Laboratory Investigation (2010) 90, 963–965
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CORRIGENDUM
Knockdown of FABP5 mRNA decreases cellular
cholesterol levels and results in decreased apoB100
secretion and triglyceride accumulation in ARPE-19 cells
Tinghuai Wu, Jane Tian, Roy G Cutler, Richard S Telljohann, David A Bernlohr, Mark P Mattson and James T Handa
Laboratory Investigation (2010) 90, 963–965; doi:10.1038/labinvest.2010.87
Correction to: Laboratory Investigation (2010) 90, 906–914
(this issue); doi:10.1038/labinvest.2009.33
The first author submitted the article, which was published
on 11 May 2009 without the knowledge of two of the authors.
Below are the amendments to the original article that reflect
the interpretations of all the authors.
INTRODUCTION
The third paragraph should read:
In this study, we found that knockdown of FABP5 gene
expression resulted in (1) decreased cellular cholesterol and
cholesterol ester, (2) increased cellular FFAs and triglycerides,
and (3) decreased secretion of apoB100 protein in ARPE-19
cells. These observations indicate that FABP5 is critical in
lipid metabolism in ARPE-19 cells, suggesting that FABP5
downregulation in the RPE/choroid complex in vivo1 might
contribute to age-related changes and early AMD.
compared with NTC control. To amplify the effect of the
knockdown of FABP5 on neutral lipid deposition, we challenged siRNA-treated cells with oleic acid, which increases
the synthesis of triglycerides.3 Figure 4 shows a marked increase in the size of lipid deposits in FABP5 siRNA-treated
cells compared with NTC control after oleic acid challenge.
Assessment of cell viability after the Nile Red binding assays
with Resazurin showed no significant difference between the
cells treated with FABP5 siRNA/oleic acid and NTC siRNAtreated cells (n ¼ 3 independent experiments; data not
shown), suggesting that the increase in Nile Red binding
in FABP5 siRNA/oleic acid-treated cells was not associated
with cell death, and thus reflected a true increase in lipid
deposits.4
Increased Cellular Lipid Deposits
We next investigated the effect of FABP5 knockdown on
neutral lipid accumulation in siRNA-treated cells by
performing the Nile Red binding assay. Cells treated with
FABP5 siRNA developed large Nile Red-stained lipid deposits
Decreased Secretion of apoB100 by FABP5 Knockdown
The availability of cellular cholesterol can influence apoB
lipoprotein particle production and secretion.5–7 We first
used western blot analysis to determine whether apoB protein
is secreted into the culture medium, and whether FABP5
inhibition influences this secretion. We found a 56% reduction of ApoB in the culture medium after FABP5 siRNA
treatment when compared with NTC controls. In contrast,
the amount of total secreted protein in the cultured medium
from FABP5 siRNA- and NTC-treated cells is comparable
under our experimental conditions. To determine whether
the apoB protein identified by western blot analysis is synthesized and secreted and not a contaminant of the culture
medium, we next evaluated apoB100 secretion using 35S-labeled apoB100 immunoprecipitation. We isolated 35S-labeled
apoB100 at 520 kDA and its degraded products with apparent
molecular weights of 250 and 75 kDa from the cultured
medium. We found that the level of newly synthesized apoB
in the culture medium from FABP5 siRNA-treated cells was
decreased by 76% (Po0.05) than that from NTC siRNAtreated cells (Figure 5). This result correlates nicely with the
western blot results, and suggests that apoB100 is not a
contaminant of the medium.
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963
RESULTS
These sections are amended as follows:
Decreased BODIPY-FA Uptake
Liao et al2 reported that BODIPY-FA uptake is closely correlated with the uptake and metabolism of radiolabeled FAs.
To determine the effect of FABP5 gene knockdown on the
influx and metabolism of FAs into siRNA-treated cells,
we performed BODIPY-FA uptake assays and found that the
mean V values for BODIPY-FA uptake at 24, 48, and 72 h
after FABP siRNA treatment were decreased by 5, 8
(Po0.05), and 6 % (Po0.05), respectively (Figure 3). These
results suggest that BODIPY-FA uptake is mediated, in part,
by FABP5. However, the biological effect appears to minimal.
