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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 11 4138-4143
Copyright © 1999 by The Endocrine Society

Original Studies

Regulation of Transforming Growth Factor-ß1 Expression by Granulocyte Macrophage-Colony-Stimulating Factor in Leiomyoma and Myometrial Smooth Muscle Cells

Nasser Chegini, Xin-Min Tang and Chunfeng Ma

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

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human myometrium and leiomyomas express granulocyte macrophage-colony-stimulating factor (GM-CSF), transforming growth factor-ß (TGFß), and their receptors. Overexpression of TGFß and, to a limited extent, GM-CSF has been associated with tissue fibrosis throughout the body, including leiomyomas. The objective of the present study was to determine the action of GM-CSF on leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) and examine whether the action of GM-CSF is mediated through the induction of TGFß1 expression. Using competitive quantitative RT-PCR and enzyme-linked immunosorbent assay, we found that LSMC express significantly higher GM-CSF messenger ribonucleic acid (mRNA; 0.6 ± 0.1 x 103 copies of mRNA/µg total RNA) and protein (0.75 ± 0.2 ng/mL) than MSMC (0.5 ± 0.1 x 102 copies of mRNA and 0.45 ± 0.07 ng/mL protein; P < 0.05). In addition, LSMC expressed significantly higher TGFß1 mRNA (1.6 ± 0.3 x 104 copies of mRNA/µg total RNA) than MSMC (2.4 ± 0.4 x 103 copies) and synthesized and secreted more TGFß1 protein (1.7 ± 0.2 vs. 0.5 ± 0.02 ng/mL); whereas MSMC contained more cell-associated TGFß1 (56.2 ± 1.2 ng/mL) than LSMC (35.2 ± 1.2 ng/mL; P < 0.05). We found that GM-CSF (0.01–100 ng/mL) has limited mitogenic activity for LSMC but not for MSMC determined by the rate of [3H]thymidine incorporation and cell proliferation assay. However, GM-CSF at 1 ng/mL increased its own production, the expression of TGFß1 mRNA, the cell-associated TGFß1 protein content in both cell types, and TGFß1 released into the culture-conditioned medium of LSMC (P < 0.05). TGFß1 also increased its own mRNA and protein expression, but had no effect on cell-associated TGFß1 in both cell types (P < 0.05). Cotreatment of LSMC and MSMC with GM-CSF and TGFß1 induced changes similar to those produced by GM-CSF in both cells. In conclusion, our data suggest that GM-CSF is not a mitogen for MSMC and LSMC, but it regulates its own expression and the expression of TGFß1 by these cells, a regulatory interaction that may account for the GM-CSF-induced tissue fibrosis that occurs in leiomyomas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEIOMYOMAS are benign uterine tumors consisting mainly of smooth muscle cells and a network of connective tissue (1, 2). At the cellular level a combination of mitotic activity, cellular hypertrophy, and accumulation of extracellular matrix (ECM) are considered to participate in leiomyoma growth (1, 2, 3, 4). At the molecular level ovarian steroids are the major regulators of leiomyoma growth, and for that reason, GnRH agonist (GnRHa) therapy, which regresses ovarian function, is often used for short term medical management of leiomyoma growth (5). Compared to myometrium, leiomyomas are reported to overexpress estrogen and progesterone receptors, and GnRHa therapy lowers their content in both tissues (5, 6).

Data suggest that locally expressed growth factors and cytokines, whose expression is regulated in part by ovarian steroids, are also important in leiomyoma growth (1, 2). Of particular interest is transforming growth factor-ß (TGFß), whose overexpression has been associated with tissue fibrosis at various sites throughout the body (7, 8). The expression of TGFßs and TGFß receptors has been documented in leiomyomas and myometrium, with elevated levels in leiomyomas (9, 10). Another cytokine that is expressed in human endometrium, myometrium (11, 12), and leiomyomas (our unpublished data) is granulocyte macrophage- colony-stimulating factor (GM-CSF), which is the major growth factor for granulocyte/macrophage proliferation, differentiation, and functional activation (13). GM-CSF has also been reported to stimulate the proliferation of nonhemopoietic cell types (13) and to induce fibrotic reaction in several tissues such as its topical administration into the sc tissue and lung in rats or its elevated expression in human epidermal cells during atrophic dermatitis (14, 15, 16, 17, 18). The mechanism(s) by which GM-CSF induces the development of fibrotic disorders in these tissues is not known; however, accumulation of {alpha} smooth muscle actin-rich myofibroblasts and induction of TGFß, which converts fibroblasts into myofibroblasts, are considered to account for GM-CSF action (14, 17, 18).

