scholarly journals Hypoxia inhibits hepcidin expression in HuH7 hepatoma cells via decreased SMAD4 signaling

2011 ◽  
Vol 300 (4) ◽  
pp. C888-C895 ◽  
Author(s):  
Timothy B. Chaston ◽  
Pavle Matak ◽  
Katayoun Pourvali ◽  
Surjit K. Srai ◽  
Andrew T. McKie ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Promoter Activity ◽  
Quantitative Polymerase Chain Reaction ◽  
Hepatoma Cells ◽  
Luciferase Reporter ◽  
Smad Signaling ◽  
Hepcidin Expression ◽  
Huh7 Cells ◽  
Hepcidin Mrna

Hepcidin negatively regulates systemic iron homeostasis in response to inflammation and elevated serum iron. Conversely, hepcidin expression is diminished in response to hypoxia, oxidative stress, and increased erythropoietic demand, though the molecular intermediates involved are incompletely understood. To address this, we have investigated hypoxic hepcidin regulation in HuH7 hepatoma cells either cultured alone or cocultured with activated THP-1 macrophages. HuH7 hepcidin mRNA expression was determined using quantitative polymerase chain reaction (Q-PCR). Hepcidin promoter activity was measured using luciferase reporter constructs containing a 0.9 kb fragment of the wild-type human hepcidin promoter, and constructs containing mutations in bone morphogenetic protein (BMP)/SMAD4, signal transducer and activator of transcription 3 (STAT3), CCAAT/enhancer-binding protein (C/EBP), and E-box-responsive elements. Hepatic expression of bone morphogenetic proteins BMP2 and BMP6 and the BMP inhibitor noggin was determined using Q-PCR, and the protein expression of hemojuvelin (HJV), pSMAD 1/5/8, and SMAD4 was determined by western blotting. Following exposure to hypoxia or H2O2, hepcidin mRNA expression and promoter activity increased in HuH7 cells monocultures but were decreased in HuH7 cells cocultured with THP-1 macrophages. This repression was attenuated by mutation of the BMP/SMAD4-response element, suggesting that modulation of SMAD signaling mediated the response to hypoxia. No changes in hepatocyte BMP2, BMP6 or noggin mRNA, or protein expression of HJV or pSMAD 1/5/8 were detected. However, treatment with hypoxia caused a marked decrease in nuclear and cytosolic SMAD4 protein and SMAD4 mRNA expression in cocultured HuH7 cells. Together these data indicate that hypoxia represses hepcidin expression through inhibition of BMP/SMAD signaling.

scholarly journals Plastrum Testudinis Extracts Promote BMSC Proliferation and Osteogenic Differentiation by Regulating Let-7f-5p and the TNFR2/PI3K/AKT Signaling Pathway

2018 ◽  
Vol 47 (6) ◽  
pp. 2307-2318 ◽  
Author(s):  
Geng-Yang Shen ◽  
Hui Ren ◽  
Jin-Jing Huang ◽  
Zhi-Da Zhang ◽  
Wen-Hua Zhao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Osteogenic Differentiation ◽  
Mrna Expression ◽  
Signaling Pathway ◽  
Quantitative Polymerase Chain Reaction ◽  
Akt Signaling ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Akt Signaling Pathway ◽  
Alizarin Red

Background/Aims: Plastrum testudinis extracts (PTE) show osteoprotective effects on glucocorticoid-induced osteoporosis in vivo and in vitro. However, the underlying molecular mechanism of PTE in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. Methods: BMSC proliferation was investigated using the Cell Counting Kit-8 assay. BMSC differentiation and osteogenic mineralization were assayed using alkaline phosphatase and Alizarin red staining, respectively. The mRNA expression levels of Let-7f-5p, Tnfr2, Traf2, Pi3k, Akt, β-catenin, Gsk3β, Runx2, and Ocn were measured using real time quantitative polymerase chain reaction. Protein levels of TNFR2, TRAF2, p-PI3K, p-AKT, p-β-CATENIN, and p-GSK3β were analyzed by western blotting. The functional relationship of Let-7f-5p and Tnfr2 was determined by luciferase reporter assays. Results: The optimum concentration for PTE was 30 μg/ml. PTE significantly promoted BMSC osteogenic differentiation and mineralization after 7 and 14 days in culture, respectively. The combination of PTE and osteogenic induction exhibited significant synergy. PTE upregulated Let-7f-5p, β-catenin, Runx2, and Ocn mRNA expression, and downregulated Tnfr2, Traf2, Pi3k, Akt, and Gsk3β mRNA expression. PTE inhibited TNFR2, TRAF2, and p-β-CATENIN protein expression, and promoted p-PI3K, p-AKT, and p-GSK3β protein expression. In addition, Tnfr2 was a functional target of Let-7f-5p in 293T cells. Conclusions: Our results suggested that PTE may promote BMSC proliferation and osteogenic differentiation via a mechanism associated with the regulation of Let-7f-5p and the TNFR2/PI3K/AKT signaling pathway.

