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Introduction
Obesity is a multifactorial disease resulted from
interactions between susceptibility genes and environmental
factors. Furthermore, obesity is an important risk factor for
other diseases, including type 2 diabetes, hypertension,
hyperlipidemia, and cardiac
infarction[1-3]. During the development of obesity, adipose tissue plays a key role in energy
homeostasis by regulating the balance between energy
storage and energy consumption in response to nutritional
status[1]. Therefore, the identification and functional
characterization of genes whose expression is differentially regulated
in obese patients relative to normal weight controls may
provide new insights into the molecular mechanisms
underlying obesity-associated pathogenesis.
In a previous study, we performed suppression
subtractive hybridization[4-5] using omental adipose tissue from obese
patients and normal weight controls. We identified 426
differentially-expressed genes in adipose tissue from obese
patients. In total, 216 genes were upregulated and 210 genes
were downregulated[6]. Among these genes, we found that
the expression of six-transmembrane epithelial antigen of the
prostate (STEAP) 4, a member of the STEAP protein family,
was significantly downregulated in the obese patients,
suggesting that STEAP4 may be associated with obesity.
Although a previous study had shown an association between
STEAP4 overexpression and human prostate
cancer[7], a link between human STEAP4 and obesity had not been reported.
Studies of the mouse homolog of STEAP4,
six-transmembrane protein of prostate (STAMP) 2 [previously called
tumor necrosis factor (TNF)-α-induced adipose-related
protein], which shares 90% amino acid identity with human
STEAP4, showed TNF-α-mediated STAMP2 induction and
adipose conversion[8]. Recently, Wellen
et al identified STAMP2 as a critical modulator of inflammation and nutrition,
suggesting a potential role for STEAP4 in human
obesity[9].
In the present study, we determined the relationship
between human STEAP4 and obesity. We analyzed: the
differential expression of STEAP4 in adipose tissue from obese
patients relative to normal controls, the STEAP4 expression
in a panel of human tissues, the subcellular localization of
STEAP4 in human adipose tissue, and the TNF-α-mediated
induction of STEAP4 in human adipose tissue. Taken
together, these findings are in agreement with previous studies of
mouse STAMP2 and suggest that STEAP4 is likely to play a
significant role in the development of human obesity.
Materials and methods
Antibody preparation Rabbit polyclonal antiserum was
generated against an N-terminal peptide of STEAP4 (AEYLAHLVPGAHVVKAC), coupled to bovine serum
albumin and subjected to antigen affinity purification (GL
Biochem, Shanghai, China).
Analysis of the differential expressions of STEAP4
mRNA and protein All human omental adipose tissues were
obtained from male patients undergoing abdominal surgery
for acute simple appendicitis. Patients were assigned to the
normal weight group or obese group according to their body
mass index (BMI) [10], which was defined as weight in
kilograms divided by the square of height in meters. When
patients' BMI =30 kg/m2, they were assigned to the obese
group (n=6; BMI 30.3±0.3; age 50.6±9.2); when BMI scores
fell between 18 and 25 kg/m2,
the patients were assigned to the normal group
(n=6; BMI 22.2±1.7; age 47.1±11.7).
Written consent was obtained from each patient, and the
experiments were conducted according to the Declaration of
Helsinki.
The differential expression of STEAP4 mRNA was
analyzed using RT_PCR. Total RNA from the omental adipose
tissues of obese and normal weight patients was extracted
using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Total
RNA samples (200 ng) were subjected to RT_PCR using
random primers with Moloney murine leukemia virus reverse
transcriptase (Promega, Madison, WI, USA), and an aliquot
(10%) of the resulting cDNA was amplified using
GAPDH and STEAP4-specific primers. The primer sequences
for STEAP4 and GAPDH were as follows: STEAP4,
5'-CGAAACTTC CCTCTACCCG-3' (sense) and 5'-ACACAAACACCTGCCGACTT-3' (antisense); GAPDH,
5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' (sense) and 5'-CATGTGGGCCATGAGGTCCACCAC-3' (antisense). After
denaturation at 94 °C for 3 min, 34 cycles of PCR
amplification were performed (30 s at 94 °C, 30 s at 58 °C, and 40 s at
72°C). A final extension step was performed at 72 °C for 7
min. Finally, 5 μL of the PCR product was loaded on a 1.5%
agarose gel.
