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Original Studies |
Expression in Human Uterine Leiomyoma and Its Down-Regulation by Progesterone1
Department of Obstetrics and Gynecology, Kobe University School of Medicine, Kobe 650-0017, Japan
Address all correspondence and requests for reprints to: Takeshi Maruo, M.D., Department of Obstetrics and Gynecology, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: maruo{at}kobe-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF) has been shown mainly to
inhibit proliferation and induce apoptosis in a variety of cells, no
information is available regarding whether human leiomyoma cells
express TNF. In the present study, we examined the expression of
TNF in leiomyomas, in comparison with that in the adjacent normal
myometrium, using immunohistochemical staining and Western immunoblot
analysis with a polyclonal antibody to human TNF. Furthermore, we
investigated the effect of sex steroid hormones on TNF expression in
leiomyoma cells cultured under serum-free, phenol red-free conditions.
Immunohistochemical staining showed that TNF expression in leiomyoma
cells was higher than that in the adjacent normal myometrial cells,
being more abundant in the proliferative phase than in the secretory,
progesterone (P4)-dominated, phase of the menstrual cycle. TNF
expression in leiomyoma cells in pregnant uterus was scarce. Western
immunoblot analyses of leiomyoma and normal myometrial tissue extracts
revealed that TNF, with a molecular mass of 17.3 kDa, was abundantly
present in leiomyoma tissue extracts, relative to normal myometrial
tissue extracts, and that TNF expression in leiomyoma cells was most
abundant in the proliferative phase of the menstrual cycle, less
abundant in the secretory phase, and least abundant in pregnant uterus;
whereas no such changes in TNF expression were noted in the normal
myometrium. In monolayer cultures of uterine leiomyoma cells under
serum-free conditions, addition of P4 (3.18 x
10-7 mol/L) resulted in a decrease in TNF
expression in the cells, relative to that in control cultures, whereas
treatment with 17ß-estradiol (3.67 x
10-8 mol/L) did not affect the TNF
expression in the cells. The concentrations of sex steroids used were
within the physiological tissue concentrations noted in leiomyoma and
myometrium. The present results suggest that the abundant expression of
TNF may be a molecular basis characteristic of leiomyomas in the
human uterus and that P4 may play a vital role in down-regulating the
expression of TNF in human uterine leiomyoma. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Homeostatic control of the net growth of tumor is the result of the dynamic balance between cell proliferation and cell death (8). It is possible that, in tumors, the death pathway may be suppressed, extending the lives of the cells (9). We have also demonstrated that the abundant expression of Bcl-2 protein, an apoptosis-inhibiting gene product, may be one of the molecular bases characteristic of leiomyomas and that P4 up-regulates Bcl-2 protein expression in leiomyoma cells (10).
Tumor necrosis factor- (TNF) is among the most versatile
cytokines that are produced not only by activated macrophages but also
by many types of cells in the female reproductive organs. Recent study
has indicated that Bcl-2 protein blocks TNF and TNF receptor
(TNF-R)-mediated apoptosis in some cells (11). It
seems, therefore, that TNF may play an important role in regulating
apoptosis in leiomyoma cells. No information is, however, available on
the expression of TNF in uterine leiomyoma. Thus, the present study
was first conducted to clarify whether human leiomyoma cells express
TNF throughout the menstrual cycle, and then to elucidate the effect
of ovarian sex steroids on TNF expression in cultured leiomyoma
cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phenol red-free DMEM (12, 13) and antibiotics
(1 x 105 U/L penicillin, 50 mg/L
streptomycin) were purchased from Life Technologies, Inc.
(Grand Island, NY). FBS, E2, and P4 were obtained from
Sigma (St. Louis, MO). Collagenase was purchased from
Wako Pure Chemical Industries Ltd. (Osaka, Japan).
Monoclonal antibodies to human cytokeratin 19, desmin, and vimentin
were purchased from Nichierei Co. (Japan) (Tokyo, Japan).
Polyclonal antibody to human TNF was purchased from Genzayme Corp./Techne (Minneapolis, MN). Human recombinant TNF
was purchased from Becton Dickinson and Co. (Bedford,
MA).
