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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 3 1350-1361
Copyright © 2003 by The Endocrine Society

Differential Expression, Regulation, and Induction of Smads, Transforming Growth Factor-ß Signal Transduction Pathway in Leiomyoma, and Myometrial Smooth Muscle Cells and Alteration by Gonadotropin-Releasing Hormone Analog

Jingxia Xu, Xiaoping Luo and Nasser Chegini

Department of Obstetrics/Gynecology, University of Florida, Gainesville, Florida 32610

Address all correspondence and requests for reprints to: Dr. Nasser Chegini, Department of Obstetrics/Gynecology, University of Florida, Box 100294, Gainesville, Florida 32610. E-mail: cheginin{at}obgyn.ufl.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The objective of this study was to further elucidate the role of TGFß and GnRH analog (GnRHa) in leiomyoma growth and regression. We examined the expression of Smads, TGFß receptor intracellular signaling molecules, in leiomyoma and myometrial smooth muscle cells (LSMC and MSMC), and determined whether TGFß and GnRHa differentially regulate their expression and induction in these cells. Using semiquantitative RT-PCR, Western blot analysis, and immunohistochemistry, we demonstrated that leiomyoma, myometrium, LSMC, and MSMC express receptor-activated Smad3, common Smad4, and the inhibitory Smad7 mRNA and protein and showed that TGFß1, in a time-dependent manner, transiently induced Smad7 expression, with Smad3 and Smad4 remaining largely unchanged. TGFß1 increased the rate of Smad and phosphorylated Smad3 (pSmad3) induction in both cell types. Pretreatment with TGFß type II receptor antisense oligonucleotide resulted in a trend toward a lower TGFß-induced pSmad3. GnRHa, in a dose- and time-dependent manner, increased the expression of Smad7 mRNA and the rapid induction of Smad3, Smad4, and Smad7 as well as pSmad3, which declined to control values at doses above 1 µM in MSMC, but not in LSMC. GnRHa-induced pSamd3 was partly inhibited by a GnRH antagonist (antide). We concluded that leiomyoma, myometrium, LSMC, and MSMC express Smads, which are differentially expressed, induced, and activated by TGFß and are altered as a result of GnRHa treatment. These results suggest that TGFß and GnRHa mediate their actions through cross-talk involving Smads and most likely other signaling pathways that result in leiomyoma growth and regression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEIOMYOMAS ARE BENIGN uterine tumors considered to originate from cellular transformation of myometrial smooth muscle cells and/or connective tissue fibroblasts during the reproductive years. The identity of factors that initiate such cellular transformation is not known; however, ovarian steroids are essential for leiomyoma growth, and GnRH analog (GnRHa) therapy, creating a hypoestrogenic condition, is often used for their medical management (1, 2). GnRHa-induced leiomyoma regression is accompanied by alterations in uterine arteriole size, blood flow, and cellular content as well as changes in the expression of several growth factors, cytokines, extracellular matrix, proteases, and protease inhibitors (reviewed in Refs.3 and4). Differential expression and autocrine/paracrine action of many of these molecules are considered to play a central role in leiomyoma growth and GnRHa-induced regression (3, 4).

TGFß, a multifunctional cytokine, is a key regulator of cellular migration, growth and differentiation, inflammation, extracellular matrix (ECM) turnover and tissue remodeling (5, 6). Overproduction of TGFß is widely accepted as a key element in tissue fibrosis, acting through a mechanism involving enhanced cell migration, expression, and deposition of various ECM with concurrent inhibition of proteases that accelerate ECM degradation (6). Leiomyoma is a fibrotic disorder in which TGFß and TGFß receptors are overexpressed compared with normal unaffected myometrium, and GnRHa-induced leiomyoma regression is accompanied by down-regulation of their expression (7). Under in vitro conditions, TGFß regulates its own expression, the expression of ECM, matrix metalloproteinases, and tissue inhibitor of matrix metalloproteinases as well as the growth of leiomyoma and myometrial smooth muscle cells (8, 9, 10, 11). In addition, GnRHa treatment has been shown to down-regulate TGFß- and ovarian steroid-induced TGFß expression in these cells (11).

TGFß mediates its biological activity from the cell surface to the nucleus through the activation of serine/threonine kinase TGFß receptors and subsequent activation of multiple intracellular signals, including Smads (reviewed in Ref.12). Smads are comprised of regulatory (Smad1, -2, -3, -5, and -8; RSmad), common (Smad4), and inhibitory (Smad6 and -7) types, of which the pathway-specific RSmads become phosphorylated by activated TGFß type I receptor kinase, associate with Smad4, and translocate into the nucleus (13, 14). The inhibitory Smad7 interacts with TGFß type I receptor and prevents the phosphorylation of RSmad that leads to interruption of TGFß receptor signaling (13, 14). Similar to TGFß, Smads are expressed by various normal and malignant cell and tissues. Gene mutation and/or alteration of Smad expression have been associated with several abnormalities, including resistance to growth inhibitory action of TGFß, matrix expression, and malignancies (15, 16, 17).