The STEAP4 protein expression was analyzed by immunoblotting, as described
previously[6]. Briefly, approximately 100 mg of fresh or frozen adipose tissue was
resuspended in 1 mL lysis buffer (50 mmol/L Tris-HCl, 1% Triton
X-100, 0.2% sodium deoxycholate, 0.2% SDS, and 1 mmol/L
EDTA at pH 7.4) and homogenized using a polytron
homogenizer at 4 °C. The lysate supernatant was collected after
centrifugation at 20 670×g for 30 min at 4 °C. Protein
concentrations were determined using a bicinchoninic acid protein
assay kit (Pierce, Rockford, IL, USA), and 30 µg of total
protein was loaded in each lane of a 10% polyacrylamide gel,
followed by SDS_PAGE. Prestained protein standards
(Fermentas, Hanover, MD, USA) were used as molecular
weight markers. The separated proteins were
electrophoretically transferred to a nitrocellulose membrane filter (Whatman,
GmbH, Dassel, Germany), and the membrane was blocked
with 5% dried milk for 2 h at room temperature. Subsequently,
the membranes were incubated at 4 °C overnight in a primary
antibody (polyclonal rabbit anti-STEAP4 antibody or
monoclonal mouse anti-GAPDH antibody; KangChen Bio-tech,
Shanghai, China) at an appropriate dilution, followed by
incubation with horseradish peroxidase (HRP)-conjugated
secondary antibodies (Santa Cruz Biotechnology, Santa Cruz,
CA, USA) for 1 h at room temperature. STEAP4 and GAPDH
bands were detected using enhanced chemiluminescence
reagents (Amersham, Piscataway, NJ, USA) and hyperfilm
exposure.
Analysis of STEAP4 expression in a panel of human
tissues RT_PCR was performed using specific primer pairs
to amplify human STEAP4 and GAPDH (primer sequences
given earlier) in a panel of human tissues. We purchased the
human multiple tissue cDNA (MTC) panel I
(Clontech, Mountain View, California, USA). The adipose tissue cDNA sample
was prepared manually from normal weight human omental
adipose tissue obtained with prior consent during
abdominal surgery for appendicitis. The human normal testis and
spleen cDNA samples were purchased from Bio/Biotech
(Shanghai, China). All PCR reactions were performed using
the following protocol: denaturation at 94 °C for 30 s,
followed by 35 amplification cycles (30 s at 94 °C, 30 s at 60 °C,
and 40 s at 72 °C), and a final extension at 72 °C for 5 min.
Negative controls (no cDNA template) were included with
both the GAPDH- and STEAP4-specific primer reactions. Gel
analysis was performed using aliquots removed at cycle 22
for GAPDH and at cycle 34 for STEAP4; 5 μL of the
amplification product was loaded on a 1.5% agarose gel.
Immunohistochemistry Human omental adipose tissues
were fixed at 4 °C overnight with 4% paraformaldehyde in
0.1 mol/L phosphate buffer at pH 7.2 and embedded in paraffin.
Next, 5 µm-thick sections were cut and mounted onto
microscope slides. The sections were deparaffinized with xylene
and hydrated in graded ethanol. After deparaffinization, the
sections were subjected to antigen retrieval in 0.01 mol/L
sodium citrate at pH 6.0 at 100 °C for 10 min in a microwave
oven. They were then incubated at 4 °C overnight with a
polyclonal anti-STEAP4 antibody (1:100 dilution). After
washing with phosphate-buffered saline, the sections were
treated with a goat antirabbit HRP-conjugated secondary
antibody (1:100 dilution) at 37 °C for 1 h. The primary
antibody was detected using a diaminobenzidine (DAB) kit
(ZhongShan Golden Bridge Biotechnology, Beijing, China)
according to the manufacturer's instructions. The sections
were counterstained with hematoxylin, observed under a light
microscope, and photographed. Negative controls were
obtained for each section by omitting the primary antibody
and by using pre-immune serum instead of the primary
antiserum.
