Keywords RAGE - engineered cell line - lentiviral - transduction - flow cytometry
Introduction
Receptor for advanced glycation end-products (RAGE) is one of the members of the immunoglobulin
superfamily and a multiligand cell surface receptor with structural homology to other
immunoglobulin-like receptors.[1 ] RAGE consists of an extracellular domain, a transmembrane domain, and a cytoplasmic
domain.[2 ] The extracellular domain of RAGE includes a variable domain (V1) and two constant
domains (C1 and C2), which are responsible for binding by different ligands.[3 ] V1 and C1 domains are generally regarded as the main interaction motifs with various
ligands. Although the cytoplasmic tail is very short, it is critical for intracellular
signal transduction.[4 ] RAGE has a variety of ligands, such as advanced glycation end-products (AGEs),[5 ] high mobility group protein B1 (HMGB1),[6 ] amyloid β protein (Aβ),[7 ] and S100 family proteins.[8 ] All these ligands interact with the extracellular domain of RAGE and activate downstream
signaling pathways, including activation of extracellular regulated protein kinases
(ERK 1/2) and phosphatidylinositol 3-kinase (PI3K).[9 ]
[10 ]
[11 ]
[12 ]
[13 ] Activated RAGE promotes the production of reactive oxygen species and proinflammatory
cytokines, and it also inhibits the expression of tumor suppressor genes.[14 ]
[15 ] Overactivation of RAGE is associated with many diseases, including diabetes complications,
atherosclerosis, neurodegenerative diseases, liver fibrosis, and tumors.[9 ]
[16 ]
[17 ]
[18 ]
[19 ]
[20 ] Blockade of ligand/RAGE receptor interaction is a potential strategy for the treatment
of these diseases, and RAGE has become a new target for drug discovery.
Several cell lines have been used in studies of RAGE ligand activities or biological
function in vitro, such as neuroblastoma cells (SH-SY5Y), breast cancer cells (MCF-7),
and human embryonic kidney cells (HEK293). However, RAGE expression is low in these
cell lines, which were not suitable for investigation of the affinity of RAGE ligands
or anti-RAGE antibodies.[21 ] The transgenic cell lines play a critical role in processes of new drug development,
such as drug screening, activity assay, and expression of recombinant protein or antibody,
etc. Establishment of an engineered cell line for expression of human RAGE (hRAGE)
was necessary to support new drug development for targeting RAGE. Mouse embryonic
fibroblast NIH3T3 was widely used in cellular signal transduction, protein–protein
interaction, and molecular activity assays because it was easy to maintain, transfect,
and grow.[22 ]
[23 ]
[24 ] In this study, NIH3T3 cells were used to generate transgenic cell lines for stable
expression of hRAGE.
Materials and Methods
Instrument
A super clean bench (Suzhou Purification Equipment Co., Ltd., China), a cell counter
(Ruiyun Biotechnology Co., Ltd.), a chemiluminescence imager (Tanon, China), a multifunctional
microplate reader (Tecan, Switzerland), a flow cytometer (Beckman Coulter, United
States), and protein electrophoresis gel equipment (Bio-Rad, United States).
Materials
NIH3T3 and HEK293T were purchased from Chinese Academy of Sciences Cell Bank (ACSCB),
anti-hRAGE rabbit polyclonal antibody and rabbit immunoglobulin G (IgG) from Shanghai
Kaijing Biotechnology Company, China, pLVX-puro-human-RAGE-HA from Universal Biological
System Co., Ltd., China, Aβ1–42 from Chutai Biotechnology Company, China, mouse anti-HA
tag antibody from Proteintech, United States, mouse anti-GAPDH antibody from Proteintech,
United States, fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (H+ L) and HRP-conjugated rabbit anti-mouse IgG (H+ L) from Jackson, United States; CCK8 Kit from Dojindo, Japan, and substrate of enhanced
chemiluminescence (ECL) from Merck Millipore, United States.
Cell Culture
All cells were maintained in high-glucose DMEM (Gibco, United States) supplemented
with 10% fetal bovine serum (FBS; Gibco) and incubated at 37 °C with 5% CO2 in a humidified cell incubator. For passaging, cells were dissociated using 0.25%
trypsin-EDTA at 80 to 90% confluence and replanted at 1:3 dilutions.
Package of Lentivirus and Transduction of Cells
The procedure of lentivirus packaging and cell transducing were performed as previously
described.[25 ] The mixture of pLVX-puro-human-RAGE-HA plasmid ([Fig. 1 ]), pVSV-G, pRev, pTat, and pGag-pol plasmids was transfected into 293T cells by lipofectamine
2000. Lentiviral particles were harvested after 2 days of transfection. The NIH3T3
cells were seeded to a six-well plate and incubated overnight. The cells were fed
with fresh medium containing lentiviral particles and polybrene whose final concentration
was 10 μg/mL. The transduced cells were selected with 10 μmol/L puromycin.