We hypothesized that overexpression of TGFß and TGFß receptor in leiomyoma leads to alteration of TGFß intracellular signaling and the underlying mechanism of TGFß action. We also hypothesized that GnRHa-induced leiomyoma regression, which results in down-regulation of TGFß and TGFß receptors, also alters the components of the TGFß signal transduction pathway. To test our hypothesis we first determined the expression and cellular localization of Smads in leiomyoma and myometrium. We then isolated leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) to determine 1) whether LSMC and MSMC express Smad3, Smad4, and Smad7; 2) whether TGFß regulates the expression and induction of Smads; and 3) if their expression and induction are altered by GnRHa.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All of the materials for collection of leiomyoma and myometrium, isolation and culturing of their smooth muscle cells, as well as semiquantitative RT-PCR, Western blotting, and immunohistochemistry were purchased from commercial sources, as previously described (18). Recombinant human TGFß1 was purchased from R Systems, Inc. (Minneapolis, MN). Affinity-purified mouse monoclonal anti-Smad4, rabbit antiphosphorylated Smad3 (pSmad3), goat anti-Smad3, and rabbit anti-Smad7 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Additional polyclonal antibody generated against Smad7 was provided by Dr. Carl-Henrik Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden). Monoclonal antibody specific to human ß-actin was purchased from Sigma-Aldrich (St. Louis, MO).

Portions of leiomyoma and matched myometrium were collected from premenopausal women who were undergoing hysterectomy for symptomatic uterine leiomyomas. These patients had not taken any medication during the previous 3 months before surgery. The tissues were collected at the University of Florida-affiliated Shands Hospital with the approval of the institutional review board. Immediately after collection, portions of leiomyoma and matched normal myometrium were snap-frozen and stored in liquid nitrogen, fixed in Bouin’s solution for immunohistochemistry, or prepared for isolation of LSMC and MSMC as previously described (19). The isolated LSMC and MSMC were cultured in DMEM/Ham’s F-12 containing antimycotic, antibiotics and 10% fetal bovine serum and incubated at 37 C in a humidified 5% CO2 incubator until reaching visual confluence. Before their use in these experiments the cell cultures were characterized using {alpha} smooth muscle actin, desmin, and vimentin antibodies as previously described (19).

To determine the expression of Smad-3, -4, and -7 mRNA and protein in leiomyoma and myometrium, total RNA and protein were extracted from these tissues and subjected to semiquantitative RT-PCR and Western blot analysis, and their cellular localization was determined using immunohistochemistry as previously described (7, 18). Total cellular RNA was isolated using TRIzol (Life Technologies, Inc., Grand Island, NY), and an equal amount of RNA (2 µg) was converted to cDNA by RT. The PCR reaction was carried out over a range of 25–35 cycles to obtain the optimal condition within the logarithmic phase of amplification with primers (Table 1Go) for Smad3, Smad4, Smad7, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH; Mastercycler-Gradient, Eppendorf Scientific, Westbury, NY). The cDNA was then subjected to 30–35 cycles of PCR at 95 C (1 min), 55–61 C (0.5 min), and 72 C (1 min) in reaction buffer containing 1.5 mM MgCl2. The PCR products were separated on 1% agarose gels containing ethidium bromide, and the images were captured on a Kodak DC290 digital camera (Eastman Kodak Co., Rochester, NY) and stored as TIFF files. The relative band density was determined using Kodak EDAS and/or NIH Image (version 1.6) densitometry software and was reported as the fold change in the ratio of Smad/G3PDH mRNA.


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  		Table 1. PCR primers used

