The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 5 1385-1389
Copyright © 1997 by The Endocrine Society
Reproductive Endocrinology |
Messenger Ribonucleic Acid Expression of Heat Shock Proteins HSP70 and HSP90 in Human Endometrium and Myometrium during the Menstrual Cycle1
Takayuki Komatsu,
Ikuo Konishi,
Manabu Fukumoto,
Kanako Nanbu,
Masafumi Koshiyama,
Masaki Mandai and
Takahide Mori
Department of Gynecology and Obstetrics (T.K., I.K., K.N., M.K.,
M.M., T.M.) and Pathology (M.F.), Faculty of Medicine, Kyoto
University, Kyoto, Japan
Address all correspondence and requests for reprints to: Ikuo Konishi, M.D., Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
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Abstract
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Heat shock proteins of 72 and 90 kDa (HSP70, HSP90) are thought to be
involved in the functional modulation of sex steroid receptors. To
examine the expression and transcriptional regulation of HSP70 and
HSP90 in both the endometrium and myometrium, messenger ribonucleic
acid (mRNA) and protein level expression of these HSPs during various
phases of the menstrual cycle were analyzed by Northern blotting and
immunohistochemistry. In the endometrium, HSP70 mRNA levels increased
during the secretory phase compared with levels during the
proliferative phase; this is consistent with the stronger
immunostaining of HSP70 in glandular cells in the secretory phase. In
the myometrium, however, mRNA and protein expression of HSP70 were
higher in the proliferative phase than in the secretory phase. This
indicates that the regulatory mechanism of HSP70 expression during the
menstrual cycle differs between the endometrial glandular cells and
myometrial smooth muscle cells and may be correlated with the period of
sex steroid-related functions of the respective cells. Expression of
the HSP90 mRNA and protein in the myometrium was higher in the
proliferative phase than in the secretory phase. In the endometrium,
HSP90 immunostaining in the glandular cells was also stronger in the
proliferative phase, although expression of HSP90 mRNA was not
significantly different between the tissues of the two phases.
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Introduction
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HEAT SHOCK proteins (HSP) are highly
conserved proteins in nearly all organisms and are induced by various
kinds of stress, including nonphysiological temperature (1). However,
recent studies have demonstrated that several HSPs are expressed even
under physiological conditions and play an important role in normal
cell function (1, 2). A 90-kDa HSP (HSP90) is well known to modulate
the function of sex steroid receptor proteins (3, 4, 5, 6), and a 72-kDa HSP
(HSP70) and others are thought to be involved in the assembly of
receptor proteins (4, 5, 6). We previously reported the
immunohistochemical localization of HSP70 and HSP90 in the human
endometrium during various phases of the menstrual cycle (7). However,
little is known about the transcriptional regulation of HSP70 and HSP90
in the normal endometrium. In addition, expression of these HSPs has
not been reported in the myometrium, which is another target for sex
steroids in the human uterus (8, 9, 10). Therefore, we examined the
messenger ribonucleic acid (mRNA) expression of HSP70 and HSP90 in both
endometrial and myometrial tissues during the menstrual cycle by
Northern blot analysis. Immunohistochemical localization of HSP70 and
HSP90 in the same specimens was also examined.
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Materials and Methods
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Specimens
Normal endometrial and myometrial tissues were obtained from 19
patients, aged 32–45 yr, who underwent total hysterectomy for the
treatment of cervical intraepithelial neoplasia. Informed consent was
obtained from each patient according to the guidelines (no. 90) of the
ethical committee of Kyoto University Faculty of Medicine. All 19
patients had had regular menstrual cycles, and none had received
hormonal therapy. At the time of operation, 7 were in the proliferative
phase, and 12 were in the secretory phase. None of the 19 specimens
showed pathological abnormalities in the endometrium or myometrium. The
menstrual cycles of the patients were estimated by endometrial dating
according to the method of Noyes et al. (11). Immediately
after the surgical procedure, endometrial tissues (mainly the
functional layer) were scraped with a scalpel, and apparently normal
myometria were obtained from the anterior wall of the uterine corpus.
Tissues were snap-frozen in liquid nitrogen and stored at -80 C.
RNA isolation and Northern blot analysis
Total RNA was prepared from normal human endometrial or
myometrial tissue by the acid guanidinium-phenol-chloroform method (12)
using Trizol Reagent (Life Technologies, Gaithersburg, MD).
Five-microgram aliquots of total RNA were separated by electrophoresis
in 1% agarose-formaldehyde gels and transferred onto nylon membranes.
The membranes were prehybridized for 1 h at 65 C in 5 x
saline-sodium phosphate-ethylenediamine tetraacetate, 1% SDS, 5
x Denhardt’s solution, and 0.5 mg/dL salmon sperm DNA. The genomic
DNA probe for human HSP70 was provided by Dr. Morimoto (Northwestern
University, Evanston, IL), and the complementary DNA probe for human
HSP90-
was purchased from Riken Gene Bank (Tsukuba, Ibaraki, Japan).