Fig. 1 The vector of pLVX-puro-human-RAGE-HA.
Western Blotting
hRAGE-NIH3T3 and NIH3T3 cells were lysed in protein lysis buffer and β-mercaptoethanol,
followed by electrophoretic separation on a 10% tris-polyacrylamide gel, and then
transferred onto a polyvinylidene fluoride (PVDF) membrane at 200 mA for 120 minutes.
The membrane was blocked with 5% nonfat dry milk in TBST for 1 hour. Membranes were
incubated in primary antibody (mouse anti-HA antibody, mouse anti-GAPDH antibody)
at 4 °C overnight and subsequently with the corresponding horseradish peroxidase-labeled
secondary antibody diluted in 5% nonfat dry milk in TBST for 45 minutes at room temperature.
The signal of blot was detected by ECL substrate and captured by a Tanon Image system.
Flow Cytometry Assay
hRAGE-NIH3T3 and NIH3T3 cells were harvested and suspended by DMEM with 10% FBS. In
total, 2.5 × 105 cells for each sample were centrifuged and washed three times using FACS (PBS, pH
7.2, 2% FBS). Various concentrations of anti-hRAGE antibody (1, 2, and 5 μg) were
added to each sample, using PBS as the control. All samples were incubated for 30 minutes
at 4°C and then washed three times with FACS. FITC-conjugated goat anti-rabbit IgG
was added to the samples which were then incubated at 4°C in the dark. After 30 minutes,
cells were washed, suspended, and analyzed by a flow cytometer.
CCK8 Cell Viability Assay
hRAGE-NIH3T3 or NIH3T3 cells were seeded in 96-well plates (5,000 cells/well) and
incubated overnight in 100 μL DMEM with 10% FBS. Then, the medium was changed and
fed with DMEM with 1% FBS for starvation. After 24 hours of starvation, the cells
were fed a complete medium (DMEM + 10% FBS) and treated with Aβ (8 μmol/L) with different
concentrations of anti-hRAGE antibody (3.125, 6.25, 12.50, 25.00, and 50.00 μg/mL)
or PBS as control. After 48 hours of treatment, cell viability was assessed by a CCK8
kit according to the manufacturer's instructions.
Statistical Analyses
Statistical analyses were performed using Prism 7.0 (GraphPad Software, San Diego,
California, United States). Significant differences (p < 0.05) were calculated by one-way analysis of variance followed by Tukey's multiple-comparison
test.
Results
Selection and Identification of hRAGE-NIH3T3 Cell Colonies
Lentivirus is a useful tool in gene delivery and has high efficiency to integrate
exogenous gene in the genome of host cells. In this study, pLVX-Puro lentivirus vector
was used as a backbone and the insert was a hRAGE-fused HA tag DNA fragment. The expected
molecular weight of recombinant hRAGE-HA expressed by NIH3T3 should be approximately
43 kDa and it could be recognized by the anti-HA tag antibody in Western blotting.
Interestingly, there were two bands on the membrane detected by the anti-HA antibody
with molecular weights of approximately 45 and 55 kDa, respectively ([Fig. 2 ]). The migration pattern of hRAGE-HA was in accordance with literature data.[26 ]
Fig. 2 hRAGE-HA expression in hRAGE-HA-transduced NIH3T3 cell lysate. The cell lysate was
analyzed with Western blotting. The anti-HA tag antibody or anti-GAPDH antibody was
used to probe the membrane. Lane 1: NIH3T3 cell lysate; lane 2: hRAGE-HA-transduced
NIH3T3 cell lysate.
Colony selection played a critical role in the construction of transgenic cells for
stable expression of exogenous genes because protein levels of every cell are distinctive
in the cell pool. Cell clones expressing the highest level of hRAGE-HA could be chosen
and used in subsequent research studies. We isolated 12 hRAGE cell clones because
of their growth capacity in culture. The selected clones were plated on six-well plates
and samples of the cells were collected for Western blotting analysis. Our data showed
that three clones have higher hRAGE levels than others ([Fig. 3 ]). These three cell clones, named RAGE-1, RAGE-7, and RAGE-10, were selected for
cell-line establishment, in vitro antibody binding, and ligand bioactivity assays.