 
For Western blotting, total protein was isolated from small pieces of leiomyoma and myometrium by homogenization (Polytron, Brinkmann Instruments, Inc.-Eppendorf, Westbury, NY) in a buffer containing 50 mM HEPES (pH 7.4), 1% Nonidet P-40, 0.5% deoxycholate, 5 mM EDTA, 1 mM sodium ortho-vanadate, 5 mM NaF, and phosphatase and protease inhibitor cocktails (Sigma-Aldrich). The lysates were centrifuged at 14,000 x g for 15 min at 4 C, the supernatants were collected, their total protein content was determined using a conventional method (Pierce Chemical Co., Rockford, IL), and aliquots were stored at -80 C until assayed. Equal amounts of sample proteins were resolved using 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane by electroblotting in a buffer containing Tris-HCl (25 mM), glycine (192 mM), and methanol (20%, v/v). After transfer, the blots were incubated in 5% powdered milk in Tween/Tris-buffered saline [10 mM Tris-HCl (pH 7.5), 0.15M NaCl, and 0.1% Tween 20] at room temperature for 1 h, then incubated with anti-Smad3, -Smad4, and -Smad7 and pSmad3 antibodies overnight at 4 C, washed with Tween/Tris-buffered saline, and exposed to corresponding horseradish peroxidase-conjugated IgG for 1 h. Immunostained proteins were visualized using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ) and captured by Kodak DC290 camera, and the band intensity was determined using Kodak and NIH image software.

For immunohistochemistry small portions of leiomyoma and myometrium were fixed in Bouin’s solution and paraffin-embedded and tissue sections 3–5 µm thick were prepared. Following standard procedures that included pretreatments with Triton X-100 and protease, the sections were incubated with anti-Smad3, -4, and -7 and pSmad2/3 antibodies at 5 µg immunoglobulin G/ml prepared in PBS, pH 7.4, containing 0.01% BSA (18). The sections were then exposed to biotinylated secondary antibodies, avidin horseradish peroxidase (Vector Laboratories, Inc., Burlingame, CA), chromogenic reaction was developed using 3,3'-diaminobenzidine, and sections were counterstained with hematoxylin. Tissue sections incubated with normal IgG instead of the primary antibodies or deletion of the primary antibodies during immunostaining served as controls.

To determine whether LSMC and MSMC express Smads, the cells were cultured in six-well dishes at an approximate density of 106 cells/well. After 48 h or until cells reached subconfluence by visual inspection, total cellular RNA and protein were isolated and subjected to semiquantitative RT-PCR and Western blot analysis as described above. The cells were lysed in the above buffer, their total protein content was determined, and an equal amount of protein was subjected to Western blot analysis. To determine whether TGFß1 regulates Smad mRNA and protein expression, serum-starved LSMC and MSMC, cultured as described above, were treated with TGFß1 at 2.5 ng/ml for 2–24 h. Total RNA and protein were isolated and subjected to semiquantitative RT-PCR and Western blot analysis, respectively. To determine whether TGFß1 induces and activates Smads, serum-starved LSMC and MSMC were treated with TGFß1 at doses of 1–5 ng/ml for 5–30 min. Total protein was isolated from TGFß-treated and untreated cells and subjected to Western blot analysis. To determine the autocrine/paracrine action of TGFß1 on Smad induction and activation, serum-starved LSMC and MSMC were treated with TGFß type II receptor antisense or sense 20-mer oligonucleotide at 1 µM for 24 h as previously described (11). The cells were washed and treated with 2.5 ng/ml TGFß1 for 15 min, and total protein was isolated and subjected to Western blot analysis.

To determine whether GnRHa alters the expression of Smads, serum-starved LSMC and MSMC were treated with GnRHa [leuprolide acetate (LA), Sigma-Aldrich] at 0.1 µM for 2–24 h. Total RNA and protein were isolated and subjected to semiquantitative RT-PCR and Western blot analysis, respectively. To determine whether GnRHa alters Smad induction, serum-starved cells were treated with 0.1 µM LA for 5–30 min or 0.001–10 µM LA for 15 min. Total protein were isolated and subjected to Western blot analysis. The specificity of LA action on Smad induction was determined by treatment of serum-starved LSMC and MSMC with the GnRH antagonist, antide (Sigma-Aldrich) at 10 µM for 15 min before exposure to 0.1 µM LA for 15 min. To determine whether LA alters TGFß-induced pSmad3, LSMC and MSMC were treated with LA (0.1 µM), TGFß1 (2.5 ng/ml), or LA plus TGFß1 for 15 min. Total cellular protein was isolated and subjected to Western blot analysis. To determine whether LA action is independent of TGFß receptor-mediated signaling, LSMC were treated with TGFß type II receptor antisense and or sense oligomers for 24 h as described above and then exposed to LA (0.1 µM) for 15 min. Total cellular protein was isolated and subjected to Western blot analysis to determine the rate of pSmad3 induction. The results were compared with TGFß1, used at 2.5 ng/ml.