Tissue collection
A total of 27 uterine leiomyomas, up to 6–8 cm in size, and myometrial tissues were collected from different patients, of which 9 were from the proliferative phase, 11 from the secretory phase of the menstrual cycle, and 7 from pregnant uterus. Uterine leiomyomas and the adjacent normal myometrial tissues were obtained from symptomatic women with regular menstrual cycles who underwent abdominal hysterectomy for medically indicated reasons, whereas uterine leiomyoma tissues during pregnancy were obtained from pregnant women who had cesarean section between 37 and 39 weeks, with varied indications such as repeated cesarean section and breech presentation, at Kobe University Hospital. The patients ranged in age from 28–41 yr, with a mean age of 35.3 yr, and none had received hormonal therapy for at least 3 cycles before surgery. Informed consent was obtained from each patient, before surgery, for the present studies. Endometrial tissues were obtained from the extirpated uteri, and the day of the menstrual cycle was determined by endometrial histological dating according to the method of Noyes et al. (14).
Immunohistochemical staining
Obtained uterine tissue specimens were fixed in 4% buffered
neutral formaldehyde solution, dehydrated, and embedded in paraffin.
Sections, 5- to 6-µm thick, were deparaffinized. Immunohistochemical
staining was performed by the avidin/biotin immunoperoxidase method
with the use of a polyvalent immunoperoxidase kit (Omnitags, Lipshow,
MI) as previously described (15). Rabbit polyclonal
antibody to human TNF was used at a dilution of 1:500 as the primary
antibody. To assure the specificity of the immunological reaction,
adjacent control sections were subjected to the same immunoperoxidase
method, except that the primary antibody was replaced by nonimmune
murine IgG (Miles, Erkhardt, IN) at the same dilution as the specific
antibody. The replacement of the specific primary antibody, with
nonimmune murine IgG, resulted in a lack of positive
immunostaining.
Cell culture
Uterine leiomyoma tissues, obtained from the uterus in the proliferative phase and secretory phase of the menstrual cycle, were washed in PBS, cut into small pieces, and digested in 2% collagenase (wt/vol) at 37 C for 3–6 h (16). The leiomyoma cells were collected by centrifugation at 460 x g for 5 min and washed several times with DMEM containing 1% antibiotic solution. The isolated leiomyoma cells were plated in 75-cm2 flasks at an approximate density of 5 x 105 cells/flask and subcultured for 120 h at 37 C in a humidified atmosphere of 5% CO2-95% air in DMEM supplemented with 10% FBS (vol/vol). The trypan blue exclusion test was used to determine the cell viability. Characterization of the cultured cells was examined using immunohistochemical staining with monoclonal antibodies to desmin, vimentin, and cytokeratin 19. As previously described (10), cells cultured for 120 h after collection from leiomyoma tissues were immunostained with the monoclonal antibody to desmin, but not immunostained with antibodies to either vimentin or cytokeratin 19, indicating a pure population of isolated cells with smooth muscle cell characteristics without either stromal or glandular epithelial cell contamination. Thereafter, the cultured cells were stepped down to serum-free, phenol red-free conditions. Treatment with E2 (3.67 x 10-8 mol/L) or P4 (3.18 x 10-7 mol/L) was begun when the cultured cells were at approximately 30–40% confluence, and monolayer cultures were maintained in serum-free DMEM for an additional 72 h. These cell culture experiments could be performed successfully with six uterine tissue specimens collected from different patients, of which three were from the proliferative phase and the other three were from the secretory phase of the menstrual cycle.
Protein extraction and Western immunoblotting
At the termination of cultures, cultured cells were incubated at 4 C for 15 min in the presence of a lysis buffer consisting of 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mmol/L Tris-HCl, and 2 mmol/L phenylmethylsulfonylfluoride, pH 7.5. Cells were subsequently scraped off the plates, the extracts were centrifuged at 13,000 x g for 30 min, and the supernatants were collected. Leiomyomas and adjacent normal myometrium tissues for protein extraction were collected immediately after hysterectomy. These tissue samples were homogenized at 4 C in the lysis buffer (5 g tissue in 1–2 mL). Homogenates were subsequently centrifuged at 13,000 x g for 30 min, and the supernatants were collected. Protein estimation of the supernatants was performed by the Bradford assay (17).
