The TRAF2 and TRAF6 expression in myomas and myometrium of women in reproduction and perimenopausal age.

Abstract

Uterine myomas represent one of the most common female diseases. Uterine myomas or fibromas are benign, hormone-responding tumours of, respectively, smooth muscles and fibroblasts and their aetiology induces a significant interest. In myomas the presence of aromatase was detected and, in addition, oestrogen was found to be synthesized in myoma cells. The studies were performed on myoma patients of generative age and those in peri-menopausal age. Expression of TRAF2 and TRAF6 proteins was examined using immunohistochemistry and Western blot approach in small and large uterine myomas isolated from women of various age. In addition, the evaluation was conducted at the periphery of every myoma. We indicated that the level of both tested proteins in myomas is higher than in control. TRAF2 level in myometrium was lower than in myomas but higher than in control. In the case of TRAF6 those changes were ambiguous. Age didn't have influence the level of expression in both tested TRAF in studied structures.

Full text

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©Polish Histochemical et Cytochemical Society
Folia Histochem Cytobiol. 2010:48(3): 407 (407-416)
10.2478/v10042-010-0039-6
Introduction
Despite the high frequency of leiomyoma manifesta-
tion scanty information is available on their develop-
ment and growth. On the basis of biochemical and
cytogenetic studies every leiomyoma is thought to rep-
resent a product of clonal expansion of an individual
myocyte [1-3]. Just as it is the case with other tumours,
probably several genetically sensitive sites exist,
which lead to dysregulation of a smooth muscle cell,
resulting in its transformation to the common pheno-
type of a leiomyoma cell.
Studies performed till now demonstrated that an
immune response is linked to activation of NF-κB
under effect of IL-1 and TNF-α [4-8]. The two
cytokines exert their effect due to activation of
a kinase which induces NF-κB and, then, kinase IκB,
which directly affects activity of NF-κB transcription
factor. The phosphorylated, C-terminal domain of IL-1
receptor-linked kinase interacts with TRAF6 protein
[9-11]. Transmission of IL-1 signal may include role of
TRAF6 and/or TRAF2 [12-14]. TRAF-6 activates
NIK kinase but it should be stressed that NIK is a com-
mon mediator of signalling pathways originating from
multiple cytokine receptors [15-17].
TRAF (TNF receptor-associated factor) family rep-
resents a family of cytoplasmic proteins capable both
of negative control of apoptosis and of induction of
genes which promote survival. They were shown to
serve as adaptor proteins for a broad range of surface
receptors, to play an important role not only in apop-
tosis but also in response to stress. The proteins may
directly interact with intracellular domains of surface
FOLIA HISTOCHEMICA
ET CYTOBIOLOGICA
Vol. 48, No. 3, 2010
pp. 407-416
The TRAF2 and TRAF6 expression in myomas
and myometrium of women in reproduction
and perimenopausal age
Andrzej Plewka1, Pawe³ Madej2, Danuta Plewka3, Gra¿yna Nowaczyk3, Micha³ Morek1,
Edyta Bogunia1, Monika Ciupiñska-Kajor4, Karolina Sieroñ-Sto³tny5
1Department of Proteomics, Medical University of Silesia, Sosnowiec, Poland
2Chair and Department of Gynaecological Endocrinology, Medical University of Silesia, Katowice,
Poland
3Department of Histology, Medical University of Silesia, Katowice, Poland
4Department of Histopathology, Medical University of Silesia, Katowice, Poland
5Department of Internal Medicine, Medical University of Silesia, Bytom, Poland
Abstract: Uterine myomas represent one of the most common female diseases. Uterine myomas or fibromas are benign,
hormone-responding tumours of, respectively, smooth muscles and fibroblasts and their aetiology induces a significant inter-
est. In myomas the presence of aromatase was detected and, in addition, oestrogen was found to be synthesized in myoma
cells. The studies were performed on myoma patients of generative age and those in peri-menopausal age. Expression of
TRAF2 and TRAF6 proteins was examined using immunohistochemistry and Western blot approach in small and large uter-
ine myomas isolated from women of various age. In addition, the evaluation was conducted at the periphery of every
myoma. We indicated that the level of both tested proteins in myomas is higher than in control. TRAF2 level in myometri-
um was lower than in myomas but higher than in control. In the case of TRAF6 those changes were ambiguous. Age didn't
have influence the level of expression in both tested TRAF in studied structures.
