The transcriptional landscape of a hepatoma cell line grown on scaffolds of extracellular matrix proteins

Abstract Background The behavior of cells in vivo is complex and highly dynamic, as it results from an interplay between intercellular matrix proteins with surface receptors and other microenvironmental cues. Although the effects of the cellular niche have been investigated for a number of cell types using different molecular approaches, comprehensive assessments of how the global transcriptome responds to 3D scaffolds composed of various extracellular matrix (ECM) constituents at different concentrations are still lacking. Results In this study, we explored the effects of two diverse extracellular matrix (ECM) components, Collagen I and Matrigel, on the transcriptional profile of cells in a cell culture system. Culturing Huh-7 cells on traditional cell culture plates (Control) or on the ECM components at different concentrations to modulate microenvironment properties, we have generated transcriptomics data that may be further explored to understand the differentiation and growth potential of this cell type for the development of 3D cultures. Our analysis infers transcription factors that are most responsible for the transcriptome response to the extracellular cues. Conclusion Our data indicates that the Collagen I substrate induces a robust transcriptional response in the Huh-7 cells, distinct from that induced by Matrigel. Enhanced hepatocyte markers (ALB and miR-122) reveal a potentially robust remodelling towards primary hepatocytes. Our results aid in defining the appropriate culture and transcription pathways while using hepatoma cell lines. As systems mimicking the in vivo structure and function of liver cells are still being developed, our study could potentially circumvent bottlenecks of limited availability of primary hepatocytes for preclinical studies of drug targets.

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The transcriptional landscape of a hepatoma cell line grown on scaffolds of extracellular matrix proteins

10.1101/2020.07.18.191395 ◽

2020 ◽

Author(s):

Souvik Ghosh ◽

Anastasiya Börsch ◽

Mihaela Zavolan

Keyword(s):

Cell Culture ◽

Extracellular Matrix ◽

Hepatoma Cell ◽

Cell Types ◽

Growth Potential ◽

Matrix Proteins ◽

Hepatoma Cell Line ◽

Sequencing Data ◽

Data Set

AbstractThe behavior of cells in vivo is complex and highly dynamic, as it results from an interplay between components of the intercellular matrix proteins with surface receptor and other microenvironmental cues. Although the effects of the cellular niche have been investigated for a number of cell types using different molecular approaches, comprehensive assessments of how the global transcriptome responds to 3D scaffolds composed of various extracellular matrix (ECM) constituents are still lacking. In this study, we explored the effect of the biomechanical parameters of Collagen I and Matrigel (ECM) on transcriptional gene regulation in a cell culture system. Using Huh-7 cells cultured on traditional cell culture plates or on the components of the ECM at different concentrations to modulate microenvironment properties, we have generated transcriptome sequencing data that may be further explored to understand the differentiation and growth potential of this cell for the development of 3D cultures. Assessment of the hepatocyte phenotype in relation to our transcriptomic data set would be very useful for the development of systems mimicking the in vivo structure and function of liver cells which still remains a challenge.

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Dissecting the Mechanisms of Hepcidin and BMP-SMAD Pathway Regulation By FKBP12

Blood ◽

10.1182/blood-2021-152172 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2008-2008

Author(s):

Alessia Pagani ◽

Mariateresa Pettinato ◽

Alessandro Dulja ◽

Silvia Colucci ◽

Mariam Aghajan ◽

...

Keyword(s):