Fig. 3 Expression of hRAGE-HA in different colony samples from colony 1 to 12. The anti-HA
tag antibody or anti-GAPDH antibody was used to probe the membrane.
hRAGE-NIH3T3 Cell Stability Assay
The exogenous genes in host cells could be inactive due to genome epigenetic modifications
(reference: doi 10.1074/jbc.M109.092007). To investigate the stability of hRAGE-HA
integrating in NIH3T3 cells, hRAGE-HA expression in selected clones was analyzed from
the 1st to the 20th generation. We found that the levels of hRAGE-HA were not significantly
different during 20 generation passages. This indicated that the hRAGE-HA was stably
expressed in hRAGE-NIH3T3 cells ([Fig. 4 ]).
Fig. 4 The hRAGE-HA stably expresses through 20 generations in hRAGE-NIH3T3. The samples
were sequentially collected at generations 1, 4, 7, 10, 13, 16, and 20. These sample
were loaded on SDS-PAGE from lane 1 to lane 7, respectively. The anti-HA tag antibody
or anti-GAPDH antibody was used to probe the membrane in Western blotting assay.
Anti-hRAGE Antibody Recognition of hRAGE-HA in Engineered Cells
Recently, over 30 therapeutic antibodies have been used in the clinic and target receptor-overproducing
cell lines were involved in all stages of therapeutic antibody development, for example,
cell-based binding and biological function assays.[27 ]
[28 ] Anti-hRAGE rabbit polyclonal antibody was used to verify whether hRAGE-HA localized
on the cell surface could be specifically recognized by anti-hRAGE antibody. Based
on the assay of FACS, we found that signals from both anti-hRAGE antibody and control
IgG were very weak on NIH3T3 cells. The data showed that anti-hRAGE antibody had very
low affinity in binding to mouse RAGE on the surface of NIH3T3 cells ([Fig. 5A ]). Meanwhile, in hRAGE-NIH3T3 cells, signals of anti-hRAGE were stronger than that
of control IgG ([Fig. 5B ]).
Fig. 5 Anti-hRAGE antibody had high affinity binding with hRAGE-NIH3T3 cells (A : NIH3T3 cells, B : hRAGE-NIH3T3 cells).
Anti-hRAGE Antibody Protection of hRAGE-NIH3T3 Cells against Aβ-Induced Apoptosis
It is well known that therapeutic antibody works as a receptor inhibitor depending
on distribution of ligands bound to receptor. We investigated hRAGE-HA biological
function induced by Aβ, which is one of the RAGE ligands and could induce cell apoptosis.[29 ] To confirm the availability of hRAGE-NIH3T3 cells for anti-RAGE molecule screening
and functional study, NIH3T3 cells or hRAGE-NIH3T3 cells were treated with Aβ or Aβ/anti-hRAGE
antibodies for 48 hours. The data showed that anti-hRAGE antibodies significantly
improved hRAGE-NIH3T3 survival under Aβ stimulation, but the antibody had no effects
on protecting NIH3T3 cells ([Fig. 6 ]). hRAGE-HA in hRAGE-NIH3T3 cells also had biological function and the engineered
cell line could be used to access biological activities of anti-hRAGE antibodies or
ligands in vitro.
Fig. 6 Anti-hRAGE antibody improvement of hRAGE-NIH3T3 cell survival following Aβ treatment.
The anti-hRAGE rabbit polyclonal antibody (PcAb) and amyloid β (Aβ) were used to treat
NIH3T3 cells (A ) or hRAGE-NIH3T3 cells (B ). Data of cell viability were normalized by the control group. Experiments were repeated
three times: p
1 value = 0.0017, p
2 value = 0.0001, p
3 value = 0.0001.
Discussion
Pulmonary fibrosis, diabetes complications, and other diseases are closely associated
with overactivation of the RAGE pathway. RAGE inhibitors have the potential to treat
these diseases.[30 ]
[31 ] We have analyzed RAGE expression in several human cancer cell lines by Western blotting
and FACS. The protein level of RAGE in those tumor cells was much lower than that
in hRAGE-NIH3T3 cells (data not shown). The hRAGE-NIH3T3 cell line should have good
sensitivity and high efficiency for screening antibodies or interaction partners of
hRAGE. We employed a lentivirus gene delivery technique to construct engineered cell
lines because the lentivirus system has high safety and efficiency in transferring
exogenous genes into host cells.[32 ] It is also used to produce chimeric antigen T-cells for clinical treatment.[33 ]
In summary, the hRAGE-transgenic NIH3T3 cell line established in this study stably
expresses hRAGE through 20 generations and it was successfully applied to analyze
specific affinity and biological activity of anti-RAGE antibody. This cell line may
be a powerful tool for the development of drugs targeting the RAGE pathway in the
future. It could also be used to study the molecular mechanism of RAGE-mediated signal
transduction in vitro.