All of the experiments were performed using at least two or three separate cell cultures prepared from different tissues and were preformed in duplicate. The results were expressed as the mean ± SEM and were statistically analyzed using nonparametric Kruskal-Wallis and Wilcoxon rank tests with SigmaStat (Jandel Corp., San Rafael, CA) software; P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Smads in leiomyoma and myometrium

Before isolating the myometrial and leiomyoma smooth muscle cells for in vitro studies, we examined whether myometrium and leiomyoma expresses Smad mRNA and protein. As shown in Fig. 1Go, leiomyoma and myometrium express mRNA (A) and protein (B) for Smad3, Smad4, and Smad7 and contain phosphorylated Smad3 (pSmad3). Smad3 and Smad4 were localized in the cytoplasm of leiomyoma and myometrial smooth muscle cells, connective tissue fibroblasts, and vasculature, whereas Smad7 was localized in both cytoplasm and nuclear regions, and pSmad3 was principally localized in the nuclear region (Fig. 1CGo). Furthermore, as shown in Fig. 2Go, LSMC and MSMC isolated from these tissues and maintained in culture also express Smad3, Smad4, and Smad7 mRNA and protein and pSmad3 (Fig. 2Go).



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  		Figure 1. A, Semiquantitative RT-PCR of Smad3, Smad4, Smad7, and G3PDH (lower bands) mRNA expression in leiomyoma (L) and matched myometrium (M) from two patients (DM, DNA marker). B, Western blot analysis of Smad3, Smad4, Smad7, and pSmad3 (arrows) in leiomyoma (L) and matched myometrium (M) from three patients. C, Immunolocalization of Smad3 (a), Smad4 (b), Smad7 (c), and pSmad3 (d) in leiomyoma associated with smooth muscle cell cytoplasm (a and b), cytoplasmic/nuclear (c), and nuclear (d) regions. Magnification, x110.

 


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  		Figure 2. Semiquantitative RT-PCR of Smad3, Smad4, Smad7 (arrows), and G3PDH (lower bands) mRNA expression using total RNA isolated from LSMC and MSMC cultured for 48 h. Western blot analysis of the cell lysates prepared from these cells using antibodies specific to Smad3, Smad4, Smad7, and pSmad3 are shown in the lower panels (protein).

 
Regulation of Smad expression by TGFß

We then determined whether the expression of Smads in MSMC and LSMC is regulated by TGFß. Treatment of MSMC and LSMC with TGFß1 (2.5 ng/ml) for 2–24 h resulted in a moderate induction of Smad7 mRNA expression in MSMC and a trend toward higher expression in LSMC (Fig. 3Go, A and B) without significantly affecting Smad3 and Smad4 mRNA expression (bar graphs not shown). TGFß1 treatment also altered Smad protein expression in MSMC and LSMC, in which it had a limited effect on Smad3 and Smad4 (not shown), but increased Smad7 expression in both LSMC and MSMC after 2–12 h of exposure, which declined after 24 h compared with that in untreated control (Fig. 3Go, C and D). The results suggest that TGFß1 has a limited regulatory effect on Smad3 and Smad4 expression in MSMC and LSMC; however, it induced Smad7, where, through a feedback interaction, it can regulate TGFß action in these cells.



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  		Figure 3. Time-dependent action of TGFß1 (2.5 ng/ml) on Smad3, Smad4, and Smad7 mRNA (A and B) and protein (C and D) expression in MSMC and LSMC. Serum-starved MSMC and LSMC were treated with TGFß1 for 2–24 h, and total RNA and protein were isolated from treated and untreated control (Crtl or time zero) and subjected to semiquantitative RT-PCR, coamplifying Smads (arrows, upper bands), and G3PDH (lower bands; M, DNA marker). The bar graph (B) shows the mean ± SEM fold change in the ratio of Smad7/G3PDH mRNA expression in MSMC and LSMC, with b, c, d, and e' statistically different from a and a', respectively (P < 0.05). C, Western blot analysis of Smad7 in MSMC and LSMC and of ß-actin in MSMC (control) in cell lysates prepared from TGFß1-treated and untreated controls, with the bar graph (D) showing the fold change in Smad7 band intensity, and b–f and b'–f' statistically different from a and a', respectively (P < 0.05).

 
Induction of Smads by TGFß

In addition to regulating Smad expression, TGFß mediates its action through the induction and activation of Smads. We found that treatment of MSMC and LSMC with TGFß1 (1, 2.5, and 5 ng/ml) for 15 min increased the rate of Smad3 and Smad7 activity, whereas it decreased Smad4 induction in MSMC at high concentration compared with that in untreated control (Figs. 4Go). TGFß also increased the rate of pSmad3 activity in both MSMC and LSMC compared with untreated controls (Fig. 4Go). TGFß1-inducted Smad3, Smad4, Smad7, and pSmad3 occurred in a time-dependent manner, with some difference between LSMC and MSMC (Fig. 5Go). TGFß1-induced pSmad3 was in part abrogated after pretreatment of the cells with TGFß type II receptor antisense, but not sense, oligonucleotide (see Fig. 11Go). These results indicate that TGFß1 rapidly induces and activates Smads in both LSMC and MSMC, with some differences in their responses that could lead to differential downstream transcriptional response to TGFß as described in other cell types (8, 13).