STAT3 mediates hepatic hepcidin expression and its inflammatory stimulation

Blood ◽  
2006 ◽  
Vol 109 (1) ◽  
pp. 353-358 ◽  
Author(s):  
Maria Vittoria Verga Falzacappa ◽  
Maja Vujic Spasic ◽  
Regina Kessler ◽  
Jens Stolte ◽  
Matthias W. Hentze ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Culture Conditions ◽  
Control Culture ◽  
Binding Motif ◽  
Iron Release ◽  
Hepcidin Expression ◽  
Inflammatory Conditions ◽  
Human Liver Cell ◽  
Hepcidin Mrna

Abstract Hepcidin is a key iron-regulatory hormone produced by the liver. Inappropriately low hepcidin levels cause iron overload, while increased hepcidin expression plays an important role in the anemia of inflammation (AI) by restricting intestinal iron absorption and macrophage iron release. Its expression is modulated in response to body iron stores, hypoxia, and inflammatory and infectious stimuli involving at least in part cytokines secreted by macrophages. In this study we established and characterized IL6-mediated hepcidin activation in the human liver cell line Huh7. We show that the proximal 165 bp of the hepcidin promoter is critical for hepcidin activation in response to exogenously administered IL6 or to conditioned medium from the monocyte/macrophage cell line THP-1. Importantly, we show that hepcidin activation by these stimuli requires a STAT3 binding motif located at position –64/–72 of the promoter. The same STAT binding site is also required for high basal-level hepcidin mRNA expression under control culture conditions, and siRNA-mediated RNA knockdown of STAT3 strongly reduces hepcidin mRNA expression. These results identify a missing link in the acute-phase activation of hepcidin and establish STAT3 as a key effector of baseline hepcidin expression and during inflammatory conditions.

scholarly journals Inhibiting Effects of Down-regulating of Fascin 1 on Proliferation and Migration in Hepatoma Cells

2021 ◽  
Author(s):  
Wanglu Gu ◽  
Guilan Wang ◽  
Xinyang Zhang ◽  
Li Chen ◽  
Jiaming Zhou
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Hepatoma Cell ◽  
Hepatoma Cells ◽  
Human Hepatoma Cell ◽  
Specific Sequence ◽  
Knock Down ◽  
Huh7 Cells ◽  
And Migration ◽  
Proliferation And Migration

Abstract Objective: To investigate the inhibiting effects of fascin 1 gene knock-down on the proliferation and migration of hepatoma cells by means of small interfering RNA (siRNA).Methods: SiRNA targeting fascin 1 gene (si-fascin) and non-specific sequence siRNA (si-NC）were constructed and transfected into human hepatoma cell lines (HepG2 and Huh7) to down-regulate the expression of fascin 1. RT-qPCR, Western blotting, and Immunofluorescence technique were used to evaluate the efficiency of si-fascin. The proliferation and migration of cells were detected by MTT method and Transwell experiments, and the protein expression of genes related to proliferation and migration in cells were detected by Western blotting. The apoptosis and pseudopodia formation of cells were observed under scanning electron microscope (SEM).Results: Compared with human normal liver cells (LO2), the expressions of fascin 1 mRNA and protein were significantly higher in HepG2 and Huh7 cells. The expression of fascin 1 was overall inhibited in HepG2 and Huh7 cells transfected by the constructed four si-fascins, among which, fascin_siR3 had the highest inhibitory efficiency, therefore was selected in this study. In HepG2 and Huh7 cells transfected by si-fascin significant knock-down target gene expression, while reducing cell proliferation, migration and the formation of pseudopods, and causes reduced protein expression associated with proliferation and migration. Conclusion: This study further confirmed that fascin 1 gene has the function of promoting hepatoma cells proliferation and migration, suggesting that downregulating the expression of fascin 1 in hepatoma cells may be one of the strategies to intervene in liver cancer.