In the endometrium, ovarian steroids may regulate the expression of GM-CSF (12, 19). However, in several cell types, including macrophages, fibroblasts, and endothelial cells, other cytokines, such as tumor necrosis factor-{alpha}, interleukin-1, and TGFß are reported to regulate GM-CSF expression (13). In the endometrium, we have shown that GM-CSF regulates its own expression and the expression of TGFß1 in epithelial and stromal cells (11).

Because human myometrium, leiomyomas, and their isolated smooth muscle cells express TGFßs, GM-CSF, and their receptors (9, 10, 11, 12), the objective of the present study was to examine the biological action of GM-CSF in myometrial and leiomyoma smooth muscle cells and determine whether GM-CSF can regulate the expression of TGFß1 in these cells. Such interactions between TGFß1 and GM-CSF may in part influence the growth and the outcome of the fibrotic nature of leiomyomas compared to adjacent normal myometrium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All of the materials and procedures used for isolation and culture of myometrial and leiomyoma smooth muscle cells (MSMC and LSMC), RT-PCR, construction of external complementary ribonucleic acid (cRNA) standard for quantitative RT-PCR, enzyme-linked immunosorbent assay (ELISA), immunocytochemistry, [3H]thymidine incorporation, and cell proliferation assays have been previously described (9, 10, 11, 20). Human-specific GM-CSF and TGFß1 ELISA kits were purchased from Prospective Diagnosis, Inc. (Cambridge, MA) and Promega Corp. (Madison, WI), respectively.

Portions of leiomyomas and unaffected myometrium were obtained from women who were undergoing hysterectomies and who were not receiving any hormonal treatment before or at the time of surgery. The collection of these tissues was approved by the University of Florida institutional review board. The MSMC and LSMC were isolated and cultured in DMEM supplemented with 10% FBS, and the purity of these cell cultures was determined using antibodies to desmin and {alpha} smooth muscle actin (10, 20).

The LSMC and MSMC were cultured in a 75-mm flask in the presence of 10% FBS until reaching approximately 90% confluence. Total cellular RNA was isolated from these cells and subjected to standard RT-PCR to determine the expression of GM-CSF and TGFß as previously described (10, 11). For quantitative RT-PCR, complementary DNA was synthesized in a series of standard reactions, each containing 2 µg total RNA isolated from each cell type and several dilutions of competitive external cRNA standard from 102–107 copy/µg for GM-CSF and 103–108 copy/µg for TGFß as previously described (9, 10, 11). A detailed procedure to calculate the final quantity of GM-CSF and TGFß messenger RNA (mRNA) expression has been previously described (9, 21).

GM-CSF and GM-CSF receptors were immunolocalized in LSMC and MSMC cultured on eight-well Lab-Tek slides using antibodies specific to human GM-CSF, GM-CSF {alpha}, and GM-CSF ß receptors at 5 µg/mL prepared in phosphate-buffered saline, pH 7.4, containing 0.1% BSA and indirect immunofluorescent microscopy (10, 11). For controls, the cell cultures were immunostained using primary antibodies preabsorbed with the respective recombinant GM-CSF or synthetic GM-CSF receptor peptides. We have previously reported the presence of immunoreactive TGFß1 and receptors in these cells (10).