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  		Figure 4. Dose-dependent action of TGFß1 (1, 2.5, and 5 ng/ml) on Smad3, Smad4, Smad7, and phospho-Smad3 (pSmad3) induction in LSMC and MSMC. Serum-starved MSMC and LSMC were treated with TGFß1 for 15 min, and cell lysates were prepared and analyzed by Western blotting, with ß-actin as the loading control. Bar graphs show the mean ± SEM fold change in Smad7 and pSmad3 band intensity, with b, c, and d significantly different from a, and b', c', and d' significantly different from a' (P < 0.05).

 


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  		Figure 5. Time-dependent action of TGFß1 (2.5 ng/ml) on Smad3, Smad4, Smad7, and pSmad3 induction in LSMC and MSMC. Serum-starved cells were treated with TGFß1 for 5, 10, and 15 min, and cell lysates from TGFß1-treated and untreated control (time zero) were prepared and analyzed by Western blotting, with ß-actin as the loading control. The bar graphs show the mean ± SEM fold change in Smads band intensity from three different experiments performed in duplicate. Smad3 and Smad4: a is significantly different from c, and a' is different from b' and c'. Smad7: b is significantly different from a, and b' and d' are different from a'. pSmad3: b, c, and d are significantly different from a, and b and c are different from a and d (P < 0.05).

 


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  		Figure 11. The effect of TGFß1 and GnRHa (LA) on pSmad3 induction after treatment of LSMC with TGFß type II receptor antisense (1 µM) and sense (1 µM) oligonucleotides. LSMC were treated with TGFß type II receptor antisense or sense oligomers for 24 h (medium contained 2% fetal bovine serum), and the cells were washed and then treated with TGFß1 (2.5 ng/ml), GnRHa (0.1 µM), or their combination for 15 min. The cell lysates from treated (+) and untreated (-) groups were analyzed by Western blot, with ß-actin serving as the loading control. The bar graph shows the rate of pSmad3 induction from a representative experiment performed twice. TGFß type II receptor antisense and sense oligonucleotide treatments slightly increased pSmad3 (possibly due to serum in the culture medium), GnRHa had a limited effect on pSmad3 induction, but it increased that in cells pretreated with TGFß type II receptor antisense oligonucleotide, but not in those treated with sense oligonucleotide. TGFß1induced pSmad3 was inhibited after pretreatment with TGFß type II receptor antisense, but not sense, oligonucleotide.

 
Alteration of Smad expression by GnRHa

Because leiomyoma and myometrium as well as LSMC and MSMC express GnRH and GnRH receptors, and GnRHa alters the expression of TGFß and TGFß receptors, we investigated whether GnRHa alters the expression, induction, and activation of Smads. We found that treatment of serum-starved LSMC and MSMC with GnRHa (LA) had a limited effect on Smad3 and Smad4 mRNA and protein expression (not shown); however, in a time-dependent manner (2–24 h), GnRHa increased Smad7 mRNA expression in MSMC, with a trend toward an increase in LSMC (Fig. 6Go). GnRHa, in a dose- and time-dependent manner, also resulted in a rapid induction of Smad3, Smad4, and Smad7 in MSMC and LSMC, which were either dose or time dependent (Fig. 7Go, A and B, and Fig. 8Go). However, GnRHa increased the rate of pSmad3 induction at a lower dose in MSMC, which returned to control levels (Fig. 7BGo) or lower (Fig. 8Go) at a dose of 1 µM or higher, whereas in LSMC, pSmad3 activity displayed a trend toward an increase, except with GnRHa at 1 µM after 5 min of incubation (Figs. 7BGo and 8Go). These results provide further support for the direct action of GnRHa on leiomyoma and myometrium, suggesting that GnRH receptor, either directly or most likely through activation of other signaling pathways, interacts with TGFß receptor signaling that alters pSmad3 induction in the absence of a significant change in Smad3 (20). Although pretreatment of MSMC and LSMC with GnRH antagonist (antide) resulted in partial reversal of GnRHa action, antide alone also increased pSmad3, despite inconsistency in its action (Fig. 9Go). This may be due to interactions of GnRHa and antide with two types of GnRH receptors in these cells that are alternatively and independently activated by GnRH agonists and antagonists (21, 22, 23, 24, 25).