TNF-α induction Human omental adipose tissues were
obtained from normal weight patients during abdominal
surgery for appendicitis. The samples were processed as
described previously[11], and the minced adipose tissue
fragments were placed in serum-free M199 (Invitrogen, USA)
containing 25 mmol/L HEPES with or without increasing
concentrations of TNF-α (5, 50, and 100 μg/L; Sigma, St Louis,
MO, USA). Cultures were maintained for 48 h. At the end of
culture, the tissues were rapidly washed in saline, frozen in
liquid nitrogen, and stored at _80 °C until the analysis.
STEAP4 mRNA and protein expressions were determined by
RT_PCR and an immunoblot analysis, respectively, as
described earlier.
Statistical analysis All data are expressed as
mean±SEM. Statistical analysis
was performed using the paired
Student's t-test of the SPSS 10.0 statistical software package (SPSS,
Chicago, IL, USA). The threshold of significance was
defined as P<0.05.
Results
Differential expression of STEAP4 in adipose tissue
from obese patients and normal weight controls
STEAP4 mRNA and protein expressions in human omental adipose
tissues from obese patients and normal weight control were
determined by RT_PCR and an immunoblot analysis, respectively. As shown in Figure 1, STEAP4 mRNA levels
were lower in adipose tissue from obese patients relative to
normal weight controls. The immunoblot analysis showed
approximately a 52 kDa band that corresponded to the
predicted size of STEAP4 (data not shown). Moreover, STEAP4
protein levels were also lower in the adipose tissue from obese
patients relative to the normal weight controls (Figure 2).
STEAP4 mRNA expression pattern in a panel of human
tissues We performed RT_PCR using a panel of human
cDNA samples to determine the tissue distribution of
STEAP4 mRNA; this study included 11 human adult tissues.
Figure 3 shows that among the human tissues tested,
adipose tissue showed the highest levels of the STEAP4
expression, followed by placenta and lung. Lower levels of
STEAP4 transcripts were detected in the skeletal muscle and
kidney, whereas STEAP4 mRNA was undetectable in the
spleen and brain.
STEAP4 protein localizes to the plasma membrane of
adipocytes Because the subcellular localization of human
STEAP4 protein in adipose tissue may provide key insights
regarding STEAP4 function, we performed immunohistochemical analyses of human adipose tissue using
anti-STEAP4 antibodies and HRP-conjugated secondary
antibodies. Figure 4 shows that the STEAP4 protein
localized to the plasma membrane of adipocytes, suggesting that
STEAP4 might be a membrane-associated protein.
TNF-α-induced modulation of STEAP4 mRNA and
protein expressions To determine whether the STEAP4 gene
expression could be induced by TNF-α, we examined the
STEAP4 expression in primary cultures of adipose tissue
treated with increasing levels of TNF-α. As shown in Figure
5, TNF-α led to a dose-dependent increase in the STEAP4
mRNA expression after 48 h of treatment. This effect was
undetectable at 5 μg/L TNF-α, and reached the maximum at
50 μg/L; a half-maximal response was obtained at 100
μg/L. Similarly, the STEAP4 protein expression also increased in
response to TNF-α treatment. The treatment of cultured
adipose tissue for 48 h with 50 μg/L TNF-α led to a stronger
increase in the STEAP4 protein expression than that induced
by 100 μg/L TNF-α; this effect was undetectable in the
presence of 5 μg/L TNF-α (Figure 6).
Discussion
Obesity is a multifactorial disease resulted from
interactions between susceptibility genes and environmental
factors. The obesity gene map reveals that putative loci
affecting obesity-related phenotypes are found on all
autosomes and on the Y chromosome. The number of genes,
markers, and chromosomal regions that have thus far been
associated with or linked to obesity phenotypes has reached
200 and continues to increase[12].