To determine GM-CSF and TGFß1 production in LSMC and MSMC, the cells were cultured in 24-well dishes at 2.5 x 105 cells/well as described above in the presence of 10% FBS for 48 h, washed with serum-free medium, and further incubated in medium containing 2% FBS for an additional 24 h. The culture-conditioned medium from untreated cells (control) and cells treated with 1 ng/mL GM-CSF, TGFß1 and GM-CSF plus TGFß1 were collected, centrifuged, and stored at -80 C until assayed. The culture-conditioned medium and the medium unexposed to cells were assayed for GM-CSF and TGFß1 using ELISA according to the manufacturer’s recommended procedure, with detection limits of 2.5 and 2.0 pg/mL, respectively (11).

To determine whether GM-CSF is a mitogen for LSMC and MSMC, the cells were cultured in 96-well microplates (Costar Corning, Cary, NC) at an approximate density of 2.5 x 103 cells/well in the presence of 10% FBS for 48 h (10). The cells were washed and made quiescent in serum-free medium for 48 h, and the quiescent cells were then incubated in medium supplemented with 2% FBS in the presence and absence of various doses of GM-CSF and 2 µCi/mL [3H]thymidine to determine the rate of DNA synthesis after 48 h of incubation (20). The rate of cell proliferation was determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (10). The culture conditions for the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays were similar to those used for [3H]thymidine incorporation. HT29 cells were cultured as previously described and used as a positive control (11).

To determine whether GM-CSF regulates the expression of TGFß1, quiescent LSMC and MSMC were incubated in medium containing 2% FBS in the presence and absence of 1.0 ng/mL GM-CSF, TGFß1, or GM-CSF plus TGFß1 for 24 h. After the incubation, the conditioned media were collected, and the cells were washed, scraped, and either used for isolation of total cellular RNA or lysed to obtain total cellular proteins (11). The levels of TGFß1 (active plus total) released into the culture-conditioned medium and the cell-associated TGFß1 in the cell lysate were determined by ELISA, and TGFß1 mRNA expression was determined by quantitative RT-PCR as described above. Statistical analysis was performed using unpaired Student’s t test and Kruskal-Wallis ANOVA with Dunn’s test using the computer software program SigmaStat (Jandel Corp., San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LSMC and MSMC express GM-CSF and CM-CSF {alpha} and ß receptor mRNA as shown by standard RT-PCR (Fig. 1Go; data for MSMC are not shown). Digestion of PCR products with respective restriction enzymes resulted in the anticipated smaller fragments (Fig. 1Go). Amplification without the RT step to detect any contaminating genomic DNA or tubes containing all the PCR components except the RT reaction mixture to check for the presence of DNA that may have carried over from a prior reaction were negative (not shown). The cells also contain GM-CSF, GM-CSF {alpha}, and GM-CSF ß receptor-immunoreactive protein (Fig. 2Go, A–C). After deletion or replacement of primary antibodies with preabsorbed antibodies, a considerable reduction in staining over these cells was observed (Fig. 2DGo). Quantitatively, LSMC express 0.6 ± 0.1 x 103 copies/µg total RNA of GM-CSF mRNA and 0.75 ± 0.2 ng/mL protein compared to MSMC, which express 0.5 ± 0.1 x 102 copies of GM-CSF mRNA and 0.45 ± 0.07 ng/mL GM-CSF protein, significantly lower than expression in LSMC (P < 0.05).



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  		Figure 1. Immunocytochemical localization of GM-CSF (A), GM-CSF {alpha} (B), and GM-CSF ß (C) receptors as well as the controls (D) in leiomyoma smooth muscle cells. Magnification, x140.

 


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  		Figure 2. RT-PCR products of the predicted size are seen for GM-CSF (286 bp; lane A), GM-CSF receptor {alpha} (546 bp; lane C), and GM-CSFß receptor (380 bp; lane E) using total RNA isolated from leiomyoma smooth muscle cells. Restriction enzyme digestion of the PCR products and an appropriate enzyme produced the expected MspI digestion for GM-CSF (183 and 103 bp, lane B), KpnI digestion for GM-CSF {alpha} receptor (376 and 170 bp; lane D), and PvuII digestion for GM-CSF ß receptor (224 and 156 bp; lane F). DNA markers are shown in lane M.