Key words: TRAF2, TRAF6, leiomyomas, small and large leiomyoma, women
Correspondence: A. Plewka, Department of Proteomics,
Medical University of Silesia, ul. Ostrogórska 30,
41-200 Sosnowiec, Poland tel.: (+4832) 3641430,
fax.: (+4832) 3641440, e-mail: aplewka@sum.edu.pl

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receptors, including TNF-α (TRAF2) or IL-1β
(TRAF6) receptors.
TRAF proteins may serve for modulation of recep-
tors capacities to mobilize various signalling path-
ways, which lead to phosphorylation and activation of
protein kinases and, then, to activation of transcription
factors of Rel and AP-1 family.
In our earlier study we evaluated expression of aro-
matase in uterine myomas [18]. Expression of the aro-
matase depends on several factors, including TRAF.
Even if neither TRAF2 nor TRAF6 directly affect
expression of aromatase, their indirect effect is evi-
dent. The two proteins are known to directly stimulate
activity of NF-κB. This nuclear factor may bind one of
the promoters, such as I.4, which mobilizes aromatase
synthesis pathway. Expression of aromatase may also be
stimulated by PII promoter. While the promoter is acti-
vated by PGE2, levels of the prostaglandin are is direct-
ly related to activity of COX-2, and activity of the
cyclooxygenase is stimulated, among others, by NF-κB.
In this aim the attempt will be undertaken to
demonstrate whether there exist differences in the
amount and distribution of TRAF2 and TRAF6 pro-
teins in myomas of various size, isolated from women
of various age, as compared to an unchanged tissue.
The evaluation will be made using Western blot tech-
nique and quantitative analysis of immunohistochemi-
cal tests conducted on tissues of a normal uterus and
a myomatous uterus.
Materials and methods
Human material. Recruitment of patients, clinical studies and
hormonal tests will be conducted in the Chair and Department of
Gynaecological Endocrinology, Medical University of Silesia in
Katowice while enzymatic and protein studies will be executed in
the Department of Proteomics.
The studies were conducted on 40 patients with myomas, at the
reproductive age (below 45thyear of age, FSH<30 mIU/ml;
Samales were taken in follicular phase of menstrual cycle) and
40 patients with myomas in the perimenopausal age (45-55 years
of life, FSH>30 mIU/ml). Inclusion criteria will involve myoma
detected by USG, qualification of the patient to hysterectomy,
informed consent to the planned studies. The exclusion criteria will
include: therapy with any drugs, including hormonal drugs in the
minimum of 3 months before inclusion to the studies, neoplastic
disease, endometrial hypertrophy, metabolic and systemic distur-
bances, nicotinism.
Myometrial samples, for use as health controls were taken
from 10 young women (<40 years old) undergoing hysterectomies
for ovary tumors and 10 perimenopausal age women (>52 years
old) undergoing hysterectomies for uterine prolapse.
In these studies we used only material from uteruses with one
large myomas or one large and few small myomas. The material
for studies was excised from uterus taken during surgery, and fixed
histopathologically. Samples of leyomyoma 1 × 1 cm and samples
of a similar size from health myometrium at the distance of at least
4 cm each were taken. Before isolation of myometrium samples the
patients were informed on the aim of the studies. The planned
investigative procedures were approved by the Medical Bioethical
Commission.
Characteristics of studied groups
Group 1. Myometrium of menstruating women, in whom hys-
terectomy was performed for reasons other than uterine leiomy-
omas; the 10 patients represented the lesion-free control.
Group 2. Leiomyomas of <3 cm in diameter, sampled from
uteri of menstruating women (n=20 women).
Group 3. Myometrium of menstruating women, sampled at the
distance of not less than 4 cm from margins of a small leiomyoma
(n=20 women; autologous control).
Group 4. Leiomyomas of >5 cm in diameter, sampled from
uteri of menstruating women (n=20 women).
Group 5. Myometrium of menstruating women, sampled at the
distance of not less than 4 cm from margins of a large leiomyoma
(n=20 women; autologous control).
Group 6. Myometrium of non-menstruating women, in whom
hysterectomy was performed for reasons different than uterine
leiomyomas (n=10 women; lesion-free control).
Group 7. Leiomyomas of <3 cm in diameter, sampled from
uteri of non-menstruating women (n=20 women).
Group 8. Myometrium of non-menstruating women, sampled
at the distance of not less than 4 cm from margins of a small
leiomyoma (n=20; autologous control).