The mouse STAMP2 gene has been reported recently to
play a role in the coordinated regulation of nutrient and
inflammatory responses in adipose
tissue[9]. However, a role for human STEAP4, the homolog of mouse STAMP2, in
obesity has not been reported. In the present study, we found
that the STEAP4 expression was downregulated in adipose
tissue from obese human patients. Although our case
number is only 6, STEAP4 downregulation occurred consistently
at both the mRNA and protein levels. These findings
suggest a close relationship between STEAP4 downregulation
and obesity in humans. Moreover, we found that STEAP4
exhibited a tissue-specific pattern of expression pattern, and
that STEAP4 is most highly expressed in human adipose
tissue. Korkmaz et al showed that STEAP4 was expressed
most highly in the placenta, lung, heart, and prostate among
16 different human tissues[7]; however, their study did not
examine the STEAP4 expression levels in adipose tissue.
We found that the STEAP4 expression was highest in
adipose tissue, followed by the placenta, lung, and heart. Taken
together, our results strongly support an active role for
STEAP4 in adipose tissue and suggest that STEAP4 might
contribute to obesity.
To explore further the role of STEAP4 in human adipose
tissue, we confirmed that human STEAP4 exhibited a plasma
membrane-associated pattern of localization in human
adipocyte tissue. Furthermore, the STEAP4 expression was
induced in a dose-dependent manner by TNF-α in cultured
human adipose tissue. TNF-α exerts a well-established, key
regulatory role in obesity and in obesity-related insulin
resistance (IR)[13]; TNF-α also markedly alters adipose tissue
development and metabolism[14-17]. Overwhelming evidence
suggests that TNF-α could regulate the expression of many
obesity-related genes, including resistin, leptin, visfatin,
adiponectin[18-20], and of particular interest, the mouse
homolog of human STEAP4, STAMP2[8]. Therefore, we
hypothesized that STEAP4 might also be regulated by
TNF-α, in a manner similar to that of STAMP2. This would implicate
STEAP4 as a key mediator of the physiological or
pathological effects of TNF-α, which may modulate several
adipocyte-associated functions, including differentiation, lipolysis,
lipogenesis, insulin sensitivity, and apoptosis. In the present
study, we demonstrated that TNF-α treatment led to a
dose-dependent induction of the STEAP4 expression in cultured
human adipose tissue, at both the mRNA and protein levels.
However, it seemed to be contradictory that in obese
patients with high serum TNF-α level, the STEAP4 expression
level was lower than the normal controls. We considered
although TNF-α was an important regulator of the STEAP4
expression in vitro, the STEAP4 expression may also be
modulated by some other factors in vivo.
The similar phenomenon was observed in the study of resistin, which is
abundantly expressed in obesity but its expression could be
suppressed by TNF-α in vitro[18,
21]. Until now, the relationship between cytokines, such as
TNF-α with the STEAP4 expression, remains an unresolved question. This work may
make it a little clearer.
The identification of a STEAP4 N-terminal domain with
associated nicotinamide-adenine dinucleotide phosphate
(NADP) oxidoreductase coenzyme
activity[7] provides an interesting clue about potential STEAP4-associated
biochemical functions. Taken together with previous studies,
including the discovery that an NADPH-dependent
H2O2-generating system was associated with human and rodent
adipocyte plasma membranes[22, 23] and that reactive oxygen
species (ROS) played a vital role in obesity and in
obesity-related IR[24], these findings suggest that STEAP4 might be
involved in the ROS-related pathological pathway and may
eventually contribute to the development of obesity and
obesity-related IR.
Our studies were based on recent findings by
Wellen et al who systematically identified functional links between
STAMP2 and obesity in mice. Our study focused on STEAP4,
the human homolog of STAMP2. In conclusion, STEAP4
was abundantly expressed in human adipose tissue and that
the STEAP4 expression was significantly downregulated in
obese patients. Furthermore, STEAP4 underwent a
dose-dependent induction in response to
TNF-α treatment. Collectively, these findings provide new insights into how
the STEAP4 and TNF-α pathways may contribute to the
development of obesity and obesity-related IR in humans.
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