Mol. Hum. Reprod. Advance Access originally published online on January 29, 2004
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Molecular Human Reproduction, Vol. 10, No. 3, pp. 181-187, 2004
© European Society of Human Reproduction and Embryology 2004
The growth arrest-specific gene CCN5 is deficient in human leiomyomas and inhibits the proliferation and motility of cultured human uterine smooth muscle cells
1Program in Cell, Molecular, and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University, 136 Harrison Avenue, Boston, MA 02111, 2Department of Animal Sciences, University of Illinois, 1207 West Gregory Drive, ASL 310, Urbana, IL 61801 and 3Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts, USA 4To whom all correspondence should be addressed at: Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA. e-mail: john.castellot{at}tufts.edu
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Uterine fibroids (leiomyomas) are a major women’s health problem. Currently, the standard for treatment remains hysterectomy, since no other treatment modalities can reduce both symptoms and recurrence. As leiomyomas are benign neoplasias of smooth muscle cells, we sought to understand the regulation of uterine smooth muscle cell mitogenesis by CCN5, a growth arrest-specific gene in vascular smooth muscle cells which is induced and maintained by heparin treatment. Using autologous human myometrial and leiomyoma smooth muscle cells, we demonstrate that the proliferation and motility of both cell types are inhibited by the overexpression of CCN5. Surprisingly, we show that even though CCN5 is induced by heparin in vascular smooth muscle cells, treatment with heparin does not induce CCN5 expression in human uterine smooth muscle cells. Furthermore, we examine CCN5 mRNA expression in 10 autologous pairs of human myometrial and leiomyoma tissues and determine that CCN5 is down-regulated in 100% of the leiomyoma tissues analysed when compared to their normal myometrial counterparts. Thus, our data strongly suggest that CCN5 may exert an important function in maintaining the normal uterine phenotype and that loss of the anti-proliferative protein CCN5 from normal myometrium may account, at least in part, for tumorigenesis.
Key words: Key words: CCN5/CCN family/leiomyomas/myometrium/WISP-2
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Introduction |
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Uterine fibroids (leiomyomas) are the most common pelvic tumour in women (Nowak, 1999, 2001). Leiomyomas are a major cause of abnormal uterine bleeding and also cause pelvic pressure, pain, and impaired fertility (Stewart, 2001). They are responsible for nearly 200 000 hysterectomies annually in the USA (Wilcox et al., 1994). The prevalence of clinically apparent uterine leiomyomas is 20–25% in the overall population (Nowak, 1999, 2001). Interestingly, black women have an incidence rate which is at least three times higher than that of white women (Cramer, 1992; Marshall et al., 1997). Although these tumours affect a large population of women and are a major women’s health problem, the precise pathophysiology of uterine fibroids remains unclear. Currently, the only definitive treatment option available is hysterectomy, as other treatment options reduce symptoms only temporarily and do not significantly alter recurrence (Moorehead and Conard, 2001).
While it clearly would be advantageous to discover new treatment options for women wishing to maintain an intact uterus without the risk of a high recurrence rate, achieving this goal requires a better understanding of the cellular and molecular mechanisms controlling the pathogenesis of uterine fibroids.
Because leiomyomas are benign tumours of myometrial smooth muscle cells (SMC), one approach to developing therapeutic rationales is to elucidate the mechanisms that regulate myometrial SMC proliferation and to search for molecules that can inhibit myometrial SMC mitogenesis. We have previously identified the glycosaminoglycan heparin as an inhibitor of human uterine SMC proliferation and motility (Mason et al., 2003), and have shown that heparin induces and maintains CCN5 expression in vascular SMC (Delmolino et al., 2001; Lake et al., 2003). CCN5 is a member of the CCN (cyr61, CTGF, nov) family of genes, which has been implicated in many different cell functions including cell proliferation, migration, differentiation, apoptosis, angiogenesis, as well as tumorigenesis and fibrotic disease (Perbal, 2001). This family consists of cysteine-rich proteins of four modular domains and includes cyr61 (CCN1), CTGF (CCN2), and nov (CCN3) (Brigstock, 1999; Lau and Lam, 1999). Interestingly, CCN5 lacks the C-terminal domain thought to be important for the mitogenic activity of CCN2 (Brigstock et al., 1997); thus, one might expect CCN5 to inhibit rather than stimulate cell proliferation. Consistent with this idea, we have demonstrated that CCN5 inhibits vascular smooth muscle cell (VSMC) proliferation and motility (Delmolino et al., 2001; Lake et al., 2003).