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  		Figure 6. Time-dependent action of GnRHa (LA) at 0.1 µM on Smad3, Smad4, and Smad7 mRNA expression in MSMC and LSMC. Serum-starved cells were treated with LA for 2–24 h, and total RNA was isolated from treated and untreated control (Ctrl, time zero) cells and subjected to semiquantitative RT-PCR coamplifying Smads (arrows, upper bands) and G3PDH (lower bands). The bar graph shows the mean ± SEM fold change in the ratio of Smad7/G3PDH mRNA expression in MSMC and LSMC determined from the band intensity from two different experiments performed in duplicate. b, c, and e are significantly different from a, and b' is different from a' (P < 0.05). M, DNA marker.

 


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  		Figure 7. The dose-dependent action of GnRHa (LA) on Smad3, Smad4, Smad7, and pSmad3 induction. Serum-starved MSMC and LSMC were treated with 0.001–10 µM LA for 15 min, and cell lysates from LA-treated and untreated control (Crtl, time zero) were analyzed by Western blotting, with ß-actin as the loading control. Bar graphs show the mean ± SEM fold change in the band intensity from three different experiments. Smad4: e is significantly different from a and c, d and e are different from a'. Smad7: b, d, and e are different from a, and c' and d' are different from a. pSmad3: d, e, and f are significantly different from a, and b', c', d', and f are different from a' (P < 0.05).

 


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  		Figure 8. Time-dependent action of GnRHa (LA) at 0.1 µM on Smad3, Smad4, Smad7, and pSmad3 activation in LSMC and MSMC. Serum-starved MSMC and LSMC were treated with 0.1 µM LA for 5, 10, and 15 min, and cell lysates from LA-treated and untreated controls (Ctrl, time zero) were prepared and analyzed by Western blotting, with ß-actin as the loading control. The bar graphs show the mean ± SEM fold change in the band intensity from three different experiments performed in duplicate. Smad3: b is significantly different from a. Smad4: b and c are different from a and d. Smad7: d and d' are significantly different from a and a', respectively. pSmad3: a and a' are significantly different from b' and c (P < 0.05).

 


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  		Figure 9. The effect of GnRH antagonist (antide), GnRHa (LA), and GnRHa plus antide on pSmad3 induction. Serum-starved LSMC were pretreated with 10 µM antide for 15 min and than treated with 0.1 µM LA for an additional 15 min. The cell lysates from treated and untreated (control) cells were analyzed by Western blotting with ß-actin as the loading control. The bar graph shows a representative of such an experiment indicating an increase in the rate of pSmad3 induction after antide, GnRHa, and GnRHa plus antide treatments. Cotreatment with GnRHa and antide resulted in a limited change in pSmad3 compared with other treatments, although it is higher than the control value.

 
To determine whether GnRHa (LA) alters TGFß1-induced pSmad3, LSMC and MSMC were cotreated with LA (0.1 µM) and TGFß1 (2.5 ng/ml) for 15 min. TGFß1 increased pSmad3 activation in these cells compared with LA-treated or untreated controls; however, cotreatment of the cells with TGFß1 and LA resulted in a reduction in TGFß-induced pSmad3, suggesting possible cross-talk between GnRH and TGFß receptor signaling pathways (Fig. 10Go). Furthermore, blocking/reducing the expression of TGFß type II receptor to prevent TGFß autocrine/paracrine action indicated that pretreatment of the cells with TGFß type II receptor antisense oligonucleotide reduced TGFß1-induced pSmad3 activation compared with sense oligonucleotide-treated or untreated control (Fig. 11Go). However, treatment of the cells with TGFß type II receptor antisense and sense oligonucleotides also resulted in a slight increase in pSmad3, possibly due to the presence of a low level of serum in the media that contain TGFß. LA treatment of these cells resulted in a reduction in pSmad3 (Fig. 11Go).



