HCT is by far the most effective way to treat the clonal disorder disease [6
]. However, patients commonly have an increase of nontransferrin-bound iron (NTBI) and show hepatic iron overload already before HCT or after transplant conditioning [7
]. Excessive iron can stimulate the formation of ROS via the Fenton reaction [9
], and progressive accumulation of ROS may damage mitochondrial and nuclear DNA through lipid peroxidation [10
]. In our previous work, we have shown that iron overload can induce apoptosis via both extrinsic and intrinsic pathways in hepatic cells [5
]. To our knowledge, the exact cellular consequences and the cellular processes of iron overload in hepatocytes are still not very clear. In this current study, we utilized proteomics technique to dissect more signaling pathways that related to hepatocytes iron overload.
The mass spectrum results showed FAC-induced HH4 cells contained 93 differentially expressed proteins and activated multiple pathways (Figure 1
A and Table 1
). Levels of protein ATP6V1H, which facilitates acidification of intracellular endosomes formed by transferrin/transferrin receptor-mediated endocytosis [11
], increased 1.6 times (Table 1
), while levels of protein EHD2, which plays an important role in regulating transferrin exit from ERC (endocytic recycling compartment) [13
], decreased three times (Table 1
) when HH4 cells were treated with 5 mM FAC at 24 h. These two significantly changed proteins indicate that HH4 cells may absorb a large number of extracellular iron ion via endocytosis.
Addition of excess iron inevitably led to iron dyshomeostasis and thus triggered differential expression of intracellular iron homeostasis associated proteins in HH4 cells (Figure 1
B). Levels of protein TF (serotransferrin) which is mainly produced by hepatocytes and capable of binding excess iron ions and delivering it into cells increased sharply in FAC-overload HH4 cells (Table 1
and Figure 3
A). Levels of protein IRP1, which is also known as cytoplasmic aconitate hydratase (ACO1), and is the trans-acting factor located in the cytoplasm that can bind with high affinity to RNA motifs related to iron homeostasis [14
], decreased 0.5 times (Table 1
) and levels of the protein ferritin, each molecule of which is capable of storing up to 4500 iron atoms [15
], increased 4.4 times (Figure 4
A) in FAC- treated HH4 cells. Meanwhile, our MS data displayed the expression of γ-secretase C-terminal fragment 59 and amyloid-like protein 2 were increased 3.3-fold and 2.9-fold in FAC treated cells, respectively (Table 1
), suggesting that full length APP protein, which can stabilize surface FPN , may participate in the efflux of iron from HH4 cells [16
]. Our MS data also showed the expression of metallothionein-2 increased 1.8 times but SOD2 expression decreased 0.6 times in iron-loaded HH4 cells. Metallothioneins (MTs), which are conserved in the animal kingdom, can chelate heavy and trace metals such as zinc, copper or iron through sulfur-based clusters, and therefore the main function of MTs is the regulation of homeostasis, like the protection against oxidative stress or metals [17
]. While SOD2 (superoxide dismutase [Mn], mitochondrial) functioned as the antioxidant is known to scavenge the ROS, specifically the hydroxyl radical [18
]. In a word, these significantly changed proteins indicate that iron overload induces biological process of di-, tri-valent inorganic cation homeostasis in HH4 cells (Figure 1
B and Table 2
In addition, hepcidin is undoubtedly the most central regulator of iron hemostasis, and our results also confirmed that FAC-induced HH4 cells showed evidently enhanced transcription levels of gene TF, TfR1/2, IL-6, IL6ST, STAT3 and hepcidin (Figure 4
), indicating that hepcidin was involved in the re-regulation of iron homeostasis in iron overload-treated HH4 cells.