Although several studies have been conducted comparing the expression of CCN5 in both normal and tumour cells, the role of CCN5 in tumour development and progression remains unclear. Several studies have suggested that CCN5 expression is present in transformed human breast cancer cell lines but not in normal breast epithelium (Saxena et al., 2001; Zoubine et al., 2001). In contrast, other studies have shown a loss of CCN5 expression upon cell transformation (Zhang et al., 1998) and down-regulation in non-breast tumours. Kumar et al. (1999) identified CCN5 expression in human osteoblasts but not osteosarcoma, and Pennica et al. (1998) have shown a decrease in RNA expression in human colon adenocarcinoma compared to autologous normal mucosa. The expression and functions of CCN5 in leiomyoma and myometrial SMC have not been examined previously.
Using matched pairs of human uterine smooth muscle cells (UtSMC), we examined whether forced expression of CCN5 would inhibit the motility and proliferation of both myometrial and leiomyoma SMC. Furthermore, we aimed to determine whether CCN5 expression is strongly down-regulated in uterine leiomyomas in vivo.
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All tissue culture plastic was obtained from Costar (USA) and Falcon (BD Biosciences, USA). Fetal calf serum (FCS) (characterized) was purchased from HyClone (USA). Dulbecco’s modified Eagle’s medium (DMEM), trypsin–EDTA, glutamine, and penicillin–streptomycin were purchased from Invitrogen (USA). Heparin was obtained from Pharmacia & Upjohn (USA).
Patients
Fibroid and myometrial tissues were obtained from pre-menopausal women with symptomatic uterine fibroids at the time of elective hysterectomy or myomectomy and who were not receiving any type of hormonal or drug therapy. Collection of tissues was obtained under a consent for use of discarded human tissue in accordance with the Human Research Committee of the Brigham and Women’s Hospital (Boston, MA). The tissue was processed for histology to determine the phase of the menstrual cycle according to the criteria of Noyes et al. (1950).
Cell culture
Fibroid and myometrial tissues were minced into 1–2 mm explants and placed in 10% FCS/DMEM containing 200 IU/ml collagenase (Invitrogen, USA). Myometrial tissue was digested for 8–10 h, and fibroid tissue for 18–20 h in a 37°C incubator in 5% CO2. Disaggregated cells were then centrifuged at 300 g for 5 min, the resulting cell pellet was resuspended in 10% FCS/DMEM, and the cells were plated into 75 ml tissue culture flasks. Cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. The purity of the cells was assessed by immunostaining for 
-actin and desmin, as described in our earlier studies (Nowak et al., 1993). Normal growth medium consisted of 10% FCS/DMEM supplemented with 4 mmol/l L-glutamine, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulphate. For heparin experiments, cells were first growth-arrested by placing them in 0.5% FCS/DMEM for 3 days. Cells were then treated with either 10% FCS/DMEM or 10% FCS/DMEM containing 500 µg/ml heparin for 4, 8 and 24 h. Cells were used in experiments no later than passage 8.
CCN5 adenoviral infections
The CCN5 and green fluorescent protein (GFP) adenoviruses used in all experiments have been previously described (Lake et al., 2003). Briefly, the CCN5 adenovirus (AdCCN5) expressed both GFP and CCN5 tagged by a nine amino acid HA epitope on the C-terminus. These two genes were under the control of separate CMV promoters. A control virus expressing only GFP (AdGFP) was also produced. Myometrial and leiomyoma SMC cells were washed twice in serum-free DMEM and then infected for 2 h in serum-free DMEM containing various concentrations of either CCN5 or GFP adenovirus. Cells were then placed in normal growth medium and allowed to grow for 2–3 days before being used in experiments.
