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Molecular Human Reproduction vol.3 no.3 pp. 233–240, 1997
The expression of transforming growth factor-βs and TGF-β
receptor mRNA and protein and the effect of TGF-βs on human
myometrial smooth muscle cells in vitro
Xin-Min Tang, Qingchuan Dou, Yong Zhao, Frederick McLean, John Davis and Nasser Chegini1
Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, FL 32610-0294, USA
1To
whom correspondence should be addressed
In this study we investigated the expression of transforming growth factor-β (TGF-β) isoform and TGF-β
receptor mRNA and protein, and the effect of TGF-β1–3 on the rate of DNA synthesis and proliferation of
human myometrial smooth muscle cells in vitro. To determine these, we utilized primary cultures of
myometrial smooth muscle cells, standard and competitive quantitative reverse transcription–polymerase
chain reaction (RT–PCR), immunocytochemistry, enzyme-linked immunoassay, radioreceptor assay, [3H]
thymidine incorporation and cell proliferation assay. Standard RT–PCR and immunocytochemistry revealed
that myometrial smooth muscle cells express TGFβ1–3 and TGF-β type I–III receptor (TGF-βR) mRNA and
protein. Quantitative RT–PCR, using an external synthetic RNA standard, indicated that the cells express 10
copies/cell of TGF-β1 and TGF-β2, less than one copy/cell of TGF-β3 and TGF-β type IR, three copies/cell of
type IIIR, and .200 copies/cell of TGF-β type IIR mRNA. The cells also synthesized and released TGF-β1 at
the rate of 7.8 6 0.7 ng/106 cells, of which 1.4 6 0.2 ng/106 cells was in an active form. The rate of [3H]
thymidine incorporation or proliferation of subconfluent quiescent smooth muscle cells was not altered by
TGF-βs (0.1–10 ng/ml) under serum-free conditions, nor in the presence of 10% fetal bovine serum (FBS).
TGF-β1–3 at 0.25–0.5 ng/ml in the presence of 2% FBS, which induces half maximal stimulation of these cells,
stimulated the rate (P ,0.05), whereas at higher doses it reduced the rate of [3H]-thymidine incorporation
compared to the controls. The effect of TGF-β was partially reversible using neutralizing antibodies specific
to TGF-β1, TGF-β2 (10 µg/ml) or TGF-β3 (3–6 µg/ml). TGF-βs had no significant effect on cell proliferation
determined by cell counting. The data indicate that human myometrial smooth muscle cells express the
necessary components of the TGF-β system, suggesting an autocrine/paracrine role for TGF-βs in myometrium.
Key words: expression/human/myometrium/TGF-β/TGF-β receptors
Introduction
In addition to the ovarian steroids, several autocrine/paracrine
growth factors which are expressed by different uterine cell
types appear to play an important role in a variety of uterine
biological activities. Unlike endometrium which undergoes a
rapid morphological and biochemical alteration during the
menstrual cycle, myometrium is a quiescent tissue consisting
of differentiated smooth muscle cells. These cells represent a
state in which they are unable to enter the cell cycle; however, they undergo cellular enlargement or hypertrophy during
pregnancy and in leiomyomata which are benign tumours
originating from myometrial smooth muscle cells (Rein et al.,
1995; Dou et al., 1996). This suggests that myometrial smooth
muscle cells retain their ability to undergo the cell cycle if
provided with appropriate stimuli such as growth factors.
Myometrium, leiomyomata and isolated myometrial smooth
muscle cells have been shown to express mRNA and protein
for several growth factors such as epidermal growth factor
(EGF), transforming growth factor (TGFs), platelet-derived
growth factors (PDGFs), insulin-like growth factor (IGFs),
and their receptors (Bohem et al., 1990; Mondoza et al., 1990;
Rein et al., 1990; Yeh et al., 1990; Rossi et al., 1992; Giudice
et al., 1993; Chegini et al., 1994, 1996; Harrison-Woolrych
© European Society for Human Reproduction and Embryology
et al., 1994; Tang et al., 1994a; Dou et al., 1996). These
growth factors, either alone or synergistically, appear to regulate
the growth of myometrial and leiomyomata smooth muscle
cells in vitro (Fayed et al., 1989; Rossi et al., 1992; Tang
et al., 1994b) and possibly influence their differentiation and hypertrophic activities during pregnancy and
leiomyomata growth.