Mass spectral analysis also indicated the participation of other biological process including response to wounding, cell apoptosis and inflammatory response in FAC-induced HH4 cells. For example, thrombospondin-1 (THBS1) which is a matricellular glycoprotein first discovered in activated platelets that plays an important role in the process of wound healing [19
], decreased 0.47 times (Table 1
and Figure 3
B), while the expression of endophilin-B1, which is involved in the regulation of apoptosis helping the maintenance of mitochondrial morphology and autophagy [20
], was apparently increased in HH4 cells after iron overload treatment (Table 1
). Among these, the most interestingly finding is the expression of inflammation mediator TLR2 increased markedly in FAC- treated HH4 cells (Table 1
). Inflammation is a fundamental biological process that stands at the foreground of acute and chronic pathological conditions [21
]. Toll-like receptors (TLRs) discovered in the 1990s are a family of very similar proteins containing leucine-rich repeats and their activation triggers a signaling cascade which leads to the production of cytokines/chemokines, further initiating an inflammatory response [22
]. So far 13 TLRs have been identified, 10 human TLRs (TLR1-10), and 12 mouse TLRs (TLR1-9, TLR11 and TLR12) [23
]. It is well known that the acute phase response to infection and inflammation can cause alterations in iron homeostasis, thereby reducing iron supplies to pathogens. A recent study found that the acute inflammatory condition mediated by TLR2 and TLR6 induced rapid hepcidin-independent hypoferremia through decreasing the messenger RNA and protein expression of ferroportin (FPN) in mice injected with TLR ligands [24
]. Although TLR2 has been implicated in the response to infection mediated by TLR2 ligands, includes molecules with diacyl and triacylglycerol moieties, proteins and polysaccharides, scanty data is available on the direct relationship between TLR2 and iron overload. Therefore, our next research validated TLR2 mRNA and protein expression in FAC treated HH4 cells (Table 1
, Figure 3
A and Figure 5
A), which is consistent with proteomics data. In addition, our experiment indicated that inhibition of TLR2 by siRNA inactivated IL-6 expression via NF-κB pathway in FAC treated hepatocytes (Figure 5
4. Experimental Section
4.1. Cell Culture
The non-transformed human hepatocyte cell line HH4 was maintained as described previously [7
]. Briefly, HH4 cells were maintained in Williams’ medium E (Gibco Laboratories, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS), 0.1% ITS, dexamethasone (0.04 μg/mL) (Sigma Chemical Co., St. Louis, MO, USA), gentamycin (50 μg/mL) (Sigma Chemical Co.), HEPES (20 mM) and sodium pyruvate (1 mM) (Gibco Laboratories).
4.2. In Vitro Iron Overload Model
The in vitro
iron overload model was established as previously described [5
]. Briefly, HH4 cells were seeded in 15 cm culture dishes at 1 × 105
cells/mL, incubated overnight at 37 °C in 5% CO2
atmosphere, and then treated with or without 5 mM FAC (Sigma Chemical Co.) for 24 h. The experiments were repeated three times.
4.3. Proteomics Analysis
4.3.1. Cell Lysis and Protein Digestion
HH4 cells were washed three times with PBS buffer, harvested by trypsinization, and centrifuged at 1000× g for 5 min. After the addition of T-PER Reagent (Thermo Scientific, Rockford, CA, USA), PMSF (1 mM) and 0.1% aprotinin, the cell pellets were lysed for 30 min on ice and centrifuged at 13, 800× g for 15 min. Then the supernatant was transferred into new tubes. The Broadford assay was used to determine the protein concentration (Beyotime Institute of Biotechnology, Shanghai, China). Proteins were reduced with 10 mM dithiothreitol (DTT) at 56 °C for 45 min and then alkylated with 10 mM iodoacetamide (IAM) (Sigma Chemical Co.) for 20 min at room temperature in the dark. The sample was diluted four times with 40 mM ammonium bicarbonate buffer. Sequencing grade trypsin (Promega, Madison, WI, USA) was added in a 1:50 (w/w) ratio and the incubation was carried out overnight at 37 °C. After an over-night digestion peptides were recovered from the filter. Peptides were desalted with Oasis 1cc cartridges and flow through from Oasis cartridge was desalted with Sep-Pak C18 1cc (Waters, Milford, MA, USA). Elutions from Oasis and Sep-Pak were combined together and concentrated in SpeedVac.
