Mol. Hum. Reprod. Advance Access originally published online on March 29, 2006
Molecular Human Reproduction 2006 12(4):245-256; doi:10.1093/molehr/gal015
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CCNs, fibulin-1C and S100A4 expression in leiomyoma and myometrium: inverse association with TGF-ß and regulation by TGF-ß in leiomyoma and myometrial smooth muscle cells
Department of Obstetrics and Gynecology, University of Florida, Gainesville, FL, USA
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, University of Florida, Box 100294, Gainesville, FL 32610, USA. E-mail: cheginin{at}obgyn.ufl.edu
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Abstract |
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Connective tissue growth factor (CTGF; CCN2) is considered to serve as downstream midiator of TGF-ß action in tissue fibrosis. We tested this hypothesis in paired leiomyoma and myometrium by evaluating the expression of TGF-ß1/TGF-ß3 and CCN2, the other members of the CCN family, CCN3 and CCN4, as well as fibulin-1C and S100A4, calcium-binding proteins that interact with CCNs. The regulatory function of TGF-ß1 on the expression of these genes was further evaluated using leiomyoma (L) and myometrial (M) smooth muscle cells (SMC). Real-time PCR, Western blotting and immunohistochemistry revealed that leiomyomas and myometrium express CCNs, fibulin-1C and S100A4, whose levels of expression with the exception of fibulin-1C were lower in leiomyomas and inversely correlated with the expression of TGF-ß1 and TGF-ß3 (P<0.05). The expression of these genes was menstrual cycle-independent and GnRHa therapy increased the expression of CCN2 in leiomyomas, while inhibiting CCN3, CCN4 and S100A4 in myometrium (P<0.05). TGF-ß (2.5 ng/ml) in a time- and cell-dependent manner, and through MAPK and Smad pathways, differentially regulated the expression of these genes in LSMC and MSMC. We concluded that CCNs, fibulin-1C and S100A4 are expressed in leiomyomas/myometrium with relative expression levels inversely correlating with TGF-ßs and influenced by GnRHa and TGF-ß regulatory actions. The results suggest that unlike other fibrotic disorders, CCN2 (CTGF), at least at tissue level, may not serve as a downstream mediator of TGF-ß action in leiomyomas.
Key words: CCNs/expression/fibulin/S100A4/TGF-ß
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Introduction |
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Transforming growth factor (TGF-ß) is a profibrotic cytokine overexpressed in a wide range of fibrotic tissues including uterine leiomyomas (Dou et al. 1996
; Blobe et al., 2000; Lee and Nowak, 2001; Ihn, 2002; Verrecchia and Mauviel, 2002; Schnaper et al., 2003; Luo et al., 2005b). TGF-ß acts as a key regulator of cell growth and differentiation, inflammation, apoptosis and tissue remodeling, processes that are central to the outcome of tissue fibrosis (Schnaper et al., 2003; Shi and Massague, 2003). In addition to TGF-ß, connective tissue growth factor (CTGF), a member of the cystine-rich secreted family of proteins (CCN), has also emerged as a regulator of connective tissue formation, angiogenesis and tissue fibrosis (Perbal, 2001, 2004; Ihn, 2002; Brigstock, 2003; Leask and Abraham, 2003). Elevated expression of CTGF (CCN2) has been documented in a number of fibrotic disorders, and in vitro studies have indicated that CCN2 may serve as a downstream mediator of TGF-ß action in tissue fibrosis (Ihn, 2002; Leask and Abraham, 2003). In addition to CCN2, the expression of other members of the CCN family include cysteine-rich 61 (CYR61/CCN1), nephroblastoma overexpressed (NOV/CCN3), and Wnt-induced secreted proteins-1 (WISP-1/CCN4), -2 (WISP-2/CCN5) and -3 (WISP-3/CCN6) has been demonstrated in a number of cells and tissues under normal and pathological conditions (Saxena et al., 2001; Xie et al., 2001, 2005; Sakamoto et al., 2002; Lin et al., 2003; Margalit et al., 2003; Soon et al., 2003; Yu et al., 2003a; Perbal, 2004). In leiomyoma and myometrium and their smooth muscle cells the expression of CCN1, CCN2 and CCN5 has been identified using conventional and microarray analysis (Sampath et al., 2001a; Weston et al., 2003; Mason et al., 2004a).
