can induce migration and invasion for malignant transformation in human normal hepatocytes. Key words: RhoC ; Hepatocyte ; Vicious transformation

Role of Hepatitis C Virus Induced Osteopontin in Epithelial to Mesenchymal Transition, Migration and Invasion of Hepatocytes

Osteopontin (OPN) is a secreted phosphoprotein which has been linked to tumor progression and metastasis in a variety of cancers including hepatocellular carcinoma (HCC). Previous studies have shown that OPN is upregulated during liver injury and inflammation. However, the role of OPN in hepatitis C virus (HCV)-induced liver disease pathogenesis is not known. In this study, we determined the induction of OPN, and then investigated the effect of secreted forms of OPN in epithelial to mesenchymal transition (EMT), migration and invasion of hepatocytes. We show the induction of OPN mRNA and protein expression by HCV-infection. Our results also demonstrate the processing of precursor OPN (75 kDa) into 55 kDa, 42 kDa and 36 kDa forms of OPN in HCV-infected cells. Furthermore, we show the binding of secreted OPN to integrin αVβ3 and CD44 at the cell surface, leading to the activation of downstream cellular kinases such as focal adhesion kinase (FAK), Src, and Akt. Importantly, our results show the reduced expression of epithelial marker (E-cadherin) and induction of mesenchymal marker (N-cadherin) in HCV-infected cells. We also show the migration and invasion of HCV-infected cells using wound healing assay and matrigel coated Boyden chamber. In addition, we demonstrate the activation of above EMT markers, and the critical players involved in OPN-mediated cell signaling cascade using primary human hepatocytes infected with Japanese fulminant hepatitis (JFH)-1 HCV. Taken together, these studies suggest a potential role of OPN in inducing chronic liver disease and HCC associated with chronic HCV infection.

Cell Adhesion-dependent Serine 85 Phosphorylation of Paxillin Modulates Focal Adhesion Formation and Haptotactic Migration via Association with the C-terminal Tail Domain of Talin

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration.

Activation of β‐adrenergic receptors increases the in vitro migration of malignant hepatocytes

Activation of adrenergic receptors (AR) has been reported to enhance the growth and invasion of various malignancies. The effects of AR agonists on malignant hepatocyte proliferation and migration have yet to be determined. PLC/PRF/5 (PLC) and Huh-7 cells were exposed to a wide range of concentrations of the AR agonists noradrenaline (NA) and isoprenaline. Cell proliferation, migration, intracellular cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) and C (PKC), matrix metalloproteinases (MMP)-2, -3, -7 and -9, and α(1) -, β(1) - and β(2) -AR expression were documented in both cell lines. Cell proliferative activity was unaltered following exposure to physiological and stress-related concentrations of AR agonists but migration was accelerated, an effect that was inhibited by the nonselective β-AR antagonist labetalol. cAMP, PKA, PKC or MMP expression remained unchanged. Although α(1) - and β(1) -AR expressions were abundant, β(2) -AR expression was limited in both cell lines. Unlike other malignancies studied to date, in this study, the proliferative activity of malignant hepatocytes was not increased by exposure to AR agonists, a finding that could be explained by downregulation of β(2) -AR expression. The increase in malignant hepatocyte migration observed remains unexplained but does not appear to involve adenyl cyclase or MMP signaling pathways.