Proliferation assay
Adenovirally infected and uninfected myometrial and leiomyoma SMC were plated into 24-well tissue culture plates (well area 1.9 cm2) at a density of 4.2x103 – 1.1x104 cells/cm2 and allowed to attach overnight. The following day, initial cell counts were taken. Medium was changed daily until the cells were confluent. Cells were counted after 2–3 days using a Coulter Counter (Beckman Coulter, USA). Controls consisted of both AdGFP-infected cells and uninfected cells. At least two independent experiments were performed in duplicate for each data-point shown. Given that the starting cell number was always the same, growth inhibition can be calculated as follows:
Migration assay
Myometrial and leiomyoma SMC were plated onto two chamber tissue culture slides (growth area 4 cm2) at a density of 1.6x104 cells/cm2 in 10% FCS/DMEM and were allowed to attach overnight. Medium was changed daily for 3 days and then cells were infected with adenovirus as described above. After 3 days, the confluent cell layer was scraped using a P200 pipette tip, creating a ‘wound’ 
300 µm wide. The cells were rinsed five times in 10% FCS/DMEM to wash away cells scraped off in the wound. Digital images (x100 magnification) were taken of the initial wound and at various times until the 10% FCS/DMEM control wound was confluent (i.e. until the cells had fully migrated into the wound from the edges), typically 19 h. All cells were fixed in 1% paraformaldehyde (Fisher Scientific, USA) when the 10% FCS/DMEM control was confluent. Hoechst 33258 (Sigma, USA) containing mounting medium was added to visualize cell nuclei, and the wounds were analysed by fluorescence microscopy. Fluorescent images were taken (magnification x100), and the number of cells migrating into the wound was quantified using Optimas imaging software (Media Cybernetics, USA). At least two independent experiments were performed in duplicate for each data-point shown. Inhibition of cell migration was calculated as above.
Western blot analysis
Protein from the various conditions described above was harvested from 100 mm tissue culture dishes, with all procedures performed at 4°C, as follows. Cells were rinsed twice with cold Tris-buffered saline (TBS: 20 mmol/l Tris pH 7.6, 137 mmol/l NaCl) and lysed with 200 µl RIPA lysis buffer [150 mmol/l NaCl, 50 mmol/l Tris pH 7.5, 1% Nonidet-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate (SDS), final pH 8.0] for 1 h. Lysates were centrifuged for 30 min at 16 100 g in an Eppendorf centrifuge (Brinkmann, USA), and supernatants were stored at –80°C. Protein estimations were performed using the BCA method (Pierce Biotechnology, USA). Extracts containing 20 µg protein were boiled in 1xSDS sample loading buffer, resolved by SDS–polyacrylamine gel electrophoresis (PAGE) (4
20% Tris–HCl gradient gels were used; Bio-Rad, USA), and blotted onto 0.2 µm pore size Immun-Blot polyvinylidene difluoride membranes (Bio-Rad) in Towbin buffer without methanol (25 mmol/l Tris, 192 mmol glycine) at 200 mA for 2 h. The blots were dried and rewetted with methanol. Membranes were blocked for 1 h in TBS containing 5% milk, and Western blots were performed using an anti-HA high affinity monoclonal antibody (1:150 dilution; Roche, Switzerland). Blots were washed and incubated with horseradish peroxidase-conjugated donkey anti-rabbit antibody (1:5000 dilution; Jackson ImmunoResearch Laboratories, USA) in TBST (TBS + 0.2% Tween 20). Bands were visualized using the NEN (USA) Renaissance enhanced chemiluminescence (ECL) detection reagents and developed as described by the vendor. Prestained protein standard markers (Bio-Rad) were used as molecular weight markers.