The CCNs interact with multiple integrin receptors to mediate their biological activities, which include stimulation of mitosis, adhesion, migration, growth arrest, apoptosis and extracellular matrix (ECM) production (Saxena et al., 2001; Sakamoto et al., 2002; Brigstock, 2003; Lin et al., 2003; Margalit et al., 2003; Soon et al., 2003; Yu et al., 2003a; Perbal, 2004). In addition to intergrins, CCN3 also interacts with calcium-binding glycoproteins, fibulin and S100A4 (Perbal et al., 1999; Li et al., 2002). Fibulins consist of five isoforms, fibulin-1 to fibulin-5, found in association with various connective tissues, basement membranes and ECM proteins (Tran et al., 1995; Argraves et al., 2003; Timpl et al., 2003). Functionally, fibulin–1C through association with ECM proteins such as fibronectin, laminin and fibrinogen has been reported to regulate cell adhesion and migration (Argraves et al., 2003; Timpl et al., 2003). S100A4 is a member of S100 proteins, one of the largest subfamily of EF-hand proteins, which selectively bind calcium and modulates calcium signalling, cell motility, cell cycle progression, intercellular adhesion and angiogenesis (Barraclough, 1998; Heizmann and Cox, 1998; Li et al., 2002). Frequent rearrangement in S100A4 gene and altered expression in several tumors has implicated S100A4 as a tumor progression/metastasis factor (Barraclough, 1998; Heizmann and Cox, 1998). We have identified several members of fibulin and S100 family among the differentially expressed genes in leiomyoma and myometrium, and in LSMC and MSMC in response to TGF-ß (Luo et al., 2005a, b). A recent study has also demonstrated the expression of several S100 genes in leiomyoma, with S100A11 displaying an anti-proliferative activity for uterine smooth muscle tumors (kanamori et al. 2004).
Since CCN2 is considered to serve as downstream regulator of TGF-ß action on tissue fibrosis, we tested this hypothesis in leiomyomas by evaluating the correlation between the expression of TGF-ß1/TGF-ß3 and CCN2 in paired leiomyoma and myometrium from proliferative and secretory phases of the menstrual cycle, and from patients who received gonadotropin releasing hormone analoge (GnRHa) therapy. Further comparison was made between the expression of TGF-ßs and that of CCN3 and CCN4, as well as fibulin-1C and S100A4. We also assessed the regulatory function of TGF-ß1 and GnRHa on the expression of these genes in leiomyoma (L) and myometrial (M) smooth muscle cells (SMC) isolated from these tissues and the involvement of Smad and/or MAPK pathways in mediating their activities.
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Materials and methods |
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All the materials for real-time PCR, Western blotting and immunohistochemistry were purchased from Applied Biosystem (Foster City, CA), BioRad (Hercules, CA) and Vector Laboratories (Burlingame, CA) respectively, leuprolide acetate (GnRHa) was purchased from Sigma-Arlich (St Louis, MO) and human recombinant TGF-ß1 and polyclonal antibody to CCN4 was purchased from R&D System (Minneapolis, MN). Polyclonal antibodies to CCN2, CCN3, fibulin-1C, S100A4 and Smad3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and monoclonal antibody to ß actin purchased from Sigma. U0126, a MEK1/2 synthetic inhibitor, was purchased from CalBiochem (San Diago, CA).
Portions of paired leiomyoma and myometrium were collected from premenopausal women (N=27) ages ranging from 29 to 41 years, undergoing hysterectomy for symptomatic uterine leiomyomas at the University of Florida affiliated Shands Hospital. Of these patients seven received GnRHa therapy for a period of three months prior to surgery. The untreated patients did not receive any medications during the 3 months prior to surgery. Based on the endometrial histology and patient last menstrual cycle, the tissues were from proliferative (N=8) and secretory (N=12) phases of the menstrual cycle. Because of the potential difference in gene expression levels between different sizes of leiomyomas, all the leiomyoma selected for the purpose of this study were between 2 and 3cm in diameter. Prior approval was obtained from the University of Florida Institutional Review Board for the experimental protocol of this study, without requiring writen informed consent.