Among these growth factors, TGF-βs are recognized for
their bifunctional activities influencing stimulation/inhibition
of cell growth, differentiation, synthesis and deposition of
extracellular matrix, and cellular hypertrophy mediated through
their specific receptors (Massague, 1990; Massague et al.,
1990; Wang et al., 1991; Ebner et al., 1993; Bassing et al.,
1994; ten Dijke et al., 1994; Wrana et al., 1994). We have
demonstrated that human myometrium and leiomyomata
express TGF-β1–3, TGF-β type I–III receptor mRNA and
protein, and contain specific [125I]TGF-β1 binding sites
(Chegini et al., 1994; Dou et al., 1996). The data suggest that
TGF-βs may be key regulators of various biological activities
of smooth muscle cells under both normal and tumour
(leiomyoma) conditions. The present study provides evidence
that myometrial smooth muscle cells in primary culture express
the necessary TGF-β and receptor system components, and
233
X.-M.Tang et al.
that exogenous TGF-βs modulate the rate of DNA synthesis
in, but not proliferation of, these cells.
Materials and methods
The sources of materials and detailed description of methodologies
such as isolation, culturing and characterization of myometrial
smooth cells, reverse transcription–polymerase chain reaction
(RT–PCR), immunocytochemistry and DNA synthesis, TGF-β and
TGF-β receptor antibodies were similar to those previously described
(Tang et al., 1994a; Dou et al., 1996). [125I]-TGF-β1 (specific activity
220 βCi/µg), methyl-[3H]-thymidine (83 Ci/mmol) and [14C]-valine
(250 mCi/mmol) were purchased from Biomedical Technologies Inc.
(Soughton, MA, USA) and Amersham Co. (Arlington Heights,
IL, USA).
Human uterine tissues from premenopausal women, ages ranging
from 21–39 years, undergoing hysterectomy for medically indicated
reasons (excluding endometrial cancer and leiomyomata) and who
were not under any hormonal treatments at the time of surgery, were
collected and used for cell culture. Institutional Review Board
approval was obtained prior to collection and use of the tissues in
this study.
Expression of TGF-βs and receptor mRNA and protein
Myometrial smooth muscle cells were isolated and grown as monolayers in Dulbecco’s modified Eagle’s medium (DMEM)/10%
fetal bovine serum (FBS) and their purity was determined as previously
described (Rossi et al., 1992; Tang et al., 1994b). The cells were
grown to 80–90% confluence in the presence of 10% FBS, and total
cellular RNA was isolated from three individual cultures and subjected
to standard and quantitative RT–PCR as previously described (Dou
et al., 1996). For standard RT–PCR, 2 µg of total cellular RNA from
each individual preparation was subjected to PCR, with reaction
conditions of 1 min at 94°C, 2 min at 58°C and 3 min at 72°C for
40 cycles (Tang et al., 1994a; Dou et al., 1996). Controls included
total RNA amplified without the reverse transcription step to detect
the presence of any contaminating genomic DNA, and tubes containing
all the PCR components except the RT reaction mixture to check
for the presence of DNA that may have been carried over from
prior reactions.
Competitive quantitative RT–PCR was carried out using external
synthetic multiprimer RNA standard templates which were constructed
to contain the complimentary sequences for human TGF-βs and TGFβ receptors as previously described (Dou et al., 1996). cDNA was
synthesized in standard reactions each containing 2 µg of total RNA
(from individual preparation), several dilutions of external RNA
standard (13108–13104 copies/reaction), as well as 2.5 µM oligo
(dT)16, 1.5 mM MgCl2, 200 µM dNTPs, 1 IU/µl human placental
ribonuclease inhibitor, 10 mM Tris–HCl (pH 8.3), 50 mM KCl, and
200 IU/µg RNA Moloney murine leukaemia virus reverse transcriptase
(MMLV–RT) in a final volume of 100 µl. The reactions were
incubated at 25°C for 10 min, 37°C for 60 min, and 92°C for 5 min,
and DNA amplification was carried out as previously described (Dou
et al., 1996). The PCR products were separated by electrophoresis
on 2% agarose gel containing 2 ng/ml of ethidium bromide and
photographed. The photographs were scanned and band intensities
were determined using NIH-Image version 1.54; (National Institute
of Health, Bethesda, MA, USA) their intensity values were normalized
for their molecular weight (Dou et al., 1996). The ratio of band
intensities within each lane was plotted against the copy number of
added RNA standard/reaction and quantity of the target messages
was determined where the ratio of template/target band intensities
was equal to one (Dou et al., 1996). The final quantification of the
234
number of molecules per cell was derived from the constant that
there is ~26 pg of mRNA per cell (Brandhorst and McConkey, 1974).