Each of 150-µg aliquots of proteins extracted from cultured cells and
uterine tissues was run on a 10% SDS polyacrylamide gel under a
reducing condition. The proteins were electrophoretically transferred
from gels to nitrocellulose membranes (18). Blots were
exposed to the polyclonal antibody to human TNF at a dilution of
1:500 in Tris buffer. The antigen-antibody complexes were detected with
the secondary antibody using the ECL chemiluminescence detection
system (Amersham Pharmacia Biotech, Arlington Heights,
IL). Negative control procedures for Western immunoblotting included
the substitution of the primary antibody with nonimmune murine IgG and
omission of the primary antibody. These negative controls prevented the
appearance of an immunoreactive TNF band. Positive control
procedures for Western immunoblotting included using human recombinant
TNF. These positive controls showed the appearance of an
immunoreactive TNF band.
These experiments were repeated, as follows, with similar
results, and the reported results are representative. Western
immunoblotting of leiomyoma and myometrium tissue extracts with a
polyclonal antibody to TNF were performed 3 times using 18 different
uterine specimens (each 6 from the proliferative phase and the
secretory phase of the menstrual cycle, and from pregnant uterus).
Experiments to investigate the effects of sex steroids on TNF
expression in cultured leiomyoma cells were performed 4 times (2
experiments with leiomyoma cells obtained in the proliferative phase,
and the other 2 experiments with leiomyoma cells obtained in the
secretory phase of the menstrual cycle).
Statistical analysis
Relative expression of TNF was expressed as the mean ±
SD. Data were analyzed by Student’s t test as
paired observations. P < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was
abundantly present in the cytoplasm of leiomyoma cells (Fig. 1
, A and B) but was scarcely present in
normal myometrial smooth muscle cells (Fig. 1, C and D). Immunostaining
for TNF in leiomyoma tissues obtained in the proliferative phase
(Fig. 1A) was more abundant than that in leiomyoma tissues obtained in
the secretory phase of the menstrual cycle (Fig. 1B). By contrast,
there was no apparent difference, in the intensity of immunostaining
for TNF in normal myometrial smooth muscle cells, between the
proliferative phase (Fig. 1C) and the secretory phase of the menstrual
cycle (Fig. 1D). TNF expression in leiomyoma tissues obtained from
pregnant uterus was also scarcely noted (Fig. 1E). Replacement of the
primary antibody with nonimmune murine IgG resulted in a lack of
positive immunostaining in the leiomyoma cells (Fig. 1F).
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with a molecular mass of approximately 17.3 kDa
was more abundant in the tissue extracts from leiomyoma in the
proliferative phase, compared with that from leiomyoma in the secretory
phase; whereas, in leiomyoma tissue extracts from pregnant uterus,
TNF was barely detectable (Fig. 2). On
the other hand, Western immunoblot analyses of proteins extracted from
normal myometrial tissues revealed that there were no significant
changes in TNF expression in normal myometrial tissues between the
proliferative phase and the secretory phase of the menstrual cycle
(Fig. 3).
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with a molecular mass of approximately 17.3 kDa and that the
addition of P4 to the serum-free medium resulted in a remarkable
decrease in the levels of expression of 17.3-kDa immunoreactive TNF,
compared with the control cultures (Fig. 4). The treatment with E2 (3.67 x
10-8 mol/L) did not affect
TNF expression in the cultured leiomyoma cells, whereas the
concomitant treatment with E2 and P4 seemed to slightly decrease the
TNF expression in the cells, relative to that in the treatment with
E2 alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in leiomyoma cells, relative to those in
normal myometrial smooth muscle cells and its down-regulation by P4.