Wnt/β-Catenin and Hippo Pathway Deregulation in Mammary Tumors of Humans, Dogs, and Cats

2020 ◽  
Vol 57 (6) ◽  
pp. 774-790
Author(s):  
Alessandro Sammarco ◽  
Chiara Gomiero ◽  
Roberta Sacchetto ◽  
Giorgia Beffagna ◽  
Silvia Michieletto ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Quantitative Polymerase Chain Reaction ◽  
Mrna Level ◽  
Hippo Pathway ◽  
Mammary Tumors ◽  
Breast Cancers ◽  
Tumor Tissues ◽  
Mrna And Protein Expression ◽  
Polymerase Chain

Mammary cancer is a common neoplasm in women, dogs, and cats that still represents a therapeutic challenge. Wnt/β-catenin and Hippo pathways are involved in tumor progression, cell differentiation, and metastasis. The aim of this study was to evaluate mRNA and protein expression of molecules involved in these pathways in human (HBC), canine (CMT), and feline mammary tumors (FMT). Real-time quantitative polymerase chain reaction (qPCR) for β-catenin, CCND1, YAP, TAZ, CTGF, and ANKRD1, western blotting for YAP, TAZ, and β-catenin, and immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), ERBB2, β-catenin, and YAP/TAZ were performed on mammary tumor tissues. The protein expression of active β-catenin was higher in tumors than in healthy tissues in all 3 species. The mRNA expression of the downstream gene CCND1 was increased in HBC ER+ and CMTs compared to healthy tissues. Membranous and cytoplasmic protein expression of β-catenin were strongly negatively correlated in all 3 species. Tumors showed an increased protein expression of YAP/TAZ when compared to healthy tissues. Notably, YAP/TAZ expression was higher in triple negative breast cancers when compared to HBC ER+ and in FMTs when compared to CMTs. The mRNA expression of β-catenin, YAP, TAZ, CTGF, and ANKRD1 was not different between tumors and healthy mammary gland in the 3 species. This study demonstrates deregulation of Wnt/β-catenin and Hippo pathways in mammary tumors, which was more evident at the protein rather than the mRNA level. Wnt/β-catenin and Hippo pathways seem to be involved in mammary carcinogenesis and therefore represent interesting therapeutic targets that should be further investigated.

Characterization of Hepcidin-Inducing Cytokines in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2001-2001
Author(s):  
Ken Maes ◽  
Alissa Huston ◽  
David Roodman ◽  
Seth Rivera ◽  
Elizabeta Nemeth ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Promoter Activity ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Advisory Committees ◽  
Hepcidin Expression ◽  
Equity Ownership

Abstract Abstract 2001 Poster Board I-1023 Introduction: Hepcidin, the principal iron-regulatory hormone, plays an important role in the development of anemia of inflammation and other iron-restricted anemias. Patients with multiple myeloma (MM) frequently present with anemia not attributable to a known mechanism. We previously found that hepcidin is increased in MM and thus could cause or contribute to the anemia of MM. The BMP and IL-6 pathways are the two known major transcriptional regulators of hepcidin. Methods: To identify cytokines that increase hepcidin in MM patients, we screened patient sera with an in vitro cellular reporter system, consisting of human hepatoma 7 cell line (HuH7) and the hepcidin promoter-firefly luciferase reporter. Using site-directed mutagenesis, the promoter was mutated at the STAT3-binding site (STAT3-BS) and/or two BMP responsive elements (BREs), sequences known to be involved in the regulation of hepcidin expression by IL-6 and BMPs, respectively. Results: As expected, recombinant IL-6 and BMP-4, -6 and -9 activated wild-type hepcidin promoter activity several fold. Of note, IL-6 and BMP-9 interacted synergistically at low doses, at the level of the promoter. Mutations in STAT3-BS abrogated the response to IL-6. Mutations in either BRE site by itself did not abolish the response to BMPs, but concurrent mutagenesis of both sites resulted in a complete loss of hepcidin response. Importantly, STAT3-BS and BREs affected hepcidin promoter response independently from each other. Using the in vitro system, we compared sera from six MM patients with previously measured serum hepcidin levels to sera from healthy controls. Sera of four patients with high hepcidin and one with low hepcidin significantly induced hepcidin promoter activity, while serum of another patient with low hepcidin did not. Mutations in STAT3-BS only abrogated the response to two patient sera, both of which had high hepcidin. Mutations in two BREs abrogated the response in all six sera as did the triple mutation involving STAT3-BS and both BREs. Conclusions: We could separately interrogate the signaling pathways by which IL-6 and BMPs induce hepcidin transcription, allowing us to discriminate between the effects of IL-6-like or BMP-like cytokines in each patient serum. BMP-like cytokines were involved in the upregulation of hepcidin in all tested MM patients, and in some patients IL-6 or related cytokines contributed as well, either independently or through a synergistic interaction. No residual activation of hepcidin promoter by other factors was noted. Antibody neutralization may identify the specific myeloma-associated cytokines that stimulate hepcidin production through the two canonical pathways. Disclosures: Roodman: Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy; Acceleron: Consultancy. Nemeth:Intrinsic LifeSciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Xenon Pharmaceuticals: Consultancy, Equity Ownership.