The data are expressed as mean 6 SEM of the band intensities. Each
corresponding point of the curves was analysed by Student’s t-test
and all points on the curves by analysis of variance. A value of
P ,0.05 was considered to be significant.
For immunocytochemistry and light microscope autoradiography,
smooth muscle cells were grown on Lab-Tek (Nunc. Inc. Naperville,
IL, USA) 8-well slides in the presence of 10% FBS for 48 h (Rossi
et al., 1992). The slides were washed with PBS, fixed and processed
for immunolocalization of TGF-βs and TGF-β type I–II receptors
using their respective antibodies, or incubated with 1 nM [125I]-TGFβ1 in the presence and absence of 100-fold excess of unlabelled
TGF-β1 for 2 h at 37°C for autoradiographic study after exposure to
autoradiographic emulsion (Rossi et al., 1992; Tang et al., 1994a;
Zhao et al., 1994).
Effect of TGF-βs on [3H]-thymidine incorporation and cell
proliferation
The smooth muscle cells were cultured either in 24- or 96-well
dishes at an approximate density of 2.53104 and 2.53103 cells/well
respectively, in the presence of 10% FBS for 48 h. The cells were
made quiescent under serum-free conditions for 48 h (Tang et al.,
1994b) and then incubated in either serum-free or 2% FBS-supplemented medium, in the presence of various doses of TGF-βs and
2 µCi/ml [3H]-thymidine. Cell proliferation was determined by cell
counting using a Coulter counter ZM (Coulter Electronics, Hialeah,
FL, USA), as previously described (Rossi et al., 1992). The specificity
of TGF-β actions on [3H]-thymidine incorporation was determined
using specific neutralizing antibodies to TGF-β1 and TGF-β2
(5–10 µg/ml), and TGF-β3 (3–6 µg/ml). The antibodies were added
to the culture medium in the presence of corresponding TGF-βs at
the initial concentrations of 3–5 µg/ml, which induce half maximal
inhibition of [3H]-thymidine incorporation into fibroblasts (R&D
information, R&D System Inc., Minneapolis, MN, USA) and endometrial stromal cells (Tang et al., 1994a).
Radioreceptor assay (RRA)
The smooth muscle cells were cultured in 24-well dishes in the
presence of 10% FBS for 48 h, washed and further incubated in the
presence of 0.5% FBS for an additional 24 h. The culture medium
was collected and assayed for TGF-β1 using a competitive RRA
measuring the binding of [125I]-TGF-β1 to CCL64 cells, as previously
described (Tang et al., 1994a). Culture media and 0.5% FBS used
for culturing were assayed before and after acidification which resulted
in the activation of latent TGF-βs (Tang et al., 1994a; Chegini et al.,
1996). The levels of TGF-β1 in the conditioned media were compared
with known concentrations of TGF-β1 standard.
Protein degradation assay
The effect of TGF-β1 on degradation of long-lived proteins in
myometrial smooth muscle cells was determined by measuring the
rate of [14C]-valine incorporation into newly synthesized proteins as
previously described (Tang et al., 1994a). Smooth muscle cells were
grown to confluence, then incubated in valine-free RPMI-based
selectamine containing 2% FBS and [14C]-valine (1β) for 18 h,
washed and incubated in serum-free medium containing 0.1% bovine
serum albumin (BSA), 15 mM valine and 1 ng/ml of TGF-β1, and
compared with 10 ng/ml of EGF, 10 ng/ml of PDGF-BB or 2% FBS.