Real-time PCR
Total RNA was extracted from fresh uterine tissue using the guanidine isothiocyanate–caesium chloride method (Chirgwin et al., 1979). Briefly, uterine tissues were minced finely using a sterile blade, homogenized in 4 mol/l guanidine isothiocyanate, and centrifuged over a cesium chloride gradient at 100 000 g for 18 h. Total RNA was then precipitated in ethanol and sodium acetate. Total RNA isolation from cell cultures of myometrial and leiomyoma SMC was performed using the RNeasy Mini kit (Qiagen, USA). In both cases, contaminating DNA was removed using the DNA-free kit (Ambion, USA), and reverse transcription was performed using the RETROscript kit (Ambion). All assays were performed according to the manufacturer’s protocol. Controls containing no reverse transcriptase were used to check for genomic DNA contamination in each sample. PCR using the HotStarTaq kit (Qiagen, USA) and examination of products on a 1.8% agarose gel confirmed the absence of genomic DNA. Primers were purchased from IDT (Coralville, USA). The sense CCN5 (GenBank accession number gi 4028582) primer was 5'-TATTAAC ACGCTGCCTGGTCTGTCT-3' (position 1054–1078), and the antisense CCN5 primer was 5'-TCGCCCGTGTGCATGTTTGATATAG-3' (position 1196–1220). These primers produced a product size of 167 base pairs (bp). The sense GAPDH (GenBank accession number gi 182976) primer was 5'-CCACCCAGAAGACTGTGGAT-3' (position 608–627), and the antisense GAPDH primer was 5'-TTCAGCTCAGGGATGACCTT-3' (position 715–734), producing a product size of 127 bp. Real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, USA). The reactions were performed on the GeneAmp 5700 Sequence Detection System (PE Applied Biosystems). No template and no reverse transcriptase samples were used as controls. The cycling conditions were: 95°C 10 min, and 40 cycles of 95°C 15 s/60°C 1 min. The standard dissociation protocol was performed to confirm the absence of primer dimers, and product size was determined by running PCR products on a 1.8% agarose gel. A standard curve (Ct versus log C0) was constructed using a dilution series of mRNA from exponentially growing myometrial SMC. This mRNA was transcribed to cDNA using the same protocol outlined above, and relative amounts of CCN5 and GAPDH were determined.
Statistical analysis
Data are presented as the mean ± SEM. Significance of difference was assessed using a Student’s t-test. Differences were considered significant when P < 0.05. Statistical analyses were performed in consultation with Professor Jerold Harmatz (Tufts University School of Medicine, Boston, MA).
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Results |
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Heparin does not induce CCN5 expression
Heparin is a widely studied inhibitor of SMC proliferation (Mishra-Gorur et al., 1998). We and others have shown that heparin inhibits myometrial and leiomyoma SMC mitogenesis and motility (Horiuchi et al., 1999; Mason et al., 2003). In addition, we have found that heparin is capable of inducing and maintaining CCN5 expression in vascular SMC (Delmolino et al., 2001; Lake et al., 2003). We therefore hypothesized that heparin exerted its anti-proliferative effect, at least in part, by inducing CCN5 in human uterine SMC.
We previously used autologous human myometrial and leiomyoma SMC to determine that both heparin-sensitive and heparin-resistant cultures exist in nature (Mason et al., 2003). For this study, we chose to use a heparin sensitive and a heparin resistant matched pair to identify whether heparin is capable of up-regulating CCN5 mRNA expression using real-time PCR. Human myometrial and leiomyoma SMC cultures were growth-arrested by serum starvation for 3 days and then released from growth arrest by the addition of 10% FCS/DMEM in the presence or absence of 500 µg/ml heparin. As expected, the relatively high levels of CCN5 mRNA seen in growth-arrested cells quickly diminished, a pattern observed in vascular SMC (Delmolino et al., 2001; Lake et al., 2003). Surprisingly, heparin did not induce or maintain CCN5 expression in any of the four cultures tested, including both heparin-resistant and heparin-sensitive myometrial and leiomyoma SMC (Figure 1). While the matched pairs chosen for this experiment were representative, caution must be used to interpret these results given the small sample size.
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CCN5 is strongly down-regulated in human leiomyomas
If CCN5 functions as an inhibitor of HUtSMC proliferation, then fibroids should show reduced levels of CCN5 compared to their normal myometrial counterparts. To establish the role, if any, of CCN5 in human uterine leiomyomas, we obtained 10 matched pairs of human myometrial and leiomyoma tissue. All three phases of the menstrual cycle were represented: proliferative (four pairs), secretory (four pairs), and menstrual (two pairs). mRNA was harvested from these tissues and reverse transcription was performed. Real-time PCR was performed using GAPDH as a control to yield a quantitative assessment of CCN5 mRNA.