Corrigendum
Figure 4 Nile Red fluorescence microscopy. ARPE-19 cells were treated with 10 nM siRNA for 6 h, then in medium containing 1% BSA for 24 h. Nile Red assay
was performed after cells incubated in medium supplemented with 0.4 mM oleic acid for an additional 24 h. (a) NTC-treated cells show few cellular lipid
droplets compared with treatment with an siRNA to FABP5 (b). Nuclei appear dark in the image. Bar ¼ 10 mm.
DISCUSSION
The following paragraphs are amended as follows:
Paragraph 2
This study has shown that knockdown of the FABP5
gene results in several significant changes in ARPE-19 cells:
(1) altered cellular lipid composition, (2) an increase in
cellular lipid droplets, and (3) decreased secretion of
apoB100. As a principal function of FABP5 is to transport
FAs in the cytoplasm to organelles, such as the endoplasmic
reticulum, the lack of FABP5 protein may disrupt the intracellular transport of FAs, resulting in an 18 % increase in
cellular levels of FFA in FABP5 siRNA-treated cells compared
with that in NTC siRNA-treated cells (Table 1). Such a
cellular accumulation of FAs can potentially alter lipolysis
and lipogenesis in FABP5 siRNA-treated cells. FABP5 appears
to facilitate FFA efflux from cells or some type of transfer of
lipid from storage droplets to secretory droplets. As most
triglycerides are found in lipid droplets within the cytosol,
and are not associated with the endoplasmic reticulum,
triglycerides must be mobilized by lipolysis, followed by
reesterification, for triglycerides to be reassembled in the
endoplasmic reticulum before being incorporated into lipoprotein particles for secretion.8,9 Importantly, these processes
may have been interrupted by the lack of FABP5, causing a
decrease in the efficacy of FA release or blocking the
reesterification of liberated FAs in the endoplasmic
reticulum. In contrast, overexpression of FABP5 has been
reported to increase lipolysis in adipose cells.10
pathway is dependent on (1) the production of apoB, which
is necessary for the assembly of lipoprotein particles containing a neutral lipid core; (2) the availability of phospholipids (mainly PC), free cholesterol to form the monolayer
surface and triglycerides, and cholesterol ester to form the
core of the particles; and (3) the availability of microsomal
triglyceride transfer protein (MTP).11 In the absence of any
one or more of these factors, apoB is diverted from the
lipoprotein particle assemble/secretion pathway and is degraded. In HepG2 liver cells, cholesterol ester reduction has
been shown to reduce the secretion of apoB-containing
lipoproteins significantly.12 Consistent with these findings,
our study data suggest that the decreased secretion of
apoB100 (Figure 6) could have been caused by a disruption
in the formation of apoB100-containing lipoprotein-like
particles, as a result of a substantial decrease in cholesterol
and cholesterol esters after FABP5 knockdown. The lower
levels of some species of phospholipids might also contribute
to the decreased secretion of apoB in siRNA-treated cells
(Table 2).
Paragraph 5
The decreased cholesterol and cholesterol ester composition
that we observed as a result of FABP5 gene knockdown led us
to examine the secretion of apoB100 in FABP5 siRNA-treated
cells. The apoB-containing lipoprotein assembly/secretion
Paragraph 6
Our experiments suggest that knockdown of the FABP5 gene
decreased cellular cholesterol, cholesterol ester, and some
species of phospholipids, the main constituents of lipoprotein-like particles. As a consequence, it may also reduce
the production of apoB-containing lipoproteins and lead to
decreased secretion of apoB as well as the accumulation of
cellular FFAs and triglycerides. Using a knockdown approach,
we were able to observe dramatic and unexpected phenotypic
changes in FABP5 siRNA-treated ARPE-19 cells. These observations suggest that FABP5 may be a key FA transporter
and is critical in lipid metabolism in human ARPE-19 retinal
pigment epithelial cells. Given the decrease in FABP5
expression by the RPE/choroid complex in a murine model
964
Laboratory Investigation | Volume 90 June 2010 | www.laboratoryinvestigation.org
Corrigendum
of early AMD,1 the findings from this study in turn, imply a
possible scenario in which the downregulation of FABP5 in
the RPE/choroid complex in vivo might contribute to
age-related changes and early AMD.
All the authors now agree that the data and the interpretations are accurate.
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