The amount of radioactive protein present in the media 1 and 4 h
after the incubation was compared with the total radioactivity added
to the media (cells 1 1 h and 4 h media) to determine the level of
TGF-βs in human myometrium
Figure 1. The reverse transcription–polymerase chain reaction (RT–PCR) products and anticipated bp fragments for transfroming growth
factor (TGF)-β1 (4 bp lane A), TGF-β2 (310 bp, lane C), TGF-β3 (524 bp, lane E), TGF-β type IIR (431 bp, lane G), TGF-β type IR
(545 bp, lane I) and TGF-β type IIIR (1104 bp, lane K) using total RNA isolated from myometrial smooth muscle cell primary cultures
initially isolated from myometrium at the mid-secretory phase of the menstrual cycle. Digestion of the PCR products with SmaI (TGF-β1),
HindIII (TGF-β2), EcoR V (TGF-β3), HincII (TGF-β type IIR), PvuII (TGF-β type IR) and BamHI (TGF-β type IIIR) resulted in the
anticipated 360, 83 bp fragments for TGF-β1 (lane B), 164, 146 bp fragments for TGF-β2 (lane D), 332, 192 bp fragments for TGF-β3
(lane F), 261, 170 bp fragment for TGF-β type IIR (lane H), 179, 366 bp fragments for TGF-β type IR (lane J) and 183, 325, 596 bp
fragments for TGF-β type IIIR (lane L). The 83 bp in lane B, 146 bp in lane D, 192 bp in lane F and 183 bp fragments in lane L are only
faintly visible due to the reduced capacity to bind to ethidium bromide. M: DNA marker.
cellular protein degradation, as previously described (Tang et al.,
1994a).
Statistical analysis
Data presented in Figures 2–5 are reported as the mean 6 SEM and
analysed using paired Student’s t-test or analysis of variance. All of
the experiments, with the exception of immunocytochemistry and
autoradiography, were repeated either two or three times, in triplicate,
using myometrial cells isolated from the indicated number of uteri.
Results
Expression of TGF-βs and receptor mRNA and protein
Initially, the expression of TGF-β1–3 and TGF-β type I–III
receptor mRNA in isolated myometrial smooth muscle cells
in primary culture was examined qualitatively by standard
RT–PCR. As shown in Figure 1, the standard RT–PCR revealed
that these cells express TGF-β1 (lane A), TGF-β2 (lane C)
and TGF-β3 (lane E) as well as TGF-β type IIR (lane G),
type IR (lane I) and type IIIR (lane K) receptor mRNA in a
representative experiment. Digestion of the PCR products with
SmaI, HindIII, EcoR V, HincII, PvuII, or BamHI for TGF-β13 and TGF-β type II, I and III receptors respectively, resulted
in anticipated smaller fragments for TGF-β1 (lane B), TGF-
β2 (lane D), TGF-β3 (lane F), and TGF-β type IIR (lane H),
type IR (Lane J), and type IIIR (Lane L) receptors. To
determine precisely the levels of TGF-βs and TGF-β receptor
mRNA expression by myometrial smooth muscle cells, we
used quantitative RT–PCR. The results indicated that the
smooth muscle cells express 10 copies/cell of TGF-β1 and
TGF-β2, less than one copy/cell of TGF-β3 and TGF-β type
IR, three copies/cell of type IIIR, and .200 copies/cell of
TGF-β type IIR mRNA (Figures 2A,B).
The myometrial smooth muscle cells also contain immunoreactive TGF-β1–3 and TGF-β type I and type II receptor
protein (Figures 3A–E). In controls, using normal immunoglobulin (Ig)G and/or non-immune rabbit serum instead of
the primary antibodies resulted in substantial reduction of
immunostaining intensity (Figure 3F). Competitive RRA further indicated that the smooth muscle cells synthesized and
released 7.8 6 0.7 ng/106 cells of total TGF-β1 into their
culture-conditioned media, of which 1.4 6 0.2 ng/106 cells
were in an active form. The level of total TGF-β1 present in
0.5% serum unexposed to cells was ~187 6 14 pg/ml, of
which 80 6 11 pg/ml were in an active form. Light microscope
autoradiography revealed that the smooth muscle cells also
contain specific [125I]-TGF-β1 binding sites as indicated by
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X.-M.Tang et al.
the presence of many silver grains over these cells, which
were significantly reduced after incubation with 100-fold
excess of unlabelled TGF-β1, but not TGF-I or PDGF-BB
(data not shown).