 
GM-CSF at 0.1–100 ng/mL did not have any significant effect on the rate of [3H]thymidine incorporation or cell proliferation of serum-deprived quiescent LSMC and MSMC (data not shown). However, in half-stimulated quiescent LSMC and MSMC, GM-CSF increased the rate of [3H]thymidine incorporation into LSMC (P < 0.05), but not MSMC, without affecting their proliferation (Fig. 3Go, A and B). GM-CSF significantly increased the rate of [3H]thymidine incorporation and proliferation of HT-29 cells, which were used as a positive control (11).



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  		Figure 3. The effects of GM-CSF at various concentrations on [3H]thymidine incorporation (•) and cell proliferation ({circ}) on quiescent myometrial (A) and leiomyoma (B) smooth muscle cells incubated in the presence of 2% FBS for 48 h. Points are the mean ± SEM of duplicated experiments performed using isolated cells from three tissues.*, Different from control (P < 0.05).

 
As MSMC and LSMC express GM-CSF, TGFß, and their receptors, we examined the possibility of self-regulation and interactions between these cytokines. Treatment with GM-CSF, TGFß1, or GM-CSF plus TGFß1 (1 ng/mL) did not have any significant effect on GM-CSF mRNA expression in LSMC and MSMC compared to that in the untreated controls (not shown). However, GM-CSF increased its own production in both MSMC and LSMC, with a similar effect after cotreatment with GM-CSF plus TGFß1 (P < 0.05) compared to the untreated control (Fig. 4Go, A and D). In addition, treatment with TGFß1 increased GM-CSF production in LSMC, but not in MSMC, compared to that in untreated controls (Fig. 4DGo; P < 0.05).



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  		Figure 4. The effects of GM-CSF (G), TGFß1 (T), andGM-CSF plus TGFß1 (G/T) at 1 ng/mL on GM-CSF (A and D), and TGFß1 active ({square}) and total ({blacksquare}; active plus latent; B, C, E, and F) released into the culture-conditioned medium (B and E) and found as cell-associated (C and F) in myometrial (A–C) and leiomyoma (D–F) smooth muscle cells, incubated in the presence of 2% FBS for 24 h. C, Untreated control. The data are presented as the mean ± SEM of duplicate assays performed using isolated cells from three tissues. * and **, Significantly different from their respective controls (P < 0.05).

 
LSMC express a significantly higher level of TGFß1 mRNA than MSMC (1.6 ± 0.5 x 104 vs. 2.4 ± 0.3 x 103 copies/µg total RNA; P < 0.05), which was significantly increased after treatment with TGFß1 in both cells (2.9 ± 0.6 x 105 vs. 2.2 ± 0.5 x 104 copies/µg total RNA, respectively; P < 0.05; Fig. 5Go). The expression of TGFß1 mRNA was also increased by GM-CSF treatment in LSMC and MSMC (7.4 ± 0.8 x 104 and 3.1 ± 0.4 x 103 copies) and was further enhanced by cotreatment with TGFß1 and GM-CSF (1.3 ± 0.4 x 105 and 2.7 ± 0.5 x 104 copies) compared to that in the untreated controls (P < 0.05; Figs. 5Go and 6Go). LSMC synthesized and released approximately 1.7 ± 0.2 ng/mL total (latent and active) TGFß1, of which 0.6 ± 0.05 ng/mL was in the active form, compared to MSMC, which synthesized and released 0.5 ± 0.02 ng/mL total and 0.25 ± 0.04 ng/mL active TGFß1 (P < 0.05; Fig. 4Go, B and E). However, a major portion of TGFß1 in LSMC and MSMC was cell-associated, accounting for 28.2 ± 3.4 and 56.2 ± 1.2 ng/mL total and 0.9 ± 0.05 and 0.7 ± 0.03 ng/mL active TGFß1, respectively (Fig. 4Go, C and F). TGFß1, but not GM-CSF, significantly increased the levels of total and active TGFß1 released into culture-conditioned medium in MSMC (Fig. 4BGo). TGFß1 and GM-CSF increased TGFß1 production in LSMC, with similar effects after cotreatment with GM-CSF plus TGFß1 in both cells (P < 0.05). In contrast, treatment with GM-CSF and GM-CSF plus TGFß1, but not with TGFß1 alone, significantly increased the cell-associated TGFß1 in MSMC and LSMC (P < 0.05; Fig. 4Go, C and F).