scholarly journals Characterization of the G protein-coupled receptor kinase 6 promoter reveals a functional CREB binding site

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247087
Author(s):  
Maike Stegen ◽  
Andrea Engler ◽  
Crista Ochsenfarth ◽  
Iris Manthey ◽  
Jürgen Peters ◽  
...  
Keyword(s):  
Protein Expression ◽  
Binding Site ◽  
G Protein ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Western Blots ◽  
G Protein Coupled ◽  
Creb Binding Site

Background G protein-coupled receptor kinase 6 (GRK6) is part of the G protein-coupled receptor kinase family, whose members act as key regulators of seven-transmembrane receptor signalling. GRK6 seems to play a role in regulation of inflammatory processes, but mechanisms of transcriptional regulation of GRK6 expression in inflammatory cell lines have not been characterized. Protein kinase C (PKC) signalling is also involved in inflammatory regulation and an impact of PKC activation on GRK6 protein expression was described previously. Thus, the aim of this study was to 1) characterize the GRK6 promoter, and 2) investigate a potential influence of PKC on GRK6 expression. Methods Five deletion constructs of the GRK6 promoter were cloned. After transient transfection into a human T cell line, promoter activity was assessed using luciferase reporter gene assays. Putative transcription factor binding sites were identified, mutated, and binding was investigated using electrophoretic mobility shift assays (EMSA). Following stimulation with a PKC activator, GRK6 expression on mRNA and protein levels was assessed by reverse transcriptase qPCR and Western blots. Results Investigation of the GRK6 promoter revealed a putative cAMP responsive element (CRE), whose mutation led to decreased promoter activity (p = 0.0006). Functionality of the CRE binding protein (CREB) binding site was verified in EMSA blots. Stimulation with a PKC activator resulted in decreased GRK6 promoter activity (p = 0.0027), mRNA (p = 0.04) and protein expression. Conclusion We characterized the human GRK6 promoter and identified promoter activity to be influenced by a CREB binding site. PKC might be one determinant contributing to altered GRK6 expression.

scholarly journals Cardiac Hepcidin Expression Associates with Injury Independent of Iron

2016 ◽  
Vol 44 (5) ◽  
pp. 368-378 ◽  
Author(s):  
G. Fenna van Breda ◽  
Lennart G. Bongartz ◽  
Wenqing Zhuang ◽  
Rachel P.L. van Swelm ◽  
Jeanne Pertijs ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Messenger Rna ◽  
Iron Homeostasis ◽  
Quantitative Polymerase Chain Reaction ◽  
Cardiac Injury ◽  
Tissue Growth ◽  
Hepcidin Expression ◽  
Sham Surgery ◽  
Hepcidin Gene