Group 9. Leiomyomas of >5 cm in diameter, sampled from
uteri of non-menstruating women, (n= 20 women).
Group 10. Myometrium of non-menstruating women, sampled
at the distance of not less than 4 cm from margins of a large
leiomyoma (n=20 women, autologous control).
Immunohistochemistry. Tissues were fixed in 4% buffered for-
malin, dehydrated and then embedded in paraffin. Section (5 μm)
were mounted on silane-coated slides. Samples were de-waxed,
and rehydrated. To unmask the antigen, sections were boiled in
0,01 M citrate buffer, pH 6, in a microwave oven for 10 min at 800
W. Endogenous peroxidase activity was quenched using 1.5% (v/v)
solution H2O2in methanol for 10 min and then washed in PBS-
Tween 20 (0,05% v/v) and blocked with 1% BSA for 60 min. The
sections were further blocked with avidin-biotin-blocking solution
according to the manufacturer's instruction. After this the slides
were incubated with rabbit anti-TRAF2 polyclonal antibody
(Abcam) or rabbit anti-TRAF6 monoclonal antibody (Abcam) in
a humidified chamber for 22 h at 4°C. After washing in PBS-
Tween 20 sections were incubated with biotinylated goat anti-rab-
bit immunoglobulins (Vector Laboratories Inc.) for 30 min, and
were incubated with avidin-biotinylated peroxidase complex (Vec-
tor) for 30 min. The bound antibodies were visualised with
diaminobenzidine (DAB) and H2O2in PBS, pH 7,5 according to
supplier's instructions (Vector). Finally, the tissues were stained
with Gill's hematoxilin, dehydrated, and cover-slipped. For nega-
tive controls rabbit IgG were used.
In each positively stained cell, the intensity of staining was
measured as the optical density of the reaction product, with the
program KS 300 VIDAS video image analyzer served by IBAS
2.5 system and a Panasonic digital camera. For each analyzed
area, 173 × 130 μm average optical density per unit area was cal-
culated. Finally, the arithmetic mean and standard deviation were
calculated.
Western blotting. Tissue samples (~500 mg) were homoge-
nized in 0.5 % Triton X-100 lysis buffer containing 20 mM Tris-
HCl (pH 7.5), 150 mM NaCl, 10 mM NaF, 2 mM dithiothreitol
(DTT), 1mM sodium orthovanadate, 1mM phenylmethylsulfonyl
floride (PMSF) and 20 M aprotinin. Homogenates were cen-
trifuged at 10,000 × g for 10 min. Concentration of protein was
determined by Bradford assay. For immunoblotting samples (25
μg of protein) were separated by 12% SDS-polyacrylamide gels,
and transferred to nitrocellulose membranes. After blocking with
5% skim milk in PBS-T (PBS with 0.1% Tween 20) for 1h, the
membranes were probed with TRAF2 or TRAF6 antibodies
(Abcam) overnight. Primary antibodies were detected with
408
A. Plewka et al.
©Polish Histochemical et Cytochemical Society
Folia Histochem Cytobiol. 2010:48(3): 408 (407-416)
10.2478/v10042-010-0039-6

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biotinylated anti-rabbit IgG made in goat (Vectastain ABC-AmP
Vector Laboratories, Inc.) (30 min) and with streptavidin and
biotinylated alkaline phospatase complex (30 min) and developed
using a nitroblue tetrazolium and 5-bromo-4-3-indolylphosphate
(Vector Laboratories, Inc.). Subsequent Western blotting analyses
were performed.
Statistical analysis. Normal distribution of the data was confirmed
by the Kolmogorov-Smirnov test. Results are presented as a mean
± SD. The Student's t-test or the Mann-Whitney U-test were per-
formed where appropriate. To estimate correlation between TRAFs
expression, myomas and age of women we used Spearman's rank
correlation test. A p value <0.05 was considered statistically sig-
nificant.
Results
Immunohistochemical studies
Optical density of cells with expression of an evaluated
protein reflects concentration of the immunocytochem-
ical reaction product in evaluated uterine structures.
TRAF2
Young women
Evaluating levels of TRAF2 in studied structures
(Fig. 1), optical density of the immucytochemical
reaction products in small myomas of young women
was found to be higher, amounting to around 185% of
the control level (Fig. 2). The same analysis targeted at
vicinity of the myomas demonstrated also higher
expression of the protein, which in this case reached
152% of the control level. Analysis of the data showed
that expression of TRAF2 at the periphery of myomas
amounted to 82% of the value noted in the myoma.