4.3.2. Nanoflow LC-MS/MS
Peptide digests dissolved in loading buffer (0.1% trifluoroacetic acid—TFA) were loaded onto an in-house packed 20 cm capillary column with 3 µm Reprosil-Pur C18 beads (Dr. Maisch GmbH, Ammerbuch, Germany) using an EASY-nLC 1000 system (Thermo Scientific). Running buffer A was 0.1% TFA in water and running buffer B was 0.1% TFA in ACN (acetonitrile). The total gradient was 120 min and the flow rate started at 300 nL/min. The detailed gradient was 6% ACN with a linear increase to 30% ACN over 105 min followed by 4 min linear increase to 90% ACN. MS data were acquired using a data-dependent top-20 method on Q Exactive (Thermo Scientific, Bremen, Germany). Spray voltage was set to 2.0 kV, S-lens RF level at 60, and capillary temperature at 275 °C. Full scan resolutions were set to 60,000 at m
200 and AGC (automatic gain control) was 3 × 106
with a maximum fill time of 20 ms. The range of full mass was set to 350–1500 m
. MS2 scan resolutions were set to 15,000 at m
200 and AGC was 5 × 104
with a maximum fill time of 45 ms. Isolation width was set at 1.6 Th. A fixed first mass of 110 was used. Normalized collision energy was set at 27. Peptide match was set to “preferred” and isotope exclusion was on. Precursor ions with single, un-assigned, charge states were removed from fragmentation selection [25
4.3.3. Data Analysis
All data were analyzed with the MaxQuant version 188.8.131.52 [28
], with the Andromeda search engine [29
]. The false discovery rate (FDR) was set at 1% for protein, peptide spectrum match. Peptides were required to have a minimum length of six amino acids and a maximum mass of 10,000 Da. Fragmentation spectra were searched by Andromeda in the Uniprot human database (version 201502; 90,300 entries) combined with 262 common contaminants [29
]. Enzyme specificity was set as C-terminal to arginine and lysine and a maximum of two missed cleavages. Second peptides search was enabled. For proteome identification, carbamidomethylation (C) was set as a fixed modification. deamidation (NQ) and oxidation (M) were set as variable modifications. To identify significantly regulated proteins, LFQ intensities were used. Proteins with p
-value < 0.05, fold change > 1.5 or < 0.67 were recognized as significant change.
Total RNA was extracted using RNA pure Tissue Kit (Cwbio, Beijing, China) according to the manufacturer’s instruction. Real-time RT-PCR amplification was performed using an Ultra SYBR mixture kit (Cwbio) on the C1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA, USA), and the results were analyzed using CFX Manager software (Bio-Rad). Target gene expression levels were quantified using the formula 2−ΔΔct
]. Primer sequences (Invitrogen Corporation, Shanghai, China) and optimal PCR annealing temperatures are listed in Table 3
4.5. Western Blot Analysis
Cells were lysed with RIPA (1% Triton X-100, 150 mM NaCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM tetrasodium pyrophosphate, 50 mM sodium fluoride, 1 mM Na3VO4, 2 mM phenylmethanesulfonyl fluoride, 0.076 U/mL aprotinin). After centrifugation at 13,800× g for 15 min, the supernatants were collected. Protein content was determined using BCA Protein Assay. Equal amounts of protein were subjected to SDS-PAGE. Proteins were electrophoretically transferred to PVDF membranes (Bio-Rad), and blocked with phosphate buffered saline/0.05% Tween-20 (PBS-T) containing 5% non-fat dry milk or BSA at room temperature (RT) for 1 h. Membranes were incubated with primary antibody TLR2, MyD88 (Abcam, Cambridge, MA, USA), p-p38 MAPK, p38 MAPK, p-NF-κB or NF-κB (Cell Signaling Technology, Boston, MA, USA) at a 1:1000 dilution in PBS-T at 4 °C overnight, washed four times with PBS-T, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime Institute of Biotechnology) in PBS-T at room temperature for 1 h. Protein was detected using an enhanced chemoluminiscence (ECL) kit (Tiangen, Beijing, China).
4.6. RNA Interference (RNAi) of Gene TLR2
For gene silencing assays, the small interfering RNA of TLR2 (siTLR2) gene and negative control siRNA (siNC) were synthesized by Invitrogen Corporation. The sense and anti-sense strands of siRNAs were: siRNA 1, 5′-GGUGAAACAAAUUCAUUGATT-3′ (sense), 5′-UCAAUGAAUUUGUUUCACCTT-3′ (antisense); siRNA 2, 5′-CCUCUCUACAAACUUUAAUTT-3′ (sense), 5′-AUUAAAGUUUGUAGAGAGGTT-3′ (antisense); siRNA 3, 5′-GCAACUCAAAGAACUUUAUTT-3′ (sense), 5′-AUAAAGUUCUUUGAGUUGCTT-3′ (antisense); hepatocytes were transiently transfected with oligofectamine according to the manufacturer’s protocol in the presence or absence of 60 nM siRNA duplex. Western blot analysis of protein expression level of TLR2 was performed to identify inhibitory effect of siRNA in HH4 cell. After 48 h transfection with indicated siRNA, cells were treated with 5 mM FAC for 24 h and then the expression of a series of inflammation-related genes was detected using quantitative real-time RT-PCR or Western blot assay.
4.7. Statistical Analysis
Results were analyzed by one-way analysis of variance (ANOVA) or 2-sample unpaired t-test using Prism 5 program (Version 5.0). Differences in results were considered significant when p values were ≤ 0.05. Results are given as mean ± SEM of at least 3 independent experiments.