Hepatoma Cell ◽

Hereditary Hemochromatosis ◽

Hepatoma Cell Line ◽

Main Role ◽

Type I ◽

Primary Hepatocytes ◽

Smad Pathway ◽

Smad Signaling

Abstract The BMP-SMAD pathway is activated when a dimeric ligand (BMP) interacts with a dimeric serine threonine kinase receptor (BMPRII) and triggers the activation of a dimeric BMP type I receptor (BMPRI). Catalytically active BMPRIs phosphorylate SMAD1/5/8 that, upon SMAD4 binding, translocate to the nucleus to regulate the expression of BMP target genes, including hepcidin. Hepcidin is the main regulator of iron homeostasis that controls body iron levels by binding and blocking the sole iron exporter ferroportin. In agreement, hepcidin expression is homeostatically activated by serum and liver iron, and its deficiency is a common hallmark of Hereditary Hemochromatosis (HH) and the major cause of iron overload in beta thalassemia. The components of the BMP-SMAD pathway relevant for hepcidin regulation are ALK2 and ALK3 (BMPRI); BMPR2 and ACVR2A (BMPRII), BMP2 and BMP6 (BMP ligands). Recently, we have identified the immunophilin FKBP12 as an inhibitor of hepcidin and demonstrated that FKBP12 binds ALK2 to avoid ligand-independent activation of the BMP-SMAD pathway. To investigate the mechanism of BMP-SMAD pathway and hepcidin regulation by FKBP12, we performed in vitro, ex vivo and in vivo studies. We found that FKBP12 sequestration by the immunosuppressive drug Tacrolimus (TAC) stabilizes ALK2-ALK2 homodimers and ALK2-ALK3 heterodimers in a transfected human hepatoma cell line. In addition, it increases the interaction of ALK2 with ACVR2A and BMPR2. To investigate the role of FKBP12 on BMP-SMAD signaling, BMPRI and II were silenced in murine primary hepatocytes. Despite FKBP12 co-immunoprecipitates only with ALK2, silencing of Alk2 and Alk3 completely blunts TAC-mediated BMP-SMAD pathway activation, suggesting that FKBP12 functionally interacts also with ALK3. Acvr2a silencing impairs TAC-dependent hepcidin upregulation, whereas Bmpr2 silencing does not. As expected, Fkbp12 silencing abrogates hepcidin upregulation by TAC, confirming the main role of this immunophilin in hepcidin regulation. In vivo, TAC treatment upregulates hepcidin in wild type and HH mouse models, but surprisingly, Fkbp12 mRNA downregulation by ASOs does not. Interestingly, Fkbp 2, 4 and 8 are highly expressed in murine hepatocytes and, according to literature data, are able to bind to TAC. Of note, Fkbp12 is the least expressed immunophilin in murine primary hepatocytes. To further investigate the FKBPs involved in TAC-dependent hepcidin regulation, Fkbp2, 4 and 8 were knockdown in murine primary HCs that were then treated with TAC. The TAC effect is preserved in siFkbp2- and siFkbp4-derived HCs, but abolished when Fkbp8 was downregulated. Overall these data suggest that: 1) FKBP12 regulates BMP-SMAD signaling by favoring ALK2-ALK3 homo and heterodimerization, and interaction with BMPRII in the absence of ligands; 2) TAC-mediated hepcidin upregulation is dependent upon ALK2, ALK3, ACVR2A, FKBP12 and FKBP8. 3) In vivo, TAC treatment upregulates hepcidin whereas Fkbp12 silencing does not, suggesting the existence of redundancy between the different FKBPs. Further studies are needed to dissect the role of FKBP8 in BMP-SMAD pathway and hepcidin regulation. Disclosures Aghajan: Ionis Pharmaceuticals, Inc.: Current Employment. Muckenthaler: Silence Therapeutics: Research Funding. Guo: Ionis Pharmaceuticals, Inc.: Current Employment.

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Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

International Journal of Molecular Sciences ◽

10.3390/ijms22010202 ◽

2020 ◽

Vol 22 (1) ◽

pp. 202

Author(s):

Josephin Glück ◽

Julia Waizenegger ◽

Albert Braeuning ◽

Stefanie Hessel-Pras

Keyword(s):

Cell Death ◽

Cell Viability ◽

Pyrrolizidine Alkaloids ◽

Defense Mechanism ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Dependent Manner ◽

Human Hepatoma Cell

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

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Cwp84, a Surface-Associated Protein of Clostridium difficile, Is a Cysteine Protease with Degrading Activity on Extracellular Matrix Proteins

Journal of Bacteriology ◽

10.1128/jb.00578-07 ◽

2007 ◽

Vol 189 (20) ◽

pp. 7174-7180 ◽

Cited By ~ 79

Author(s):

Claire Janoir ◽

Séverine Péchiné ◽

Charlotte Grosdidier ◽

Anne Collignon

Keyword(s):