Comparing optical density in large myomas mani-
festing TRAF2 expression with the control level the
demonstrated expression of the protein was found to
be equal to 196% of the control level. Similarly to
small myomas, TRAF2 level measured at the periph-
ery was slightly lower than that in the myomas,
amounting to 182% of the control level. This corre-
sponded to 93% of TRAF2 expression level noted in
large myomas.
In women of reproductive age, TRAF2 expression
level in large uterine myomas resembled the value
noted in small myomas. Evaluation of the protein
expression at the direct periphery of the large myomas
demonstrated a similar expression level to that noted at
the periphery of small myomas.
Women at perimenopausal age
Evaluation of TRAF2 level in studied structures (Fig. 1)
showed that in women of perimenopausal age optical
density of the reaction product in small myomas was
increased to almost 165% of the control level (Fig. 2).
Analysis of myoma periphery demonstrated the level
higher than in the control (150% of the control level).
Analysis of the data disclosed that expression of TRAF2
at the periphery of small myomas constituted 92% of the
value observed inside the myomas.
Evaluating optical density in large myomas with
TRAF2 expression as compared to the control, the for-
mer was found to manifest higher expression of the
protein, amounting to almost 175% of the control
level. In contrast to small myomas, activity of TRAF2
measured at the periphery was lower than inside the
myoma and amounted to around 147% of the control
level.
Analysis of TRAF2 expression in uterine myomas
of women of post-reproductive age disclosed that in
large myomas expression of TRAF2 was comparable
to the expression documented in small myomas. At the
direct vicinity of large myomas the expression resem-
bled that observed around small myomas.
Effect of age
Evaluating content of TRAF2 reaction product, a level
of the protein in control samples obtained from young
women was slightly lower than in women of peri-
menopausal age women. A similar observation was
made upon quantitation of the protein expression in
small myomas and at their periphery in women of the
two age groups.
Analysis of the quantitative data related to large
myomas demonstrated that in women of peri-
menopausal age TRAF2 expression level was lower
and amounted to 89% of the level detected in young
women. Also at the periphery of large myomas the
level was distinct in the two age groups: in young
women it amounted to 124% of the value observed in
the older group.
TRAF6
Young women
Upon evaluation of TRAF6 levels in the studied struc-
tures (Fig. 3), in young women optical density of the
immunocytochemical reaction product was increased in
small myomas, reaching around 235% of the control
level (Fig. 4). The same analysis targeted at the myoma
periphery also in this case demonstrated expression of
the protein higher than in the control, reaching the level
of 185%. Analysis of the data demonstrated that expres-
sion of TRAF6 at the periphery of myoma amounted to
78% of the value noted in the myoma.
Comparing optical density of large myomas with
TRAF6 expression with the control level, the protein
expression was found to represent 288% of the control
value. Similarly to small myomas, TRAF6 expression
level measured at the periphery was slightly lower than
that in myomas and corresponded to 166% of the con-
409
TRAF2 and TRAF6 expression in myomas and myometrium
©Polish Histochemical et Cytochemical Society
Folia Histochem Cytobiol. 2010:48(3): 409 (407-416)
10.2478/v10042-010-0039-6

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trol level or 58% of the expression of TRAF6 noted in
large myomas.
Quantitative analysis of TRAF6 expression level in
uterine myomas of women in reproductive age, in
large myomas the expression level was higher than
that observed in small myomas, amounting to 123% of
the protein expression level in small myomas. In direct
vicinity of large myomas the expression level was
lower than the level noted around small myomas, cor-
responding to 90% of the latter level.
Women of perimenopausal age
Evaluation of TRAF6 expression level in the studied
structures (Fig. 3) disclosed that in women of peri-
menopausal age optical density of the reaction product
increased in small myomas to almost 210% of the con-
trol level (Fig. 4). At periphery of the myomas expres-
sion of the protein also was higher than in the control,
reaching the level equivalent to 195% of the control.
Analysis of the data showed that TRAF6 expression at
periphery of small myomas reached 95% of the value
observed in the myomas.
Evaluation of optical density of large myomas with
TRAF6 expression showed that the expression was
higher, corresponding to 240% of the control level. Sim-
ilarly to small myomas TRAF6 activity measured at
periphery was slightly lower than that in the myomas,
amounting to around 222% of the control value.