Isolation and culture of leiomyoma and myometrial smooth muscle cells
Leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) were isolated and cultured as previously described (Chegini et al., 2002). Prior to use in these experiments the primary cell cultures were characterized using antibodies to a smooth muscle actin, desmin and vimentin (Chegini et al., 2002). LSMC and MSMC were cultured in 6-well plates at an approximate density of 106 cells/well in DMEM-supplemented media containing 10% FBS. After reaching visual confluence the cells were washed in serum-free media and incubated for 24 h under serum-free, phenol red-free conditions. These cells were used for the following experiments.
The expression of CCNs, fibulin-1C and S100A4 and regulation by TGF-ß and GnRHa
To determine the influence of TGF-ß1 and GnRHa on CCNs, fibulin-1C and S100A4 expression in LSMC and MSMC the cells were cultured as above and treated with TGF-ß1 (2.5 ng/ml) or GnRHa (0.1µM) for 2, 6 and 12 h (Ding et al., 2004). After the time-course experiments we determined whether TGF-ß and GnRHa mediate their actions through MAPK and Smad pathways. For the involvement of MAPK pathway, LSMC and MSMC cultured as above were pretreated with U0126 (20 µM), a synthetic inhibitor of MEK1/2, for 2 h, followed by treatment with TGF-ß1 (2.5 ng/ml) and/or GnRHa (0.1µM) for 2h as previously described (Ding et al., 2004). To evaluate the involvement of Smad pathway, LSMC and MSMC were cultured until reaching 70–80% confluence and then were transfected with 200 pmol of Smad3 SiRNA using 10 µl transfectamine 2000 reagent according to the manufacturer’s instructions (Inveritogen, Carlsbad, CA), for 48 h. The cells were washed and treated with TGF-ß1 (2.5 ng/ml) for 2 h. Smad3 SiRNA was designed using Dharmacon Inc (Lafayette, CO) tool with the target sequence of 5'-UCCGCAUGAGCUUCGUCAAAdTdT-3' as previously described (Kim et al., 2004). Untreated or cells treated with scrambled Smad3 SiRNA were used as a negative control. The concentration of TGF-ß1, GnRHa, U0126 and Smad3 SiRNA, and the incubation period employed in this study were selected based on our previous studies (Chegini et al., 2002; Ding et al., 2004; Levens et al., 2005; Luo et al., 2005b). Total RNA was isolated from the treated and untreated controls cells, as well as total cellular protein isolated from SiRNA transfected and control cells, were subjected to real-time PCR and Western blotting.
Real-time PCR
Total RNA was isolated using Trizol Reagent (invitrogen). Following standard protocol the level of CCNs, fibulin-1C and S100A4, as well as TGF-ß1 and TGF-ß3 mRNA expression, was determined by real-time PCR using Taqman and ABI-Prism 7700, and Sequence Detection System 1.91 software (Applied Biosystems) as previously described (Ding et al., 2004; Luo et al., 2005b). The results were analysed using the comparative method following normalization of expression values to the 18S rRNA expression as previously described (Ding et al., 2004).
Western Blotting, ELISA and Immunohistochemistry
Small portions of paired leiomyoma and myometrium were selected and homogenized in homogenizing buffer, centrifuged at 10 000 xg and the supernatants were collected and their total protein content was determined as previously (Chegini et al., 2003; Xu et al., 2003). An equal amount of protein was subjected to SDS-PAGE, transfered to polyvinyldiene difluoride membrane and the blots were incubated with CCN2, CCN3, CCN4, fibulin-1C, S100A4 and ß-actin antibodies according to the manufacturer’s recommendation for 1–2 h. The membranes were exposed to corresponding HRP-conjugated IgG and immunostained proteins were visualized using enhanced chemiluminesence reagents (Amersham-Pharmacia, Piscataway, NJ), and band densities were determined as previously described (Chegini et al., 2003; Xu et al., 2003).
Equal amounts of tissue homogenates were also subjected to ELISA determining the level of TGF-ß1 (Chegini et al., 1999, 2002). The homogenates were assayed both before and after acidification resulting in activation of TGF-ß according to the protocol recommended by the manufacturer (Promega, Madison, WI) and described in detail in our previous studies (Chegini et al., 1999, 2002).