Probes were labeled by the random primer technique with
[32P]deoxy-CTP to a specific radioactivity of 0.5–1
x 106 cpm/mL. Hybridization with the labeled probe was
performed overnight at 65 C. After hybridization, membranes were washed
at room temperature in 2 x SSC (standard saline citrate) plus
0.1% SDS, followed by 0.1 x SSC plus 0.1% SDS at 65 C for 15
min, and then subjected to autoradiography. The membranes were washed
in boiled 0.1 x SSC plus 0.1% SDS and used again for Northern
blotting with human glyceraldehyde-3-phosphate dehydrogenase
complementary DNA probe, the expression level of which was similar in
most tissues. Radioactivity was measured in a BAS 2000 Bioimage
Analyzer (Fujix, Tokyo, Japan).
Immunohistochemistry
Immunostaining of HSP70 and HSP90 was performed on cryostat
sections by the avidin-biotin-peroxidase complex method with mouse
anti-72 kDa HSP monoclonal antibody (diluted 1:500; SPA-810, StressGen
Biotechnologies, Victoria, Canada) and mouse anti-90 kDa HSP monoclonal
antibody (diluted 1:500; SPA-830, StressGen), using a Vectastain Elite
ABC kit (Vector Laboratories, Burlingame, CA), as described previously
(7). The grade of staining intensity was estimated by two independent
observers as negative, weakly positive, or strongly positive.
Statistical analysis
Statistical analysis of the HSP mRNA levels was performed using
the Mann-Whitney U test. The values of HSP expression represent the
relative expression of these genes and glyceraldehyde-3-phosphate
dehydrogenase expression.
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Results
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Expression of HSP70 in the endometrium and myometrium
In the endometrium, HSP70 mRNA levels during the secretory phase
were significantly higher than those during the proliferative phase
(Fig. 1
). Immunohistochemically, glandular cells in the
functionalis of the endometrium were negative for HSP70 during the
proliferative phase, but they exhibited strong immunoreactivity for
HSP70 during the secretory phase. On the other hand, stromal cells in
the functionalis during both the proliferative and secretory phases
were negative or very weakly positive for HSP70.
On the contrary, HSP70 mRNA levels in the myometrium were
significantly greater during the proliferative phase compared to those
during the secretory phase (Fig. 2). Although
immunoreactivity for HSP70 was observed in myometrial smooth muscle
cells throughout the menstrual cycle, the staining intensity of HSP70
was also strong in the cells during the proliferative phase and very
weak during the secretory phase (Fig. 3). This contrasts
with the change in the staining intensity in endometrial glandular
cells, in which the staining intensity for HSP70 was much stronger in
the secretory phase than that in the proliferative phase.
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Figure 3. Immunohistochemical staining of HSP70 in
myometrial smooth muscle cells in the proliferative phase (A) and the
secretory phase (B) of the menstrual cycle. There was weaker staining
of HSP70 in the smooth muscle cells in the secretory phase than in the
proliferative phase, in contrast to changes in HSP immunostaining in
the endometrial glands. Magnification, x200.
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Expression of HSP90 in the endometrium and
myometrium
There was no significant difference in endometrial HSP90
mRNA levels between the proliferative and secretory phases (Fig. 4). Immunoreactivity for HSP90 was observed in both the
glandular and stromal cells in the functional layer throughout the
menstrual cycle. However, the staining intensity for HSP90 in the
glandular cells was strong in the proliferative phase and weak in the
secretory phase.
In the myometrium, HSP90 mRNA expression during the proliferative phase
was high, and the levels of HSP90 mRNA varied during the secretory
phase (Fig. 5). Statistically, there was a significant
difference in the HSP90 mRNA level between the proliferative and
secretory phases. Immunoreactivity for HSP90 was observed in the
myometrial smooth muscle cells throughout the menstrual cycle. The
staining intensity of HSP90 was strong in the proliferative phase and
weak in the secretory phase (Fig. 6).
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Figure 6. Immunohistochemical staining of HSP90 in
myometrial smooth muscle cells is stronger in the proliferative phase
(A) than in the secretory phase (B) of the menstrual cycle.
Magnification, x200.
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Discussion
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This study demonstrated the mRNA and protein levels of both HSP70
and HSP90 in the human endometrium and myometrium during the menstrual
cycle. Changes in HSP expression in the normal endometrium have been
reported at the protein level immunohistochemically (7) and by Western
blot analysis (13), as well as at the mRNA level by reverse
transcription-PCR (14). However, there have been no reports on HSP
expression in the human myometrium. Our study showed that the
expression of HSP90 mRNA in the myometrium is significantly higher in
the proliferative phase than in the secretory phase.
