Dataset Information

Identification of the Secreted Proteins Originated from Primary Human Hepatocytes and HepG2 Cells.

ABSTRACT: The liver plays a pivotal role in whole-body carbohydrate, lipid, and protein metabolism. One of the key regulators of glucose and lipid metabolism are hepatokines, which are found among the liver secreted proteins, defined as liver secretome. To elucidate the composition of the human liver secretome and identify hepatokines in primary human hepatocytes (PHH), we conducted comprehensive protein profiling on conditioned medium (CM) of PHH. Secretome profiling using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) identified 691 potential hepatokines in PHH. Subsequently, pathway analysis assigned these proteins to acute phase response, coagulation, and complement system pathways. The secretome of PHH was compared to the secreted proteins of the liver hepatoma cell line HepG2. Although the secretome of PHH and HepG2 cells show a high overlap, the HepG2 secretome rather mirrors the fetal liver with some cancer characteristics. Collectively, our study represents one of the most comprehensive secretome profiling approaches for PHH, allowing new insights into the composition of the secretome derived from primary human material, and points out strength and weakness of using HepG2 cell secretome as a model for the analysis of the human liver secretome.

SUBMITTER: Franko A

PROVIDER: S-EPMC6723870 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Identification of the Secreted Proteins Originated from Primary Human Hepatocytes and HepG2 Cells.

Franko Andras A Hartwig Sonja S Kotzka Jörg J Ruoß Marc M Nüssler Andreas K AK Königsrainer Alfred A Häring Hans-Ulrich HU Lehr Stefan S Peter Andreas A

Nutrients 20190803 8

The liver plays a pivotal role in whole-body carbohydrate, lipid, and protein metabolism. One of the key regulators of glucose and lipid metabolism are hepatokines, which are found among the liver secreted proteins, defined as liver secretome. To elucidate the composition of the human liver secretome and identify hepatokines in primary human hepatocytes (PHH), we conducted comprehensive protein profiling on conditioned medium (CM) of PHH. Secretome profiling using liquid chromatography-electrosp ...[more]

PMID: 31382615

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Project description:Betaine-homocysteine methyltransferase (BHMT) regulates homocysteine levels in the liver. We previously reported that the alteration of BHMT is associated with alcoholic liver steatosis and injury. In this study, we tested whether BHMT protects hepatocytes from homocysteine-induced injury and lipid accumulation. Both BHMT transfectants of HepG2 cells and primary mouse hepatocytes with suppressed BHMT were generated. Comparisons were made between the cell models with respect to their response to homocysteine treatments. Homocysteine metabolism was impaired in HepG2 cells, and the expression of BHMT in HepG2 cells ameliorated the impairment and stabilized the levels of intracellular homocysteine after the addition of exogenous homocysteine. BHMT expression inhibited homocysteine-induced glucose-regulated protein 78 (GRP78) and C/EBP-homologous protein (CHOP) and homocysteine-induced cell death. A betaine treatment protected primary mouse hepatocytes from a homocysteine-induced increase in GRP78 and cell death but not a tunicamycin-induced increase. Homocysteine induced greater CHOP expression (2.7-fold) in BHMT small interfering RNA (siRNA)-transfected cells than in a control (1.9-fold). Homocysteine-induced cell death was increased by 40% in the siRNA-treated cells in comparison with the control. Apolipoprotein B (apoB) expression was higher and triglycerides and cholesterol were lower in HepG2 expressing BHMT. In primary mouse hepatocytes, homocysteine induced the accumulation of triglycerides and cholesterol, which was reduced in the presence of betaine. Betaine partially reduced homocysteine-induced sterol regulatory element binding protein 1 expression in HepG2 cells and increased S-adenosylmethionine in primary mouse hepatocytes.The BHMT/betaine system directly protects hepatocytes from homocysteine-induced injury but not tunicamycin-induced injury, including an endoplasmic reticulum stress response, lipid accumulation, and cell death. This system also exhibits a more generalized effect on liver lipids by inducing ApoB expression and increasing S-adenosylmethionine/S-adenosylhomocysteine.