Background: Hepcidin regulates systemic iron homeostasis by downregulating the iron exporter ferroportin. Circulating hepcidin is mainly derived from the liver but hepcidin is also produced in the heart. We studied the differential and local regulation of hepcidin gene expression in response to myocardial infarction (MI) and/or chronic kidney disease (CKD). We hypothesized that cardiac hepcidin gene expression is induced by and regulated to severity of cardiac injury, either through direct (MI) or remote (CKD) stimuli, as well as through increased local iron content. Methods: Nine weeks after subtotal nephrectomy (SNX) or sham surgery (CON), rats were subjected to coronary ligation (CL) or sham surgery to realize 4 groups: CON, SNX, CL and SNX + CL. In week 16, the gene expression of hepcidin, iron and damage markers in cardiac and liver tissues was assessed by quantitative polymerase chain reaction and ferritin protein expression was studied by immunohistochemistry. Results: Cardiac hepcidin messenger RNA (mRNA) expression was increased 2-fold in CL (p = 0.03) and 3-fold in SNX (p = 0.01). Cardiac ferritin staining was not different among groups. Cardiac hepcidin mRNA expression correlated with mRNA expression levels of brain natriuretic peptide (β = 0.734, p < 0.001) and connective tissue growth factor (β = 0.431, p = 0.02). In contrast, liver hepcidin expression was unaffected by SNX and CL alone, while it had decreased 50% in SNX + CL (p < 0.05). Hepatic ferritin immunostaining was not different among groups. Conclusions: Our data indicate differences in hepcidin regulation in liver and heart and suggest a role for injury rather than iron as the driving force for cardiac hepcidin expression in renocardiac failure.

scholarly journals Interleukin-1β modulates endogenous thyroid hormone receptor α gene transcription in liver cells

2007 ◽  
Vol 194 (2) ◽  
pp. 257-265 ◽  
Author(s):  
J Kwakkel ◽  
W M Wiersinga ◽  
A Boelen
Keyword(s):  
Thyroid Hormone ◽  
Mrna Expression ◽  
Gene Transcription ◽  
Hormone Receptor ◽  
Proinflammatory Cytokine ◽  
Hepg2 Cells ◽  
Promoter Activity ◽  
Thyroid Hormone Receptor ◽  
De Novo ◽  
Hepatoma Cells

One of the main characteristics of nonthyroidal illness (NTI) is a decrease in serum triiodothyronine, partly caused by a decrease in liver deiodinase type 1 (D1) mRNA and activity. Proinflammatory cytokines have been associated with NTI in view of their capability to decrease D1 and thyroid hormone receptor (TR)β1 mRNA expression in hepatoma cells. Proinflammatory cytokine induction leads to activation of the inflammatory pathways nuclear factor (NF)κB and activator protein (AP)-1. The proinflammatory cytokine interleukin (IL)-1β decreases thyroid hormone receptor (TR)β1 mRNA in an NFκB-dependent way. The aim of this study was to unravel the effects of IL-1β on endogenous TRα gene expression in an animal model and in a liver cell line. The TRα gene product is alternatively spliced in TRα1 and TRα2, TRα2 is capable of inhibiting TRα1-induced gene transcription. We showed that both TRα1 and TRα2 mRNA decreased not only after lipopolysaccharide administration in liver of mice, but also after IL-1β stimulation of hepatoma cells (HepG2). Using the NFκB inhibitor sulfasalazine and the AP-1 inhibitor SP600125, it became clear that the IL-1β-induced decrease in TRα mRNA expression in HepG2 cells can only be abolished by simultaneous inhibition of NFκB and AP-1. The IL-1β-induced TRα1 and TRα2 mRNA decrease in HepG2 cells is the result of decreased TRα gene promoter activity, as evident from actinomycin D experiments. Cycloheximide experiments showed that the decreased promoter activity is independent of de novo protein synthesis and therefore most likely due to posttranslational modifications such as phosphorylation or subcellular relocalization.

Impact on clinical prognosis using DNA methylation of the SLC16A3 promoter and expression of the human lactate transporter MCT4 in renal cancer.