Immuohistochemistry also confirmed that the HSP90 protein is localized
in the smooth muscle cells, and the staining intensity is stronger in
the proliferative phase than in the secretory phase.
HSP90 is known to bind sex steroid receptors such as estrogen receptors
(ER) and progesterone receptors (PR). In myometrial smooth muscle
cells, expression of both ER and PR is known to be high during the
proliferative phase. In the secretory phase, ER expression is
down-regulated, although PR expression is maintained (9, 10). HSP90 is
known to mask the functional domains to maintain an inactive state (6),
but this receptor-associated protein is also thought to be a necessary
component for the maintenance of the appropriate conformation required
for hormone-binding activity of the receptor (15). Therefore, the
fluctuation of HSP90 expression in the myometrium may parallel the
change in sex steroid receptor levels during the menstrual cycle in
smooth muscle cells. This is the case for HSP90 expression in the
endometrial glands; immunostaining of HSP90 in the glandular cells was
stronger in the proliferative phase than in the secretory phase. In
this study, however, HSP90 mRNA levels were not significantly different
between the proliferative and secretory endometria. This discrepancy
may be due to either the small sample size or the heterogeneity of
endometrial tissue that contains many different kind of cells, such as
epithelial, stromal, and immune cells (16).
This study demonstrated that expression of HSP70 mRNA in the
endometrium is significantly increased during the secretory phase. This
concurs with a recent report on HSP70 mRNA by the reverse
transcription-PCR method (14). Our immunohistochemical results suggest
that the increased HSP70 mRNA in the secretary phase represents the
strong induction of HSP70 protein in the glandular cells during the
secretory phase. In our previous study, overexpression of HSP70 in the
normal endometrial glands was also observed during treatment with
medroxyprogesterone acetate (17) and was usually associated with
down-regulation of sex steroid receptors (7, 17). In the neoplastic
glandular cells of well differentiated carcinomas, however, HSP70 was
not induced, and the constitutive expression of ER and PR was present
even in the presence of progesterone (17). These findings suggest that
strong expression of HSP70 may be related to the down-regulation of sex
steroid receptors. Overexpression of HSP70 has been found in poorly
differentiated endometrial carcinomas and epithelial ovarian carcinomas
with p53 protein expression and is associated with the loss of sex
steroid receptors (17, 18).
Our study also showed changes in HSP70 expression in the
myometrium during the menstrual cycle. Both HSP70 mRNA and protein
levels were significantly higher in the proliferative phase of the
menstrual cycle, and strikingly, this was in contrast to changes in
HSP70 expression in the endometrial glands. Myometrial smooth muscle
cells maintain the expression of PR even in the secretory phase (9, 10), and progesterone has been known to play a role in the
proliferative activity (10) and in the morphological and functional
differentiation of the smooth muscle cells in the secretory phase and
during pregnancy (19). Therefore, the expression of HSP70 seems to be
inversely correlated with the hormonal regulation of the function and
proliferation of myometrial smooth muscle cells. This finding may be
consistent with previous in vivo and in vitro
studies which suggested that HSP70 may act as a repressor of steroidal
effects (5, 7).
In conclusion, the expression of both HSP70 and HSP90 is
differently regulated in the endometrium and myometrium and changes
during the menstrual cycle. In the mouse or sheep uterus, treatment
with estradiol has been shown to increase HSP90 mRNA and protein levels
(20, 21, 22, 23). Progesterone has also been shown to transiently increase
HSP70 mRNA expression in human breast cancer cell lines possessing PRs
(14). Therefore, HSP expression may be regulated by sex steroids.
However, the biological roles of HSP70 and HSP90 in the human
endometrium and myometrium remain undetermined, and further analysis is
needed to clarify them.
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Footnotes
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1 This work was supported by Grant-in-Aid 05454447 for
Scientific Research from the Ministry of Education, Science, and
Culture, Japan. 
Received December 20, 1996.
Revised January 31, 1997.
Accepted February 5, 1997.