Effect of TGF-βs on [3H]-thymidine incorporation, cell
proliferation and protein degradation
TGF-βs at 0.01–10 ng/ml appeared not to have a significant
effect on the rate of [3H]-thymidine incorporation and proliferation of the smooth muscle cells incubated under serum-free
conditions or in the presence of 10% FBS (data not shown).
However, incubation of quiescent smooth muscle cells with
2% FBS induced half the maximal stimulation of [3H]thymidine incorporation and proliferation compared with lower
(0.5–1%) or higher (5–10%) FBS concentrations as previously
described (Tang et al., 1994b). Treatment of the quiescent
smooth muscle cells with TGF-β1, TGF-β2 or TGF-β3 at
doses ranging from 0.25–1 ng/ml in the presence of 2%
FBS resulted in a significant stimulation of [3H]-thymidine
incorporation with maximal effect occurring at 0.5 ng/ml
compared with 2% FBS control (P ,0.05, Figures 4A, C and
E). At higher doses, TGF-β1 and TGF-β2, but not TGF-β3
were found to inhibit the rate of [3H]-thymidine incorporation
with maximal effect occurring at 5 ng/ml (P ,0.05). TGFβ1–3 at all the concentrations tested had no significant effect
on cell proliferation determined by cell counting (data not
shown) compared with 2% FBS. Specific neutralizing antibodies to TGF-β1 (10 µg/ml), TGF-β2 (10 µg/ml) or TGF-β3
(6 to 10 µg/ml) partially reversed the effect of TGF-β1 and
TGF-β2 (0.5 ng/ml) on [3H]-thymidine incorporation (Figures
4B, D and F), reaching the control values induced by 2% FBS.
TGF-β1, but not EGF or PDGF-BB, significantly increased
the rate of protein degradation (P ,0.05) measured by [14C]valine incorporation into newly synthesized proteins compared
with untreated control (Figure 5).
Discussion
We have previously shown that human myometrium (smooth
muscle cells) throughout the menstrual cycle expresses TGFβ1–3 mRNA and protein, with TGF-β3 being the least
expressed (Chegini et al., 1994). The present study confirms
and further demonstrates that isolated myometrial smooth cells
in primary culture express a significantly higher level of TGFβ1 and TGF-β2 mRNA than TGF-β3. These smooth muscle
cells also contain TGF-β1–3 immunoreactive proteins which
are synthesized and released into the culture medium as
demonstrated for TGF-β1. A major portion of TGF-β1 released
into the culture-conditioned medium was in latent or biologically inactive form which was significantly less than that
produced by endometrial glandular epithelial or stromal cells
(Tang et al., 1994b). Factors which regulate the expression of
Figure 2. (A) Competitive quantitative reverse transcription–
polymerase chain reaction (RT–PCR) of total RNA isolated from
myometrial smooth muscle cells in primary culture. Total RNA as
well as the external synthetic RNA standard at serial dilutions of 104–
10–1 molecules were mixed, reverse transcribed and co-amplified by
standard PCR using TGF-β1–3, TGF-β type IR–IIIR receptor primers
for 40 cycles. The cDNAs were separated on 2% agarose gels, stained
with ethidium bromide and photographed. Top bands represent
products generated from specific messages in the total RNA, and
lower bands are the products generated from serial dilutions of
external RNA standard shown from right to left at dilutions of 104–
10–1 copies/cell. The copy number is determined where the intensity
of input external standard RNA is equal to intensity of the sample
RNA. The far left lanes are the DNA markers. (B) The ratio of
external synthetic RNA standard template (T) to sample (S) band
intensities was calculated by digitally scanning photographs similar to
that shown in Figure 2A. Ethidium bromide stained gels were
photographed, scanned and the band intensities determined as
described the text. The intensity values were then normalized for their
molecular weight, and the ratio of T/S was calculated. The log ratio
was plotted against the log input copy number of the template (serial
dilutions corresponding to 104–10–1 molecules), and copy number
determined where the intensity of input RNA was equal to intensity of
the unknown sample. The level of TGF-β1 (d), TGF-β2 (,), TGFβ3 (.) and TGF-β type IIR (u) TGF-β type IIIR (u) mRNA
expression are shown. Equations of best fit lines as follows; TGF-β1:
y 5 0.482 (x) – 0.015 with r2 5 0.999; TGF-β2: y 5 0.604 (x) – 0.022
with r2 5 0.942; TGF-β3: y 5 0.520 (x) – 0.707 with r2 5 0.999;
TGF-β IIR: y 5 0.548 (x) – 0.001 with r2 5 0.942; and TGF-β IIIR:
y 5 0.612 (x) – 0.129 with r2 5 0.996.