Extracellular Matrix ◽

Clostridium Difficile ◽

Cysteine Protease ◽

Specific Inhibitor ◽

Extracellular Matrix Proteins ◽

Maximum Activity ◽

Matrix Proteins ◽

Specific Antibodies ◽

Type Iv

ABSTRACT Clostridium difficile pathogenicity is mediated mainly by its A and B toxins, but the colonization process is thought to be a necessary preliminary step in the course of infection. The aim of this study was to characterize the Cwp84 protease of C. difficile, which is highly immunogenic in patients with C. difficile-associated disease and is potentially involved in the pathogenic process. Cwp84 was purified as a recombinant His-tagged protein, and specific antibodies were generated in rabbits. Treatment of multiple-band-containing eluted fractions with a reducing agent or with trypsin led to accumulation of a unique protein species with an estimated molecular mass of 61 kDa, corresponding most likely to mature autoprocessed Cwp84 (mCwp84). mCwp84 showed concentration-dependent caseinolytic activity, with maximum activity at pH 7.5. The Cwp84 activity was inhibited by various cysteine protease inhibitors, such as the specific inhibitor E64, and the anti-Cwp84-specific antibodies. Using fractionation experiments followed by immunoblot detection, the protease was found to be associated with the S-layer proteins, mostly as a nonmature species. Proteolytic assays were performed with extracellular matrix proteins to assess the putative role of Cwp84 in the pathogenicity of C. difficile. No degrading activity was detected with type IV collagen. In contrast, Cwp84 exhibited degrading activity with fibronectin, laminin, and vitronectin, which was neutralized by the E64 inhibitor and specific antibodies. In vivo, this proteolytic activity could contribute to the degradation of the host tissue integrity and to the dissemination of the infection.

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Characterization of Collagen I Fiber Thickness, Density, and Orientation in the Human Skin In Vivo Using Second-Harmonic Generation Imaging

Photonics ◽

10.3390/photonics8090404 ◽

2021 ◽

Vol 8 (9) ◽

pp. 404

Author(s):

Marius Kröger ◽

Johannes Schleusener ◽

Sora Jung ◽

Maxim E. Darvin

Keyword(s):

Extracellular Matrix ◽

Second Harmonic Generation ◽

Harmonic Generation ◽

Skin Diseases ◽

Second Harmonic ◽

Skin Aging ◽

Collagen I ◽

Fast Fourier Transformation ◽

Occurrence Matrix

The assessment of dermal alterations is necessary to monitor skin aging, cancer, and other skin diseases and alterations. The gold standard of morphologic diagnostics is still histopathology. Here, we proposed parameters to distinguish morphologically different collagen I structures in the extracellular matrix and to characterize varying collagen I structures in the skin with similar SAAID (SHG-to-AF Aging Index of Dermis, SHG—second-harmonic generation; AF—autofluorescence) values. Test datasets for the papillary and reticular extracellular matrix from images in 24 female subjects, 36 to 50 years of age, were generated. Parameters for SAAID, edge detection, and fast Fourier transformation directionality were determined. Additionally, textural analyses based on the grey level co-occurrence matrix (GLCM) were conducted. At first, changes in the GLCM parameters were determined in the native greyscale images and, furthermore, in the Hilbert-transformed images. Our results demonstrate a robust set of parameters for noninvasive in vivo classification for morphologically different collagen I structures in the skin, with similar and different SAAID values. We anticipate our method to enable an automated prevention and monitoring system with an age- and gender-specific algorithm.

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The extracellular matrix of the developing cornea: diversity, deposition and function*

Development ◽

10.1242/dev.103.supplement.195 ◽

1988 ◽

Vol 103 (Supplement) ◽

pp. 195-205

Author(s):

J. B. L. Bard ◽

M. K. Bansal ◽

A. S. A. Ross

Keyword(s):

Extracellular Matrix ◽

Chondroitin Sulphate ◽

Corneal Stroma ◽

Experimental System ◽

Collagen I ◽

And Function ◽

20 Nm

This paper examines the role of the extracellular matrix (ECM) in the development of the cornea. After a brief summary of the corneal structure and ECM, we describe evidence suggesting that the differentiation of neural crest (NC) cells into endothelium and fibroblasts is under the control of ocular ECM. We then examine the role of collagen I in stromal morphogenesis by comparing normal corneas with those of homozygous Movl3 mice which do not make collagen I. We report that, in spite of this absence, the cellular morphology of the Movl3 eye is indistinguishable from that of the wild type. In the 16-day mutant stroma, however, the remaining collagens form small amounts of disorganized, thin fibrils rather than orthogonally organized 20 nm-diameter fibrils; a result implying that collagen I plays only a structural role and that its absence is not compensated for. It also suggests that, because these remaining collagens will not form the normal fibrils that they will in vitro, fibrillogenesis in the corneal stroma differs from that elsewhere. The latter part of the paper describes our current work on chick stromal deposition using corneal epithelia isolated with an intact basal lamina that lay down in vitro ∼3μm-thick stromas of organized fibrils similar to that seen in vivo. This experimental system has yielded two unexpected results. First, the amount of collagen and proteoglycans produced by such epithelia is not dependent on whether its substratum is collagenous and we therefore conclude that stromal production by the intact epithelium is more autonomous than hitherto thought. Second, chondroitin sulphate (CS), the predominant proteoglycan, appears to play no role in stromal morphogenesis: epithelia cultured in testicular hyaluronidase, which degrades CS, lay down stromas whose organization and fibrildiameter distribution are indistinguishable from controls. One possible role for CS, however, is as a lubricant which facilitates corneal growth: it could allow fibrils to move over one another without deforming their orthogonal organization. Finally, we have examined the processes of fibrillogenesis in the corneal stroma and conclude that they are different from those elsewhere in the embryo and in vitro, perhaps because there is in the primary stroma an unidentified, highly hydrated ECM macromolecule that embeds the fibrils and that may mediate their morphogenesis.