Analysis of TRAF6 expression in uterine myomas
of women in post-reproductive age demonstrated that
in large myomas TRAF6 expression was higher as
compared to the expression observed in small
myomas. In a direct vicinity of large myomas it was
found to be slightly higher than the level noted in
vicinity of small myomas.
Effect of age
Comparison of the product content in the reaction for
TRAF6 showed levels of the protein in control sam-
ples originating from young women was slightly high-
er than the level observed in perimenopausal women.
A similar observation was made during quantitative
evaluation of the protein expression in small myomas.
Periphery of small myomas demonstrated similar val-
ues of TRAF6 expression in the two age groups of
women.
Analysis the quantitative data related to large
myomas demonstrated that TRAF6 expresssion level in
women of perimenopausal age was lower, amounting to
83% of the level demonstrated in young women. The
reaction product measured in vicinity of large myomas
was also distinct in the two age groups: in young women
it amounted to 75% of the value observed in the older
group.
Western blot analysis
TRAF2
Upon comparison of TRAF2 expression in myometri-
um of women of various age the samples originating
from women in perimenopausal age were found to be
slightly higher, although the difference failed to reach
statistical significance.
For a better and a more legible evaluation of
changes in TRAF2 expression (and the remaining pro-
teins evaluated by Western blot analysis) a quantitative
analysis was performed exclusively in the same age
groups, in every case accepting the expression value
noted in a pure control as 100%.
Expression of TRAF2 measured at the periphery of
myomas originating from uteri of young females was
higher than that in the control, amounting to 115% and
120% (Fig. 5; Table 1) in, respectively small and large
myomas although the differences proved insignificant.
The TRAF2 expression level detected in small
myomas isolated from uteri of young women was
clearly higher than in the control and amounted to
135%, and the difference was significant. Even higher
was TRAF2 expression level detected in large
myomas, in which the expression level reached 140%
of the control level.
Upon analysis of differences in TRAF2 expression
between myomas and their direct vicinity expression
of the protein in small myomas was significantlly
higher than that in the surrounding myometrium.
A similar difference in the level of TRAF2 expression
was detected in cases of large myomas.
Analysis of differences in TRAF2 expression in
uteri of perimenopausal women demonstrated simi-
lar but slightly more accentuated differences:
expression of the protein measured at the periphery
of myomas was also higher than that in the control.
The levels amounted to 125% and 120% in small and
large myomas, respectively (Fig. 5; Table 1), but also
in this case the differences proved to be insignifi-
cant.
Expression level of the evaluated TRAF2 protein
in small myomas isolated from uteri of peri-
menopausal women was higher than that in the con-
trol and reached 145%, and the difference was sig-
nificant. In large myomas of the age group TRAF2
expression level was even higher, amounting to
160% of the control level.
Evaluating TRAF2 expression level in myomas
and in the surrounding myometrium it was shown
that in the older age group expression of TRAF2 in
small myomas was significantly higher than that in
the surrounding myometrium. An even higher differ-
ence, of 135% order was disclosed during analysis of
differences in TRAF-2 expression levels in large
myomas.
410
A. Plewka et al.
©Polish Histochemical et Cytochemical Society
Folia Histochem Cytobiol. 2010:48(3): 410 (407-416)
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411
TRAF2 and TRAF6 expression in myomas and myometrium
©Polish Histochemical et Cytochemical Society
Folia Histochem Cytobiol. 2010:48(3): 411 (407-416)
10.2478/v10042-010-0039-6
Fig. 1.
perimenopausal age women (F-J) with rabbit anti-TRAF2 polyclonal antibodies. A and F
– healthy myometrium, B and G – small myomas, C and H – periphery of small myomas,
D and I – large myomas, E and J – periphery of large myomas (original magnification
×200).
Immunohistochemical staining of uterini samples from reproductive (A-E) and
Fig. 2. Quantitative evaluation of
TRAF2 expression in immunohisto-
chemical staining. Date shown repre-
sent mean ± SD.
Differences in TRAF2 expression: *–
in reproductive age women and peri-
menopausal age women; a – in small
myomas and healthy myometrium; b –
in periphery of small myomas and
healthy myometrium; c – in large
myomas and healthy myometrium; d –
in periphery of large myomas and
healthy myometrium; e – in small
myomas and its periphery; f – in small
myomas and its periphery, are statisti-
cally significant.