For immunohistochemical studies, tissue sections were prepared from formalin-fixed and paraffin-embedded leiomyoma and myometrium and subjected to standard processing. The sections were then exposed to antibodies to CCN2, CCN3, CCN4, fibulin-1C, and S100A4 at 5µg of IgG/ml for 2–3 h at room temperature. Following further processing, including incubation with biotinylated secondary antibodies and avidin-conjugated HRP (ABC Elite kit), the chromogenic reaction was detected with 3,3'-diaminobenzidine tetrahydrochloride solution. Omission of primary antibodies, or incubation of tissue sections with non-immune rabbit and/or goat IgGs instead of primary antibodies at the same concentration during immunostaining, served as controls (Chegini et al., 2003; Xu et al., 2003).
All the in vitro experiments were performed at least three times in duplicate using independent cell cultures. Where appropriate the results are expressed as mean ± SEM and statistically analysed using unpaired Student t-test and ANOVA, with Tukey test. A probability level of P<0.05 was considered significant.
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Expression of CCNs, fibulin-1C and S100A4 in leiomyoma and myometrium
Using real-time PCR we validated the expression of CCN3, CCN4, fibulin-1C and S100A4, and confirmed the expression of CCN2 mRNA in paired leiomyoma and myometrium from proliferative and secretory phases of the menstrual cycle. The results indicated that leiomyomas express significantly lower levels of CCN2, CCN3 and S100A4, and higher levels of fibulin-1C as compared to myometrium (Figure 1A-E, P<0.05). There was a trend toward a lower expression of CCN4 mRNA in leiomyomas as compared to myometrium, but these levels did not reach statistical significance. Additionally, the relative level of CCNs, fibulin-1C and S100A4 mRNA expression in leiomyomas and myometrium was menstrual cycle-independent, although a higher level of expression was detected in individual paired leiomyoma and myometrium from the secretory phase as compared to the proliferative phase, their mean values did not reach statistical significance (P<0.06; Fig. not shown). In patients who received GnRHa therapy the relative expression of CCN3, CCN4, and S100A4 was significantly reduced in myometrium, but not in leiomyomas, with increased CCN2 expression in both tissues as compared to the untreated group (P<0.05; Figure 1A).
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Leiomyoma and myometrial tissue extracts also contained immunoreactive CCN2, CCN3 and CCN4, and fibulin-1C proteins (Figure 2A); however, S100A4 was undetectable using this antibody under the conditions of our study. Despite considerable variations in CCNs and fibulin-1C immunoreactive band densities, changes in their mean density values showed a trend supporting their respective mRNA expression in these tissues (Figure 2B). Immunohistochemically, CCN2, CCN3, CCN4, fibulin-1C and S100A4 were localized in leiomyoma and myometrial tissue sections with staining associated with smooth muscle cells, connective tissue fibroblasts and vasculature (Figure 3A-J). The immunostaining was mostly cytoplasmic with a considerable heterogeneity in intensity among various cell types without substantial difference in their staining intensity between tissues from proliferative and/or secretory phase of the menstrual cycle. Incubation with non-immune rabbit (Figure 3K) and goat (Figure 3L) sera resulted in a considerable reduction in staining intensity indicating the specificity of the reactions.
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Relationship with TGF-ß isoforms expression
To allow for comparative analysis between the expression of TGF-ßs and CCNs in paired leiomyoma and myometrium we reexamined the expression of TGF-ß1 and TGF-ß3 in these tissues. Real-time PCR confirmed our previous observations that leiomyomas expressed elevated levels of TGF-ß1 and TGF-ß3 mRNA as compared to myometrium, with a significantly higher level of TGF-ß1 as compared to TGF-ß3 in both tissues (P<0.05; Figure 4A). In addition, leiomyomas produced a significantly higher level of total and active TGF-ß1 protein as compared to myometrium (P<0.05, Figure 4B). Comparatively, the relative expression of CCN2 (CTGF) in paired leiomyoma and myometrium was inversely correlated with not only the expression of TGF-ß1, but also with the expression of TGF-ß3 (Figure 5). A similar correlation was also observed between the expression of TGF-ßs and that of CCN3 and S100A4, but not CCN4, while fibulin-1C expression displayed a direct correlation with TGF-ß1 and TGF-ß3 expression in leiomyomas (Figure 5).