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  		Figure 5. Competitive quantitative RT-PCR analysis of TGFß1 mRNA expression in leiomyoma smooth muscle cells treated with GM-CSF, TGFß1, or GM-CSF plus TGFß1 at 1 ng/mL concentration and in untreated controls (Ctrl). The upper bands are the PCR products generated from the specific message in total cellular RNA, and the lower bands from the external cRNA (shown from right to left at dilutions corresponding to 108 to 103 copies of RNA). The far left lanes are the DNA markers.

 


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  		Figure 6. The bar graph shows the level (mean ± SEM) of TGFß1 mRNA expression in leiomyoma (LSMC; {square}) and myometrial (MSMC; {blacksquare}) smooth muscle cells treated with GM-CSF, TGFß1, and TGFß plus GM-CSF and in untreated controls (CTRL). * and **, Significantly different from their respective controls (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously reported that human myometrium (12) and leiomyomas (unpublished data) express GM-CSF and GM-CSF receptor mRNA and protein. We further extended these observations, and in the present study demonstrated that LSMC and MSMC in vitro continue to express GM-CSF and GM-CSF receptor mRNA and protein. Our results indicate that LSMC express approximately 10- and 2-fold higher mRNA and protein for GM-CSF mRNA and protein, respectively, compared to MSMC, suggesting that GM-CSF expression may be regulated differently at mRNA and protein levels in these cells. Even if MSMC and LSMC produce substantially higher levels of GM-CSF, our data suggest that GM-CSF has no mitogenic activity for MSMC and has a limited effect for LSMC; however, GM-CSF enhances its own production by these cells. The lack of GM-CSF mitogenic activity for MSMC is not surprising, as myometrium is a quiescent tissue and undergoes limited alteration during the menstrual cycle. However, we expected to see some mitogenic activity for GM-CSF in LSMC, because of leiomyoma growth compared to that of myometrium, even though limited mitotic activity has been reported in leiomyomas during growth (1, 2). Despite GM-CSF’s major growth-promoting activity for granulocyte/macrophage proliferation, it also enhances the proliferation of several nonhemopoietic cell types, including osteoblasts, smooth muscle, endothelial, and epithelial cells in vitro (13).

In addition to its various biological activities (13), GM-CSF has been reported to induce fibrotic reaction in several tissues, including its administration into sc tissue and lung and its elevated expression in human epidermal cells in atrophic dermatitis (14, 15, 16, 17, 18). Accumulation of {alpha} smooth muscle actin-rich myofibroblasts has been suggested to account for GM-CSF-induced tissue fibrosis through a mechanism involving TGFß expression (14, 15, 16, 17, 18, 22). TGFß has been shown to influence the conversion of fibroblasts into myofibroblasts, and its overexpression is key regulator of induction of tissue fibrosis in various sites throughout the body due to either pathological processes or surgical injury (7, 8, 22, 23). A characteristic of myofibroblasts is coexpression of desmin and {alpha} smooth muscle actin with vimentin, a protein that is expressed by fibroblasts (22). Using differential display, we recently demonstrated that vimentin is among several genes that are differentially expressed in leiomyomas compared to adjacent normal myometrium (24), and in addition to {alpha} smooth muscle actin and desmin, leiomyoma smooth muscle cells contain immunoreactive vimentin (our unpublished data). Although a detailed analysis of vimentin expression in leiomyomas is needed, these observations suggest for the first time that leiomyomas may consist of myofibroblasts derived from transformation of myometrial smooth muscle cells, or from conversion of myometrial connective tissue fibroblasts into myofibroblasts. Because leiomyomas overexpress TGFß mRNA and protein (9), we sought to determine whether GM-CSF can regulate the expression of TGFß1 in a similar fashion proposed in other fibrotic tissues (14, 15, 16, 17, 18) and in the endometrium (11).