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References
|
|---|
-
Lindquist S, Craig EA. 1988 The heat shock
proteins. Annu Rev Genet. 22:631–677.[CrossRef][Medline]
-
Welch WJ. 1992 Mammalian stress response: cell
physiology, structure/function of stress proteins, and implications for
medicine and disease. Physiol Rev. 72:1063–1081.[Free Full Text]
-
Kost SL, Smith DF, Sullivan WP, Welch WJ, Toft DO. 1989 Binding of heat shock proteins to the avian progesterone receptor. Mol Cell Biol. 9:3829–3838.[Abstract/Free Full Text]
-
Schwalter DB, Sullivan WP, Maihle NJ, et al. 1991 Characterization of progesterone receptor binding to the 90- and 70-kDa
heat shock proteins. J Biol Chem. 266:21165–21173.[Abstract/Free Full Text]
-
Bagchi MK, Tsai SY, Tsai MJ, O’Malley BW. 1991 Progesterone enhances target gene transcription by receptor free of
heat shock proteins hsp90, hsp56, and hsp70. Mol Cell Biol. 11:4998–5004.[Abstract/Free Full Text]
-
Smith DF, Toft DO. 1993 Steroid receptors and
their associated proteins. Mol Endocrinol. 7:4–11.[CrossRef][Medline]
-
Koshiyama M, Konishi I, Nanbu K, et al. 1995 Immunohistochemical localization of heat shock proteins HSP70 and HSP90
in the human endometrium: correlation with sex steroid receptors and
Ki-67 antigen expression. J Clin Endoclinol Metab. 80:1106–1112.[Abstract]
-
Garcia E, Bouchard P, De Brux J, et al. 1988 Use
of immunocytochemistry of progesterone and estrogen receptors for
endometrial dating. J Clin Endocrinol Metab. 67:80–87.[Abstract]
-
Lessey BA, Killam AP, Metzger DA, Haney AF, Greene GL,
McCarty KS. 1988 Immunohistochemical analysis of human uterine
estrogen and progesterone receptors throughout the menstrual cycle. J Clin Endocrinol Metab. 67:334–340.[Abstract]
-
Kawaguchi K, Fujii S, Konishi I, et al.
Immunohistchemical analysis of oestrogen receptors, progesterone
receptors and Ki-67 in leiomyoma and myometrium during the menstrual
cycle and pregnancy. Virchows Arch A. 419:309–315.
-
Noyes RW, Hertig AT, Rock J. 1950 Dating the
endometrial biopsy. Fertil Steril. 1:3–10.
-
Chomczynsky P, Sacchi N. 1987 Single-step method of
RNA isolation by acid guanidinium thiocyanate-PhOH-chloroform
extraction. Anal Biochem. 162:156–159.[Medline]
-
Tabibzadeh S, Kong QF, Satyaswaroop PG, Babaknia A. 1996 Heat shock proteins in human endometrium throughout the menstrual
cycle. Hum Reprod. 11:633–640.
-
Tang PZ, Gannon MJ, Andrew A, Miller D. 1995 Evidence for oestrogenic regulation of heat shock protein expression in
human endometrium and steroid-responsive cell lines. Eur J Endocrinol. 133:598–605.[Abstract]
-
Bresnick EH, Dalman FC, Sanchez ER, Pratt WB. 1989 Evidence that the 90-kDa heat shock protein is necessary for the
steroid binding conformation of the L cell glucocorticoid receptor. J Biol Chem. 264:4992–4997.[Abstract/Free Full Text]
-
Tabibzadeh S. 1990 Proliferative activity of
lymphoid cells in human endometrium throughout the menstrual cycle. J Clin Endocrinol Metab. 70:437–443.[Abstract]
-
Nanbu K, Konishi I, Komatsu T, et al. 1996 Expression of heat shock proteins HSP70 and HSP90 in endometrial
carcinomas: correlation with clinicopathology, sex steroid receptor
status, and p53 protein expression. Cancer. 77:330–338.[CrossRef][Medline]
-
Koshiyama M, Konishi I, Mandai M, et al. 1995 Immunohistochemical analysis of p53 protein and 72 kDa heat shock
protein (HSP72) expression in ovarian carcinomas: correlation with
clinicopathology and sex steroid receptor status. Virchows Archiv. 425:603–609.[Medline]
-
Fujii S, Konishi I, Katabuchi H, Okamura H. 1990 Ultrastructure of smooth muscle tissue in the female reproductive
tract: uterus and oviduct. In: Motta PM, ed. Ultrastructure of smooth
muscle. Boston: Kluwer; 197–220.
-
Ramachandran C, Catelli MG, Schneider W, Shyamala
G. 1988 Estrogenic regulation of uterine 90-kilodalton heat shock
protein. Endocrinology. 123:956–961.[Abstract]
-
Olazábal UE, Pfaff DW, Mobbs CV. 1992 Estrogenic regulation of heat shock protein 90 kDa in the rat
ventromedial hypothalamus and uterus. Mol Cell Endocrinol. 84:175–183.[CrossRef][Medline]
-
Shyamala G, Gauthier Y, Moore SK, Catelli MG, Ulrrich
SJ. 1989 Estrogenic regulation of murine uterine 90-kilodalton
heat shock protein gene expression. Mol Cell Biol. 9:3567–3570.[Abstract/Free Full Text]
-
Wu WX, Derks JB, Zhang Q, Nathanielsz PW. 1996 Changes in heat shock proteins-90 and -70 messenger ribonucleic acid in
uterine tissues of the ewe in relation to parturition and regulation by
estradiol and progesterone. Endocrinology. 137:5685–5693.[Abstract]
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Abstract
Introduction