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  		Figure 10. The effect of GnRHa (LA), TGFß1, and GnRHa plus TGFß1 on LSMC and MSMC on pSmad3 induction. Serum-starved cells were treated with TGFß1 (2.5 ng/ml), LA (0.1 µM), or TGFß1 plus LA for 15 min. The cell lysates from treated and untreated controls (-) were analyzed by Western blotting, with ß-actin as the loading control. The bar graph show the mean ± SEM fold change in pSmad3 induction from three different experiments performed in duplicate. b, c, and d are significantly different from a, and b', c', and d' are significantly different from a' (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The expression and action of TGFß isoforms and TGFß receptors have been documented in leiomyoma and myometrium and their isolated smooth muscle cells (9, 10, 26, 27). Recent studies have established that TGFß receptors mediate their action through multiple pathways, including Smads that mediate TGFß receptor signals from the cell surface to the nucleus, resulting in transcriptional activation of TGFß-responsive genes (for review, see Ref.13). In the present study we demonstrated that leiomyoma and myometrial smooth muscle cells express some member of the Smad family, the regulatory Smad3, common Smad4, and inhibitory Smad7. In leiomyoma and myometrium, Smad3, -4, and -7 were localized in both cytoplasmic and nuclear regions of various cell types, including LSMC and MSMC, with nuclear localization of phosphorylated Smad3. Because binding of TGFß leads to phosphorylation of TGFß type I receptor and causes a rapid phosphorylation of Smad3, which interacts with Smad4, and translocation into the nucleus, nuclear localization of pSmad3 in leiomyoma and myometrial smooth muscle cells suggests that an autocrine/paracrine action of TGFß may result in activation of Smads in these cells. Smad7 is primarily localized in the nucleus in the absence of ligand, but accumulates in the cytoplasm upon receptor activation. The presence of both cytoplasmic and nuclear Smad7 in leiomyoma and myometrial smooth muscle cells further points to partial activation of the Smad pathway, possibly by an autocrine/paracrine action of TGFß. We further demonstrated that isolated MSMC and LSMC prepared and maintained in culture also express Smad3, -4, and -7. Constitutive expression of Smads in leiomyoma and myometrium as well as their isolated smooth muscle cells indicates that these cells possess the necessary components of the TGFß signaling pathway that can be recruited and activated by TGFß.

We demonstrated that Smad expression, induction, and activation were differentially regulated by TGFß1 in LSMC and MSMC. TGFß1 had a limited effect on the expression of Smad3 and Smad4; however, it increased Smad7 mRNA expression in MSMC, with a limited effect on LSMC, while increasing Smad7 protein in both cells. Differential regulation of Smad expression has been reported in several other cell types, including the induction of Smad7 expression in skin fibroblasts and Mv1Lu and HaCaT cell lines, as well as inhibition of Smad3 expression in skin fibroblasts, but not in MDA-MB468, a human breast cancer cell line deficient in Smad4 (28, 29). Differential regulation of Smad7 by TGFß is considered to act as a feedback mechanism to control TGFß signaling (29, 30, 31). Such regulation of Smad7 by TGFß in MSMC and LSMC may result in changes in Smad7 antagonistic action that lead to alteration of cell proliferation, apoptosis, and ECM accumulation in leiomyoma compared with myometrium (32, 33, 34, 35, 36). Despite limited regulation of Smad3 and Smad4, their constitutive expression in LSMC and MSMC indicates that these cells, like many other cell types, contain the necessary signaling components to respond to TGFß action. However, factors that regulate TGFß expression, i.e. ovarian steroids, may also influence Smad expression, thus regulating TGFß-mediated local action in leiomyoma and myometrium.

Although overproduction of TGFß is widely accepted as a key factor in tissue fibrosis, Smad3 is proposed as a major player in TGFß signaling pathways that lead to fibrogenesis (37, 38). We found that TGFß1 increased the rate of pSmad3 induction in LSMC and MSMC. The action of TGFß was more effective in LSMC compared with MSMC, possibly due to a higher TGFß receptor expression in leiomyoma and LSMC, resulting in enhanced activation of Smads. Because reduction/inhibition of TGFß type II receptor expression resulted in lowering of TGFß1-induced pSmad3 in LSMC, the data suggest that components of this pathway are at least involved in TGFß signaling. Furthermore, Smad7 functions as a dominant intracellular regulator of the TGFß signaling pathway through its interaction with activated TGFß type I receptor (12, 13). However, the stability of Smad7-TGFß type I receptor interaction prevents receptor-mediated phosphorylation of Smad3 causing the disruption of TGFß mediated signaling (12, 13). Therefore, a balance between agonistic pSmad3 and inhibitory Smad7 as well as predominance in their expression/activation may become critical in regulating the cellular response to TGFß in leiomyoma and myometrium during growth and regression. Inhibition of Smad7 is reported to increase the cellular responsiveness to TGFß (28, 35), whereas it elevated expression resulted in inhibition of bleomycin-induced lung fibrosis (31).