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Expression and regulation by TGF-ß1 and GnRHa in LSMC and MSMC
Real-time PCR further indicated that LSMC and MSMC, isolated from the above tissues and maintained under a defined culture condition, express CCNs, fibulin-1C and S100A4 mRNA (Figure 6A–E). TGF-ß1 (2.5 ng/ml) in a time (2, 6 and 12 h) and cell-dependent manner increased the expression of CCN2 by 2–25-fold (A), and CCN4 by two-fold (C), while inhibiting the expression of CCN3 (Figure 6A) in LSMC and MSMC (P<0.05). The effect of TGF-ß1 on fibulin-1C (Figure 6D) and S100A4 (Figure 6E) mRNA expression was limited, by moderately inhibiting them in LSMC and MSMC, while increasing the expression of fibulin-1C in MSMC (P<0.05).
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As shown in Figure 7 GnRHa (0.1 µM) in time- (2, 6 and 12 h) and cell-dependent manners inhibited the expression of CCN2 (A), CCN3 (B), CCN4 (C) and fibluin-1C (D) in LSMC and MSMC (P<0.05). GnRHa moderately increased the expression of S100A4 (E) in LSMC after 2 and 6 h, with inhibition after longer treatment period (P<0.05), while inhibiting S100A4 expression in MSMC (P<0.02).
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Involvement of MAPK and Smad pathways in TGF-ß and GnRHa-mediated action
Pretreatment of LSMC and MSMC with MEK1/2 inhibitor, U0126, inhibited the basal expression of CCNs and S100A4 as compared to untreated controls (Figure 8). U0126 had a limited effect on TGF-ß-mediated action on CCN2 expression (Figure 8A), but inhibited TGF-ß1 action on CCN3 expression in MSMC (Figure 8B). It also inhibited the expression of CCN4, fibulin-1C and S100A4 in both LSMC and MSMC (Figure 8C–E; P<0.05). Pretreatment with U0126 also altered GnRHa-mediated action on CCNs, fibulin-1C and S100A4 expression in LSMC and MSMC in cell-specific manner (Figure 8; P<0.05).
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Transfection of LSMC and MSMC with Smad3 SiRNA, but not scrambled SiRNA, significantly reduced the expression of Smad3 mRNA and protein, without effecting the expression of ß-actin (Figure 9). Smad3 SiRNA transfection also had a limited effect on the expression of CCN2, CCN4, fibulin-1C and S100A4; however, it increased the expression of CCN3 in both LSMC and MSMC (Figure 10A-E). Treatment of Smad3 SiRNA-transfected LSMC and MSMC with TGF-ß1 (2.5 ng/ml) significantly increased the expression of CCNs, fibulin-1C and S100A4 as compared to TGF-ß-treated control cells (Figures 10A-E, P<0.05).
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Discussion |
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In the present study we demonstrated that CCN2, CCN3, CCN4, fibulin-1C and S100A4 (a) are expressed in leiomyomas and myometrium during the menstrual cycle; (b) their expression is altered due to GnRHa therapy; and (c) regulated by TGF-ß and GnRHa in LSMC and MSMC in vitro, at least in part, through the activation of MAPK and Smad signaling pathways. Western blotting and immunohistochemical localization further indicated that leiomyoma and myometrial smooth muscle cells, connective tissue fibroblasts and vasculature are the potential site of expression of these genes. With respect to tissue- and the menstrual cycle-dependent expression of CCNs, our results with CCN2 expression is in agreement and confirmed the previous report that CCN2 is expressed at lower levels in leiomyomas as compared to myometrium (Weston et al., 2003
). These results are also consistent with previous reports regarding CCN1 and CCN5 expression profiles in leiomyomas and myometrium, although elevated levels of CCN5 were detected in these tissues during the proliferative phase (Sampath et al., 2001a; Mason et al., 2004a). We found that the expression of CCN3 and S100A4 in leiomyomas was similar to that observed with CCN2, with elevated expression of fibulin-1C as compared to myometrium. Despite the differences in relative expression of these genes between leiomyomas and myometrium, their expression was menstrual cycle-independent with a trend towards a higher expression during the secretory phase. Furthermore, with the exception of CCN2, the expression of these genes in myometrium, but not leiomyomas, was significantly reduced in patients who received GnRHa therapy.