236
TGF-βs in human myometrium
TGF-βs in human uterus have not yet been investigated;
however, due to the higher levels of expression of TGF-βs
during the secretory phase of the menstrual cycle, progesterone
is believed to influence TGF-β expression (Chegini et al.,
1996; Dou et al., 1996). Due to distinct differences in TGF-β
promoters, as well as 59 and 39 untranslated regions, each
isoform can be regulated differently in response to various
stimuli (Romeo et al., 1993; Roberts, 1995). Furthermore, our
preliminary data suggest that TGF-β1 up-regulates its own
expression in isolated myometrial smooth muscle cells (Tang
et al., 1996).
At the protein level, TGF-β is released by a variety of cells
in culture either as a small latent complex, consisting of TGFβ non-covalently binding to latency-associated protein, or, as
a large complex, formed by covalent addition of latent TGFβ binding protein to latency-associated protein (Olofsson et al.,
1992; Roberts 1995). The latent TGF-βs, after being released
by the cells, become sequestered by extracellular matrix;
therefore, disassociation from these complexes is essential
for bioavailability of TGF-βs and binding to their receptors
(Olofsson et al., 1992; Roberts 1995). The mechanisms which
regulate TGF-β activation in vivo are unknown; however
in vitro, plasmin, transglutamase, mannose-6-phosphate receptor, binding to thrombospondin, steroids such as retinoic acid,
tamoxifen and 1,25-dihydroxy vitamin D3 have been shown
to result in TGF-β activation (Laiho et al., 1986; Lyons et al.,
1990; Sato et al., 1990; Roberts 1995). We have recently
shown that 17β oestradiol medroxyprogesterone acetate (MPA)
up-regulates the synthesis of total (active 1 latent) TGFβ1 production by myometrial smooth muscle cells, while
gonadotrophin-releasing hormone (GnRH) agonist and GnRH
antagonist inhibit the proportion of TGF-β1 released as an
active form (Chegini et al., 1996). GnRH analogues also
inhibit the effect of 17β oestradiol with MPA on total and
active TGF-β1 production (Chegini et al., 1996).
Like myometrium (Chegini et al., 1994), myometrial smooth
muscle cells also express TGF-β type I–III receptor mRNA
and protein, and contain specific [125I]-TGF-β1 binding sites,
Figure 3. Immunocytochemical localization of transforming growth factor (TGF)-β1 (A), TGF-β2 (B), TGF-β3 (C), TGF-β type IR (D) and
IIR (E) in myometrial smooth muscle cells maintained in primary culture and incubated in the presence of 10% fetal bovine serum for 48h.
Controls using normal immunoglobulin (Ig)G instead of the primary antibodies resulted in a considerable reduction in immunostaining of
the cells (F). Original magnification 3160.
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X.-M.Tang et al.
Figure 4. The dose response effect of transforming growth factor (TGF)-β1 (A), TGF-β2 (C) and TGF-β3 (E) on [3H]-thymidine
incorporation (s-s, n-n and u-u), and isoform specific neutralizing antibodies (AB) to TGF-β1 (B), TGF-β2 (D) and TGF-β3 (F) on
[3H]-thymidine incorporation induced by TGF-β1–3 (0.5 ng/ml), by quiescent myometrial smooth muscle cells incubated in the presence of
2% FBS (control) for 48 h. The antibodies were added individually at 10 µg/ml (AB10) in assays involving TGF-β1 and TGF-β2, and
3 µg/ml (AB3) or 6 µ g/ml (AB6) in the case of TGF-β3. The data are expressed as mean 6 SEM of percentage change from triplicate
experiments repeated twice on myometrial smooth muscle cells from three uteri of early secretory phase of the menstrual cycle.