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In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction

Blood ◽

10.1182/blood.v81.3.617.617 ◽

1993 ◽

Vol 81 (3) ◽

pp. 617-623 ◽

Cited By ~ 140

Author(s):

J Fandrey ◽

HF Bunn

Keyword(s):

Polymerase Chain Reaction ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Mrna Levels ◽

Chain Reaction ◽

Human Hepatoma Cell ◽

Competitive Polymerase Chain Reaction ◽

Polymerase Chain

Abstract The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15–0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.

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Changes in distribution of extracellular matrix proteins during wound repair in corneal endothelium.

Journal of Histochemistry & Cytochemistry ◽

10.1177/36.4.3279112 ◽

1988 ◽

Vol 36 (4) ◽

pp. 409-416 ◽

Cited By ~ 16

Author(s):

S R Gordon

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Corneal Endothelium ◽

Wound Repair ◽

Matrix Proteins ◽

Post Injury ◽

Cytoplasmic Staining ◽

Wound Area ◽

Diffuse Cytoplasmic Staining

The distribution of fibronectin (FN) and laminin (LM) in non-injured and injured rat corneal endothelium in vivo was investigated by light microscopy using immunoperoxidase cytochemistry. In non-injured tissues, both FN and LM have distinct pericellular staining patterns and exhibit some diffuse cytoplasmic staining. After a circular freeze injury, cells migrating into the wound area at 24 hr lack the characteristic pericellular staining observed in non-injured cells but show cytoplasmic staining for both extracellular matrix glycoproteins. Endothelial cells on the periphery of such preparations do not partake in wound repair and retain their pericellular staining patterns. Forty-eight hours after injury, cells have filled in the wound area but are disorganized. They display intracellular FN and LM staining but do not demonstrate any pericellular staining. When observed 10 days after injury, a uniform monolayer has formed but neither FN nor LM is detected pericellularly. By 14 days post injury, endothelial cells in the wound area display pericellular FN patterns but not LM patterns. This may reflect differences in the function of each glycoprotein in maintaining the attachment of the endothelium to Descemet's membrane.

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The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4405 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4405-4414 ◽

Cited By ~ 135

Author(s):

C M DiPersio ◽

D A Jackson ◽

K S Zaret

Keyword(s):

Extracellular Matrix ◽

Transcription Factors ◽

Cell Line ◽

Cell Morphology ◽

Transcriptional Activation ◽

Hepatoma Cell ◽

Collagen Gel ◽

Hepatoma Cell Line ◽

Transcriptional Enhancer ◽

Mrna Accumulation

The extracellular matrix (ECM) promotes tissue morphogenesis, cell migration, and the differentiation of a variety of cell types. However, the mechanisms by which ECM causes differentiated gene expression have been unknown. In this report, we show that culturing the hepatocyte-derived cell line H2.35 on an ECM gel changes cell morphology and selectively stimulates the transcription of a subset of liver-specific genes, including serum albumin. Transcriptional activation by ECM also occurs with transfected plasmids bearing the transcriptional enhancer of the albumin gene. ECM substrates of different composition activated the albumin enhancer only when the ECM promoted a cuboidal, differentiated cell morphology. Enhancer activation by the ECM was mediated by two liver transcription factors, HNF3 alpha and eH-TF, which appear to be regulated differently by matrix. Specifically, we found that a collagen gel substratum caused a selective increase in the factor HNF3 alpha at the levels of mRNA accumulation and DNA-binding activity in nuclear extracts, both in H2.35 cells and in the hepatoma cell line HepG2. We conclude that the ECM can stimulate cell differentiation by selectively activating transcriptional regulatory factors and that such regulation occurs coordinately with ECM-promoted changes in cell shape.

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