Since GnRHa therapy suppresses both estrogen and progesterone production, tissue-specific changes in the expression of CCNs, fibulin-1C and S100A4 observed in leiomyoma and myometrium of patients who received GnRHa therapy may be due to the influence of the ovarian steroid action. Estrogen has been shown to regulate the expression of CCN5 in rat uterus (Mason et al., 2004b) and in human breast cancer cell lines (Sampath et al., 2001b), and the expression of CCN1 in myometrial but not in leiomyoma’s explant cultures (Sampath et al., 2001a). Considering that leiomyomas express elevated levels of both estrogen and progesterone receptors, differential expression of CCNs, fibulin-1C and S100A4 in leiomyoma compared to myometrium may not only be due to the ovarian steroid action, but also locally produced factor(s), whose expression is regulated by ovarian steroids. Among these locally produced regulators are the TGF-ß family whose expression is well characterized in leiomyoma and myometrium (Dou et al., 1996; Chegini et al., 1999, 2002, 2003; Arici and Sozen, 2000; Lee and Nowak, 2001).
Transforming growth factor-ß is a profibrotic cytokine, which is overexpressed in a number of fibrotic tissues, including leiomyomas. Similar to TGF-ß, elevated expression of CCN2 has been documented in a number of fibrotic tissues and is considered a key regulator of connective tissue formation, angiogenesis and tissue fibrosis (Ihn, 2002; Brigstock, 2003; Leask and Abraham, 2003; Perbal, 2004). Results generated mostly from in vitro studies have led to the hypothesis that TGF-ß action in tissue fibrosis is indirect and mediated through the induction of CCN2 (Ihn, 2002; Leask and Abraham, 2003). We demonstrated that the expression of CCN2 in leiomyomas is inversely correlated with not only TGF-ß1, but also with TGF-ß3 expression. Such an inverse correlation was also found between the expression of TGF-ß1 and TGF-ß3, and that of CCN3 and S100A4. However, we found that under in vitro conditions TGF-ß1 increased the expression of CCN2 in LSMC and MSMC. TGF-ß1 also increased the expression of CCN4, while inhibiting the expression of CCN3 in these cells. With respect to TGF-ß isoforms, since they bind to the same receptor system, and share similar signaling molecules, both TGF-ß1 and TGF-ß3 may have similar regulatory functions on the expression of CCNs, fibulin and S100A4 in LSMC and MSMC. However, as shown in this and our previous studies, TGF-ß1 is a dominant isoform in leiomyomas (Dou et al., 1996; Chegini et al., 1999, 2002, 2003), although TGF-ß3 relative expression has been reported to be higher in leiomyoma compared to TGF-ß1 (Arici and Sozen, 2000; Lee and Nowak, 2001). Comparatively, TGF-ß1 has also been identified as a dominant isoform in other fibrotic tissues, and the fibrotic activity associated with TGF-ß3 has been found to be due to, and mediated through, the induction of TGF-ß1 (Blobe et al., 2000; Ihn, 2002; Yu et al., 2003b). Gene expression profiling has also revealed a lower expression of CCN2 in hypertrophic scars, which express elevated levels of TGF-ß1 as compared to normal skin (Tsou et al., 2000). These observations in leiomyoma and hypertrophic scars suggest that CCN2 may not serve as a common downstream mediator of TGF-ß action in all fibrotic disorders (Ihn, 2002; Leask and Abraham, 2003). The biological significance of CCNs expression and regulation in leiomyoma and myometrium requires detailed investigation considering their wide range of biological activities in other cell types.
CCNs, fibulin-1C and S100A4 regulate several cellular activities, including cell migration, cell motility, cell cycle progression, cell growth, calcium signaling, intercellular adhesion and extracellular matrix (ECM) production (Barraclough, 1998; Heizmann and Cox, 1998; Li et al., 2002; Sakamoto et al., 2002; Argraves et al., 2003; Brigstock, 2003; Margalit et al., 2003; Timpl et al., 2003; Perbal, 2004; Garrett et al., 2006). These events are central to leiomyoma growth and regression, suggesting key regulatory functions for CCNs, fibulin and S100A4 in pathophysiology of leiomyoma. Fibulin-1C and S100A4 also interact with several ECM proteins such as fibronectin and selectively bind calcium respectively, regulating calcium signalling and angiogenesis (Argraves et al., 2003; Margalit et al., 2003). Presence of a calcium-binding EGF-like domain in fibulin-1C and CCN3 enables their interactions with extracellular domain of heparin-binding EGF (HB-EGF), a key growth factor involved with cell and ECM interactions which is expressed in leiomyoma and myometrium (Tran et al., 1997; Grigorian et al., 2001; Brooke et al., 2002; Argraves et al., 2003; Timpl et al., 2003). A recent study has demonstrated the expression of several members of S100 family, including S100A4 and S100A11, in leiomyomas and myometrium (Kanamori et al., 2004). Unlike our observation with S100A4, leiomyomas were found to express elevated levels of S100A11 as compared to myometrium, and blocking S100A11 expression in human uterine smooth muscle tumor cells resulted in cellular apoptosis (Kanamori et al., 2004). S100A4 has also been reported to regulate the expression of matrix metalloproteinases, proteolytic enzymes involved in ECM turnover, and are expressed in leiomyoma and myometrium (Merzak et al., 1994; Bjornland et al., 1999; Ma and Chegini, 1999; Grigorian et al., 2001).