We found that LSMC express approximately 10- and 3-fold more TGFß1 mRNA and protein, respectively, than MSMC, and its expression was further enhanced by GM-CSF in LSMC by 6- and 2-fold. Interestingly, the effects of GM-CSF on TGFß1 mRNA expression and protein production by these cells were somewhat different. GM-CSF increased the expression of TGFß1 mRNA in LSMC, with a marginal effect on MSMC, whereas TGFß1 protein released into their culture-conditioned medium was less affected by GM-CSF treatment. We also found that MSMC and LSMC contain a relatively high level of cell-associated TGFß1, which was higher in MSMC than in LSMC, and GM-CSF increased its content in both cell types. With the exception of our recent report on endometrial epithelial and stromal cells (11), we are not aware of any other report describing the differences between cell-associated TGFß1 and TGFß1 released into culture-conditioned medium of any other cell type and do not know what allows these cells to retain such a high level of TGFß1 intracellularly. Overall, because LSMC release a relatively higher level, but retain lower levels, of TGFß1 compared to MSMC, such a condition in vivo may create an environment favorable for cellular transformation, accumulation of ECM, and development of leiomyomas. Indeed, we have demonstrated that regardless of size, leiomyomas produce twice as much TGFß1 as normal adjacent myometrium (unpublished data). In addition, we found that the level of TGFß1 production is significantly lower in large leiomyomas (>5 cm in diameter) compared to smaller tumors and normal myometrium obtained from GnRHa-treated subjects than untreated groups (unpublished data). Interestingly, it is reported that GnRHa therapy regresses the large leiomyomas more effectively than smaller tumors, and the therapy appears to regress the nonmyoma tissues more than myomas (25). However, we did not find any difference in the level of TGFß1 production in smaller myomas (<5 cm in diameter) and myometrium in GnRHa-treated and untreated tissues (unpublished data).

Other cytokines, such as tumor necrosis factor-{alpha} and interleukin-1, which are expressed in the human uterus, are also reported to induce GM-CSF expression in macrophages, fibroblasts, and endothelial cells (13). In human endometrial epithelial and stromal cells we have shown that GM-CSF and TGFß1 up-regulate their own expression and interact to increase the expression of TGFß1 in epithelial and stromal cells (11). However, the interactions between GM-CSF and TGFß1 in endometrial cells appear to be different than those in MSMC and LSMC. GM-CSF and TGFß have also been shown to regulate the expression of integrins (26, 27), which in the uterus are implicated in endometrial receptivity for embryo implantation and endometriosis and are expressed in leiomyomas (28, 29). If GM-CSF and TGFß regulate integrin expression in myometrium or leiomyomas, they may further enhance the interactions between the ECM and cellular compartments in these tissues that undergo significant tissue remodeling during pregnancy and during leiomyoma growth and regression (1, 2, 30, 31, 32). Up-regulation of GM-CSF receptor expression by TGFß has been considered a possible mechanism for synergistic interaction between GM-CSF and TGFß (33). Because GM-CSF and TGFß1 interactions occur at a compartment-specific manner in the uterus, it is important to further examine such interactions in detail in normal and pathological conditions affecting this tissue.

In conclusion, we provide further evidence that LSMC and MSMC express GM-CSF and TGFß1 and found that, despite limited mitogenic activity, GM-CSF interacts with TGFß1 to regulate its own expression and the expression of TGFß1 in LSMC. Considering that overexpression of TGFß is associated with establishment of tissue fibrosis, regulatory interactions between GM-CSF and TGFß in leiomyomas may play a key role in the initiation and maintenance of this fibrotic disorder.

Received April 8, 1999.

Revised July 27, 1999.

Accepted August 9, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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