Recent study has shown that many types of cells in the female
reproductive organs transcribe and translate the TNF gene
(19). TNF is now recognized as a multifunctional
cytokine that elicits a wide spectrum of cellular responses in various
cells (20). The TNF gene is located in the class 32 III
region of the major histocompatibility complex, and codes for a 26-kDa
membrane-bound form, which releases a 17.3-kDa soluble form upon
cleavage from pro-TNF (21, 22, 23). Numerous studies have
reported on the ability of TNF to induce apoptosis of various cell
types. Danforth et al. (20) have shown that
TNF inhibits growth of the estrogen receptor-positive human breast
cancer cell line MCF-7 in the G0G1 phase of the cell cycle, in a
dose-dependent manner. We have also demonstrated that dominant exposure
of cultured porcine granulosa cells to TNF induced apoptosis of the
cells (24, 25). Pusztai et al.
(26) suggested that, in human uterus, increased expression
of TNF messenger RNA (mRNA) in the endometrium, in the late
secretory phase, led to the inhibition of endometrial proliferation and
the induction of apoptosis in those cells. Several pathways have been
implicated in the effect of TNF. TNF activates phospholipase A2
with release of arachidonic acid from cells that are lysed by TNF.
TNF has also been shown to increase intracellular hydroxyl radicals
in the fibroblast cell line L–M. An additional possible mechanism
includes activation of lysomal enzymes, endonucleases, proteases, and
ADP ribosylation (20). On the other hand, Terranova
et al. (19) reported that when implantation
occurs, TNF targeted to invading trophoblast cells, stimulating
their proliferation and differentiation. Accordingly, it is likely that
TNF may have important roles in the regulation of apoptosis in the
female reproductive organs other than placenta.
The basic machinery for apoptosis constitutes a complex interaction
between distinct pro- and antiapoptotic molecules. The nature of these
interactions is not completely understood. It is clear, however, that
the caspases, a family of cysteine proteases with aspartase activity,
play an important role in mediating the apoptotic process. Although
TNF mediates apoptosis via activation of caspases, Bcl-2 protein (an
antiapoptotic molecule) inhibits TNF-induced apoptosis in some cells
(11). We have recently demonstrated that the abundant
expression of Bcl-2 protein in leiomyoma cells may be one of the
molecular bases characteristic of leiomyomas (10). Taking
these findings into account, the cross-talk between TNF and Bcl-2
protein may contribute to homeostatic control of leiomyoma growth and
play a vital role in deciding whether a leiomyoma cell will follow a
proliferative pathway or an apoptotic pathway.
We have recently demonstrated that Bcl-2 protein expression in
leiomyoma cells predominated in the secretory phase of the menstrual
cycle, compared with that in the proliferative phase, and that Bcl-2
protein expression in cultured leiomyoma cells was up-regulated by P4
(10). It is, therefore, likely that P4 may act as a
growth-promoting factor in regulating leiomyoma growth through the
enhanced inhibition of apoptosis of leiomyoma cells. In this respect,
it is of great interest that the treatment with P4 (3.18 x
10-7 mol/L) resulted in
the decrease in TNF expression in cultured leiomyoma cells, relative
to that in control cultures, whereas the treatment with E2 (3.67
x 10-8 mol/L) did not
affect the TNF expression in the cells. Because Eiletz et
al. (27) reported that P4 levels in human myometrium
and leiomyoma tissues were as high as 10–70 ng/g protein,
whereas E2 levels in human myometrium and leiomyoma tissues ranged from
4–10 ng/g protein, the concentrations of sex steroids used in the
present study seem to be within the range of physiological tissue
concentrations. The fact that TNF expression in cultured leiomyoma
cells was reduced by P4 is consistent with our immunohistochemical
observation of lower expression of TNF in leiomyomas in the
secretory, P4-dominated, phase of the menstrual cycle, compared with
that in the proliferative phase of the menstrual cycle. From these
results, it is suggested that the molecular basis for P4 action in the
regulation of leiomyoma growth may include the inhibition of TNF
expression by P4 in leiomyoma cells. Furthermore, several studies
indicated that the increased size of leiomyoma during pregnancy could
be attributable to the increase in serum P4 levels, as well as the
up-regulation of P4 receptor expression in leiomyoma cells, but not
attributable to the increase in serum E2 levels (28). In
agreement with this notion, TNF expression in leiomyoma cells
obtained from pregnant uterus was negligible. This supports the
hypothesis that TNF expression in uterine leiomyoma may be regulated
by P4. Furthermore, Horiuchi et al. (29) have
recently reported that human CG directly promotes the proliferation of
cultured myometrial and leiomyoma cells, with the latter showing the
greater response. Taking all into account, it seems likely that human
CG and P4 may act in combination to increase leiomyoma growth during
pregnancy.