We found that TGF-ß action in regulating the expression of CCNs, fibulin-1C and S100A4 in LSMC and MSMC, at least in part, is mediated through the activation of MAPK and Smad signalling pathways. With respect to TGF-ß-mediated action through MAPK, U0126, a specific MEK1/2 inhibitor, reduced both basal and TGF-ß-induced CCN4, fibluin-1C and S100A4, but not CCN2 expression. The result, at least with CCN2 expression, is consistent with findings in other cells types, where U0126, but not MEK1 inhibitor PD98059, reduced the basal and TGF-ß-induced CCN2 expression (Chen et al., 2002; Leask and Abraham, 2003). In addition to MEK1/2, activation of c-jun NH(2)-terminal kinase, but not p38 MAPK, has been reported to mediate TGF-ß-induced CCN2 expression (Xie et al., 2005) and CCN4 inhibition in NCI-H295R, adrenocortical cell line (Lafont et al., 2002). Regarding Smad signalling, the activation of this pathway has been found essential in TGF-ß-induced CCN2 expression in many cell types; however, in scleroderma fibroblasts TGF-ß action occurred independent of Smad activation (Ihn, 2002; Leask and Abraham, 2003). It is possible that TGF-ß-induced CCN2 expression in LSMC may also occur in a Smad-independent manner, since inhibition/reduction of Smad3 expression increased TGF-ß-induced CCN2 expression. Similar alteration also occurred in the expression of CCN3, CCN4, fibulin-1C and S100A4. This affect was cell- and target-specific since lowering/inhibiting Smad3 expression did alter TGF-ß-induced CCN2 expression in MSMC, while significantly reduced the expression of plasminogen activator inhibitor (PAI-1), a well-known target of TGF-ß, in both LSMC and MSMC (unpublished observation). Although the results support the specificity of Smad3 activation in mediating TGF-ß-induced CCN2 expression in LSMC, the reason for the differences with other cells types is unclear. However, leiomyomas and LSMC overexpress Smad3, (Chegini et al., 2003; Xu et al., 2003) a condition that may result in lowering and/or suppression of CCN2 and other genes expression, and lowering/inhibition of Smad3 expression reversed all or some of the Smad inhibitory action. Alternatively, the activation of other signaling pathways and their crosstalk with Smad and MAPK may also result in differential regulation of these genes, including CCN2 in LSMC and MSMC (Leask and Abraham, 2003; Shi and Massague, 2003; Xu et al., 2003; Ding et al., 2004; Levens et al., 2005; Luo et al., 2005b).
From these results we concluded that paired leiomyoma and myometrium express CCNs, fibulin-1C and S100A4 in a menstrual cycle-independent manner, and with the exception of fibulin-1C, their expression is inversely correlated with the expression of TGF-ß1 and TGF-ß3. Although TGF-ß through the activation of Smad and MAPK pathways regulated the expression of these genes in LSMC, based on their tissue expression we propose that unlike other fibrotic disorders CCN2 (CTGF) may not serve as a downstream mediator of TGF-ß action in leiomyomas.
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Acknowledgements |
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This work was supported by NIH grant HD37432.
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Notes |
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Presented in part at the 52nd annual meeting of the Society for Gynecological Investigation, Los Angles, CA, March 2005.
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References |
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Submitted on August 15, 2005; resubmitted on December 22, 2005; accepted on January 9, 2006.
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