*Significantly different from the control analysed by analysis of variance and/or Student’s t-test (P ,0.05–0.005).
Figure 5. Protein degradation induced by transforming growth
factor (TGF)-β1 (1 ng/ml) and compared with epidermal growth
factor (EGF) (10 ng/ml), platelet-derived growth factor (PDGF)-BB
(10 ng/ml) and 2% fetal bovine serum (FBS; control) in smooth
muscle cells from secretory phase of the menstrual cycle. The data
are expressed as mean 6 SEM of percentage change from triplicate
experiments repeated twice on myometrial smooth muscle cells
from two different uteri. The effect of TGF-β1 was significantly
different from the control value analysed by Student’s t-test
(P ,0.05).
238
with a significantly higher level of type II than I or III mRNA
expression. Presence and interactions of TGF-β type I and II
receptors are required for TGF-β binding and signal transduction (Wang et al., 1991; Ebner et al., 1993; Bassing et al.,
1994; ten Dijke et al., 1994; Wrana et al., 1994). This suggests
an autocrine/paracrine role for TGF-βs in the myometrial
micro-environment. It has been shown that TGF-β1 and
TGF-β3 bind type I and II receptors with higher affinity than
TGF-β2, whereas they bind type III receptor with a similar
affinity (Chen et al., 1993; Ebner et al., 1993; Lopez-Casillas
et al., 1993; Moustakas et al., 1993). In myometrial smooth
muscle cells the maximal stimulatory effect of TGF-β1 and
TGF-β3 on DNA synthesis occurred at lower doses than
TGF-β2. Furthermore, TGF-β1 and TGF-β2 at higher doses
inhibited, while TGF-β3 retained, the rate of DNA synthesis
induced by 2% FBS. A similar phenomenon has also been
reported for vascular smooth muscle cells, in which TGF-β1
at 0.01–0.1 ng/ml elicited maximal effect on DNA synthesis
and proliferation, whereas at higher doses was less potent
(Battegay et al., 1990). TGF-βs appear not to have any effect
on myometrial smooth muscle cell proliferation.
TGF-βs in human myometrium
Stimulation of DNA synthesis, but not proliferation (cell
number), of myometrial smooth muscle cells by TGF-βs
suggests that the cells receive signals for cell mass and DNA
synthesis associated with cell cycle progression, but not cell
division. Such a phenomenon often occurs during cellular
hypertrophy resulting from incomplete growth stimulation, and
is regulated to some extent by growth factors such as TGF-β
with the ability to arrest cells in the G1/S phase of the cell
cycle (Brodsky and Uryvaeva, 1977; Baserga, 1984; Geisterfer
et al., 1988; Owens et al., 1988; Turner et al., 1988; Battegay
et al., 1990). Cellular hypertrophy occurs in terminally differentiated cells such as smooth muscle cells, and in the uterus
during pregnancy (uterine enlargement) and in leiomyomata,
which is characterized by enlargement of existing smooth
muscle cells with little or no change in cell number (Rein
et al. 1995). In addition, TGF-β may be involved not only in
regulating cell growth and proliferation, but also in controlling
the production of prostaglandins, and hence, controlling
myometrial contractility (Faber et al., 1996). The rate of
degradation of newly synthesized long-lived protein in
myometrial smooth muscle cells was significantly increased
by TGF-β1 but not EGF or PDGF-BB in our experiment,
which was different from the observations in endometrial
stromal cells by Tang et al. (1994a). The identity and role of
these proteins in myometrial smooth muscle cells are unknown;
however, they may be involved in cell cycle progression.
In summary, the results provide further evidence that human
myometrial smooth muscle cells express all the necessary
components of TGF-β, suggesting that TGF-βs through an
autocrine/paracrine manner modulate various biological activities of myometrial smooth muscle cells.
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Received on August 12, 1996; accepted on December 23, 1996
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