Clinically GnRHa is often used for medical management of leiomyoma’s growth. Our results demonstrated the first evidence that GnRHa (leuprolide acetate) differentially alters the expression, induction and activation of Smads in LSMC and MSMC, although the biological significance of these finding and how GnRHa alter Smads is not clear from our study. GnRHa-induced leiomyoma regression results in down-regulation of TGFß and TGFß receptors in vivo as well as TGFß1 production and estradiol-induced TGFß expression by LSMC in vitro (7, 8, 11). Our results indicated that GnRHa alters the expression of Smad7 and pSmad3 activity in LSMA and MSMC; in other cell types GnRHa leads to changes in the Smad-mediated transcriptional response (29, 39, 40, 41). Although increased Smad7 expression could transiently act as a TGFß self-regulating feedback loop, a sustainable Smad7 expression by GnRHa could prolong the antagonistic action of Smad7. Interestingly, IFN-{gamma}-induced Smad7 expression is reported to promote Smad7-Smurf2 complex formation and consequently increase TGFß receptor turnover (42). We found that GnRHa’s action on pSmad3 was somewhat dependent on down-regulation of TGFß receptor expression, which also involves TGFß’s autocrine/paracrine action. Although our results are the first to demonstrate specific changes in the TGFß signaling pathway by GnRH, more details about how GnRH receptor-mediated action leads to alteration of Smad expression and activation are required.

Signaling by TGFß receptors is not exclusively Smad dependent, as TGFß also activates MAPK, protein kinase C (PKC), and calcium/calmodulin, inducing Smad-independent transcriptional responses (12, 13, 43, 44, 45, 46). These pathways also become activated by GnRH receptors (47, 48). Because of the multifaceted activation of several signaling pathways by TGFß and GnRH receptors, understanding the cross-talk between Smad-dependent and Smad-independent pathways is necessary to sort out their complex mechanisms of action in leiomyoma. We have demonstrated that leiomyoma and myometrium expresses extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERKs, whose expression and activation are altered because of GnRHa therapy (18) or by TGFß1 and GnRHa in vitro (Xu, J., X. Luo, and N. Chegini, in preparation). This suggests that TGFß and GnRH can activate multiple signaling pathways in LSMC and MSMC, including their potential cross-talk through MAPK and Smads. Stimulation of receptor tyrosine kinase pathways via ERK2 has been shown to lead to increase phosphorylation of Smad2 (15, 49, 50). Smad3 can also act as a substrate for ERK2 (15), and ERK2-dependent phosphorylation of Smad2 has been shown to increase nuclear localization and activity of Smad2 (49). In addition, calcium/calmodulin that becomes activated by GnRH receptor alters Smad function in part due to calmodulin binding to Smad amino terminal (50, 51). Inhibition of calmodulin is reported to increase activin-dependent induction of target gene expression in mink lung epithelial cells, whereas overexpression of calmodulin resulted in decreased activin- and TGFß-dependent induction of transcriptional reporter genes that provide support for the functional consequence of calmodulin in TGFß receptor signaling (50). Our preliminary results also indicate that GnRHa and TGFß1 activates PKC and calcium/calmodulin in LSMC and MSMC, suggesting that GnRH receptor activation of PKC, MAPK, and/or calmodulin could differentially regulate Smads and hence TGFß signaling in leiomyoma (15, 45, 49, 50, 51).

Because leiomyoma growth is dependent on ovarian steroid actions, and a GnRHa-induced hypoestrogenic state results in leiomyoma regression, ovarian steroids could also serve to regulate the expression of Smads in a fashion similar to TGFß and TGFß receptor expression in these tissues. However, data supporting the influence of ovarian steroid on Smad expression are limited, with reports indicating differential regulation of Smads by estrogen in human breast cancer cell lines and by androgen in prostate cancer (52, 53, 54). Accumulating evidence also indicates that estrogen and progesterone receptors activate MAPK and PKC; thus, cross-talk among ovarian steroids, GnRH, and TGFß receptors may be critical to regulate the availability of Smads resulting in leiomyoma growth and regression. Because TGFß has a key regulatory action in tissue fibrosis such as leiomyoma, and GnRHa therapy causes leiomyoma regression, identification of GnRH receptor signal transduction pathways that result in interruption of TGFß action is of great clinical value in this and other uterine abnormalities that are responsive to TGFß actions.

Taken together, the results of the present study indicate that leiomyoma, myometrium, and their smooth muscle cells express Smad mRNA and protein, where they are differentially expressed, induced, and activated by TGFß and altered as a result of GnRHa treatment. These results suggest that TGFß and GnRH mediate action through cross-talk involving Smads, and most likely other signaling pathways result in leiomyoma growth and regression.


   Footnotes
 
This work was supported by NIH Grant HD-37432. This work was presented in part at the 48th Annual Meeting of the Society for Gynecological Investigation, Los Angeles, California, March 2002.

Abbreviations: ECM, Extracellular matrix; ERK, extracellular signal-regulated kinase; GnRHa, GnRH analog; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; LA, leuprolide acetate; LSMC, leiomyoma smooth muscle cell; MSMC, myometrial smooth muscle cell; PKC, protein kinase C; RSmad, regulatory Smad.

Received August 19, 2002.

Accepted December 9, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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