2014 ◽  
Vol 32 (4_suppl) ◽  
pp. 452-452
Author(s):  
Jens Bedke ◽  
Pascale Fisel ◽  
Stefan Winter ◽  
Stephan Kruck ◽  
Marcus Scharpf ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Protein Expression ◽  
Mrna Expression ◽  
Promoter Activity ◽  
Epigenetic Mechanism ◽  
Monocarboxylate Transporter ◽  
Clinical Prognosis ◽  
Cpg Sites ◽  
Promoter Dna Methylation ◽  
Promoter Dna

452 Background: The monocarboxylate transporter 4 (MCT4) is a metabolic target in tumor biology because it mediates lactate transport across membranes resulting in antiapoptotic effects. Cell experiments support the importance of MCT4 in clear cell renal cell carcinoma (ccRCC). In this study, we assessed the prognostic potential of MCT4 expression in ccRCC and its epigenetic regulation by DNA methylation as novel predictive marker for patient outcome using independent ccRCC cohorts. Methods: MCT4 protein expression was quantified in 207 ccRCC and corresponding nontumor tissues. Data of an independent ccRCC cohort from The Cancer Genome Atlas (TCGA) were analyzed on MCT4 mRNA (n = 482) and DNA methylation (n = 283) level. The findings on MCT4 expression and DNA methylation in the SLC16A3 promoter were validated in a third cohort (n = 64). Promoter activity assays were conducted in four RCC cell lines. Results: MCT4 protein expression was upregulated (p < 0.0001) in ccRCC and showed significant association with cancer-related death. Upregulation of MCT4 mRNA expression (p < 0.00001) was confirmed in the TCGA cohort. Single CpG sites correlated inversely with mRNA expression and were associated with overall survival in Kaplan-Meier analyses [HR = 0.39; 95% confidence interval (CI), 0.24-0.64; P[log-rank] = 1.23e(-04)]. Promoter activity studies confirmed MCT4 regulation by DNA methylation. The significant correlation between MCT4 protein and gene expression or DNA methylation at single CpG sites was validated in a third cohort. Again, higher methylation at individual CpG sites was associated with prolonged survival [HR = 0.05; 95% CI, 0.01-0.40; P[log-rank] = 6.91e(-05)]. Conclusions: This study identified SLC16A3 promoter DNA methylation as a novel epigenetic mechanism for MCT4 regulation in ccRCC. First evidence of a biological rationale for prognosis and clinical outcome is supported by this specific SLC16A3 promoter DNA methylation.

scholarly journals Regulation of Plasminogen Gene Expression by Interleukin-6

Blood ◽  
1997 ◽  
Vol 89 (7) ◽  
pp. 2394-2403 ◽  
Author(s):  
G. Ronald Jenkins ◽  
Dietmar Seiffert ◽  
Robert J. Parmer ◽  
Lindsey A. Miles
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Interleukin 6 ◽  
Luciferase Activity ◽  
Hepatoma Cells ◽  
Luciferase Reporter ◽  
Plasmin Activity ◽  
Luciferase Reporter Gene ◽  
Human Hepatoma Cells

Abstract Plasmin, the primary fibrinolytic enzyme, has a broad substrate spectrum and participates in other biological processes dependent upon proteolytic activity. Consequently, plasmin activity is tightly regulated by plasminogen activators and protease inhibitors. In this study, we examined whether regulation of plasminogen gene expression also might provide a new mechanism for controlling this system. We examined the effects of recombinant human interleukin-6 (rhIL-6), a pleiotropic cytokine, on plasminogen mRNA expression in primary murine hepatocytes and Hep3B human hepatoma cells. In primary hepatocytes, rhIL-6 and hydrocortisone separately increased plasminogen mRNA expression, but hydrocortisone did not markedly enhance the response to rhIL-6. Hep3B hepatoma cells exhibited more modest responses to rhIL-6. We used the polymerase chain reaction to amplify a 1,067-bp fragment of the human plasminogen promoter/5′ flanking region. This fragment was cloned upstream of a luciferase reporter gene. Hep3B cells transiently transfected with this construct provided ∼100-fold higher luciferase activity compared to cells transfected with control plasmids, and luciferase activity was increased ∼4.5-fold when these cells were treated with rhIL-6. Furthermore, mice injected with rhIL-6 exhibited increases in hepatic plasminogen mRNA. Circulating plasminogen levels were significantly higher in the mice injected with rhIL-6 compared to mice injected with saline. Mice injected with lipopolysaccharide (an inducer of IL-6 in vivo) also showed increased hepatic plasminogen mRNA. Thus, plasminogen gene expression can be modulated by rhIL-6, suggesting a new mechanism for regulating biological systems that use plasmin.

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