Cycle-associated fluctuations between sex steroid hormones and
TNF-gene have been described in the endometrium. Tabibzadeh et
al. (30) reported that TNF in situ
hybridization signals in human and mouse endometrial epithelial cells
and stromal cells gradually increased during the estrogen-driven
proliferative phase, temporally decreased at the early secretory phase,
and thereafter rose to high intensity at the mid to late secretory
phases. Using in situ hybridization techniques, Roby
et al. (31) also demonstrated that
administration of E2 in ovariectomized mice stimulated two waves of
TNF message, with the first occurring only 1 h post treatment
and the second being delayed until 72 h post treatment, whereas
TNF in situ hybridization signals after treatment with P4
were highest at 24 h. Each of the two hormones has a distinct
temporal profile for the stimulation of TNF expression in the
endometrium. Based on TNF expression patterns in cycling human
endometrium, Hunt (32) proposed the hypothesis that low
levels of estrogen-stimulated TNF during the early proliferative
phases of the menstrual cycle may promote DNA synthesis in endometrial
epithelial and stromal cells, whereas high levels of TNF produced by
endometrial cells targeted by E2 plus P4 may culminate in cytolysis and
menstruation. Thus, it should be noted that the effects of sex steroid
hormones on TNF expression vary among the different cell types, even
in the uterus. Indeed, unlike leiomyoma cells, in normal myometrial
smooth muscle cells there were no differences in cellular levels of
TNF expression between the proliferative phase and the secretory
phase of the menstrual cycle. This suggests that no cyclic changes in
TNF expression exist in the normal myometrium throughout the
menstrual cycle.
TNF binds to and facilitates multimerization of two distinct
cellular receptors, termed p55/p60 (Type I, b) and p75/p80 (Type II, a)
to reflect their molecular masses. The receptor genes have been cloned
and sequenced in several species (33, 34). TNF-RI is
primarily associated with cytotoxicity (35, 36).
Transgenic mice lacking TNF-RI are insensitive to
lipopolysaccharide-induced shock (37, 38) and are less
efficient killers of certain intracellular microorganisms.
Although TNF-RII seems to be primarily associated with lymphocyte
proliferation (35, 39), this receptor may also be involved
in transducing a cytotoxic signal. Interestingly, knockout of the
TNF-RII has minimal phenotypic effect (40). The
expression of TNF-Rs in leiomyoma cells still remains to be
elucidated.
In conclusion, we have demonstrated increased expression of TNF in
leiomyomas, relative to the normal myometrium in the same individual
uterus, and that the increased TNF expression in leiomyoma cells
declined in the secretory, P4-dominated phase of the menstrual cycle,
compared with that in the proliferative phase. Consistent with these
findings, TNF expression in leiomyoma cells cultured in
vitro under serum-free, phenol red-free conditions was
down-regulated by P4. Because it is now evident that TNF expression
is closely linked to inducing apoptosis in various cells, it seems
likely that P4 may participate in the regulation of leiomyoma growth
and apoptosis through inhibiting TNF expression in leiomyoma
cells.
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Footnotes |
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Received September 12, 2000.
Revised December 28, 2000.
Accepted February 2, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the female reproductive tract. Proc Soc
Exp Biol Med. 209:325–342.[CrossRef][Medline]
modulates oestradiol responsiveness of MCF-7 breast cancer
cells in vitro. J Endocrinol. 138:517–528.—a primary mediator of the host response. Annu Rev
Immunol. 7:625–655.[Medline]
mRNA expression in endometrial cells by TNF and
by oestrogen withdrawal. Mol Hum Reprod. 5:1141–1149. gene in the female reproductive tract. Reprod
Fertil Dev. 5:141–153.[CrossRef][Medline]
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