Project description:Macroautophagy, a catabolic process of cellular self-digestion, is an important tumor cell survival mechanism and a potential target in antineoplastic therapies. Recent discoveries have implicated autophagy in the cellular secretory process, but potential roles of autophagy-mediated secretion in modifying the tumor microenvironment are poorly understood. Furthermore, efforts to inhibit autophagy in clinical trials have been hampered by suboptimal methods to quantitatively measure tumor autophagy levels. Here, we leveraged the autophagy-based involvement in cellular secretion to identify shed proteins associated with autophagy levels in melanoma. The secretome of low-autophagy WM793 melanoma cells was compared to its highly autophagic metastatic derivative, 1205Lu in physiological 3-dimensional cell culture using quantitative proteomics. These comparisons identified candidate autophagy biomarkers IL1B (interleukin 1, ?), CXCL8 (chemokine (C-X-C motif) ligand 8), LIF (leukemia inhibitory factor), FAM3C (family with sequence similarity 3, member C), and DKK3 (dickkopf WNT signaling pathway inhibitor 3) with known roles in inflammation and tumorigenesis, and these proteins were subsequently shown to be elevated in supernatants of an independent panel of high-autophagy melanoma cell lines. Secretion levels of these proteins increased when low-autophagy melanoma cells were treated with the autophagy-inducing tat-BECN1 (Beclin 1) peptide and decreased when ATG7 (autophagy-related 7) was silenced in high-autophagy cells, thereby supporting a mechanistic link between these secreted proteins and autophagy. In addition, serum from metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from patients with low-autophagy tumors. These results suggest that autophagy-related secretion affects the tumor microenvironment and measurement of autophagy-associated secreted proteins in plasma and possibly in tumors can serve as surrogates for intracellular autophagy dynamics in tumor cells.

Project description:Primary human hepatocytes (PHH) are a main instrument in drug metabolism research and in the prediction of drug-induced phase I/II enzyme induction in humans. The HepG2 liver-derived cell line is commonly used as a surrogate for human hepatocytes, but their use in ADME and toxicity studies can be limited because of lowered basal levels of metabolizing enzymes. Despite their widespread use, the transcriptome of HepG2 cells compared to PHH is not well characterized. In this study, microarray analysis was conducted to ascertain the differences and similarities in mRNA expression between HepG2 cells and human hepatocytes before and after exposure to a panel of fluoroquinolone compounds. Comparison of the naÃ¯ve HepG2 cell and PHH transcriptomes revealed a substantial number of basal gene expression differences. When HepG2 cells were dosed with a series of fluoroquinolones, trovafloxacin, which has been associated with human idiosyncratic hepatotoxicity, induced substantially more gene expression changes than the other quinolones, similar to previous observations with PHH. While TVX-treatment resulted in many gene expression differences between HepG2 cells and PHH, there were also a number of TVX-induced commonalities, including genes involved in RNA processing and mitochondrial function. Taken together, these results provide insight for interpretation of results from drug metabolism and toxicity studies conducted with HepG2 cells in lieu of PHH, and could provide further insight into the mechanistic evaluation of TVX-induced hepatotoxicity. Experiment Overall Design: HepG2 cells were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in Minimum Essential Medium (Invitrogen Life Technologies, Carlsbad,CA) with 10% Fetal Bovine Serum under a humidified 5% CO2 atmosphere using T-162 plastic culture flasks. The cells were split when they reached approximately 70-85% confluence after washing with sterile phosphate buffered saline and detachment of the cells with trypsin (Invitrogen Life Technologies, Carlsbad, CA). The HepG2 cells were cultured in 6-well plastic plates upon exposure to quinolone compounds. Primary human hepatocytes, obtained from In Vitro Technologies (IVT, Baltimore, MD) in 6-well type I collagen coated plates, were cultured with 2 mL of Hepatocyte Incubation Media (IVT) at 37Â°C with 5% CO2 for 24 hours after receipt. Experiment Overall Design: For the genomic experiments, quinolone compounds, dissolved in 0.1 N KOH (Sigma Chemical Co., St. Louis, MO), were added to the wells with fresh media at levels of 100 ÂµM (HepG2) or 400 ÂµM (primary human hepatocytes) for 24 hours using at least two technical replicates. Trovafloxacin was dosed using two separate preparations of HepG2 cells and two separate donors of human hepatocytes. Vehicle control cells were dosed with an equivalent volume of 0.1N KOH as the experimental samples. For intracellular comparison (HepG2 cells vs PHH), naÃ¯ve cells were harvested using TRIzolâ?¢ reagent (Invitrogen Life Technologies, Carlsbad, CA). Experiment Overall Design: Total RNA was isolated from the TRIzolâ?¢ extracts using the standard procedure from the manufacturer. O.D. at 260 nm determined RNA concentrations. RNA quality was accessed using an Agilent Technologies bioanalyzer before proceeding to microarray sample preparation. Microarray analysis was performed using the standard protocol provided by Affymetrix Inc. (Santa Clara, CA) and as previously described, starting with 5 Âµg of total RNA (Richert et al., 2006). Experiment Overall Design: Fragmented, labeled cRNA was hybridized to an Affymetrix human genome U133A array, which contains sequences corresponding to roughly 22,200 transcripts at 45Â°C overnight. The arrays were washed, developed, and scanned.

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Dataset Information

Identification of the Secreted Proteins Originated from Primary Human Hepatocytes and HepG2 Cells.

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Identification of the Secreted Proteins Originated from Primary Human Hepatocytes and HepG2 Cells.

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