In all three phases of the menstrual cycle, CCN5 is down-regulated in leiomyoma compared to matched myometrium in 10 of 10 uteri (Figure 2). The average level of CCN5 overexpression in the myometrium compared to leiomyoma is 7-fold in the proliferative phase and 5-fold in the secretory phase. In contrast, CCN5 mRNA levels are only slightly higher in the menstrual phase. While this difference is statistically significant for all 10 autologous pairs studied (P < 0.05), the difference may not be biologically significant in the menstrual phase because the CCN5 levels are similar in this phase.
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Adenoviral CCN5 constructs force expression of CCN5 in uterine SMC
Previously we used an adenoviral CCN5 construct to overexpress CCN5 in vascular SMC (Lake et al., 2003). This adenovirus contains CCN5 and green fluorescent protein (GFP) under control of separate CMV promoters. In addition, the CCN5 construct contains an HA tag. Thus, expression can be easily monitored in cultures for infection using a fluorescence microscope, and CCN5 experession can be compared from one experiment to another using a Western blot with an anti-HA antibody. We typically obtain >90% infection in cultures. By varying the multiplicity of infection (MOI) of the CCN5 adenoviral construct, different expression levels of CCN5 can be achieved in human uterine SMC. Using Western blot analysis of cultures of human myometrial SMC, we demonstrate a dose-dependent increase in CCN5 protein levels with increasing MOI of CCN5 adenovirus (Figure 3). By comparison, growth-arrested SMC express approximately the same amount of CCN5 as 50–100 MOI AdCCN5 (data not shown).
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AdCCN5 inhibits myometrial and leiomyoma SMC proliferation
Because we have identified that CCN5 levels are decreased in leiomyomas compared to matched normal myometrium and our previous studies have demonstrated that CCN5 inhibits vascular SMC proliferation (Delmolino et al., 2001; Lake et al., 2003), we hypothesized that CCN5 may act as a growth inhibitor of both myometrial and leiomyoma SMC and that loss of this gene in leiomyomas may contribute to tumorigenesis. For these studies, cultures of myometrial and leiomyoma SMC were infected with different concentrations (MOI) of AdCCN5. Cells were plated and allowed to grow for 2–3 days. Forced expression of CCN5 inhibits the proliferation of exponentially growing myometrial SMC in a dose-dependent manner (Figure 4). At an MOI of 200, cells are inhibited by CCN5 by >60%, and at an MOI of 50, cells are inhibited by
30%. There is no difference in the amount of inhibition seen at 2 days or 3 days.
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At MOI 50, CCN5 inhibits both myometrial and leiomyoma SMC proliferation to a similar extent (21% for myometrial SMC, 25% for leiomyoma SMC). A slight amount of inhibition was seen at MOI 50 with AdGFP, but these cells were always >85% of uninfected control. The
25% inhibition seen for AdCCN5 in both myometrial and leiomyoma SMC is an additional effect over the slight amount of inhibition seen with AdGFP. Interestingly, the amount of inhibition with CCN5 at MOI of 50 is similar to the amount observed with 300 µg/ml heparin (Mason et al., 2003).
CCN5 inhibits myometrial and leiomyoma SMC motility
Because motility is a function critical to leiomyoma pathogenesis, we examined the effect of CCN5 on cell migration. To determine whether CCN5 has an anti-migratory effect on uterine SMC, we performed a scratch wound assay using uninfected as well as AdCCN5- and AdGFP-infected cells. Uninfected control cells migrate and fill the wound within 20 h, and AdGFP has only a small inhibitory effect (<20%). In contrast, forced CCN5 expression inhibits myometrial SMC motility (Figure 5). At MOI 10, the motility of myometrial SMC infected with AdCCN5 is inhibited by 33% when compared to AdGFP-infected cells.
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Further experiments using both myometrial and leiomyoma SMC cultures reveal that CCN5 inhibits cell migration in both cell types (Table I). When compared to the uninfected control cells, 49% inhibition is observed for myometrial SMC and 71% for leiomyoma SMC at MOI 10. AdGFP was used as a control and in both cases the inhibitory effect was minimal (<20%).
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In this communication, we examine the possibility that CCN5 plays an important role in myometrial and leiomyoma SMC function in culture and in vivo. We show for the first time that CCN5 inhibits myometrial and leiomyoma SMC proliferation and motility. We also present the new and potentially important observation that CCN5 is strongly down-regulated in human leiomyomas. Unexpectedly, our data show that CCN5 is not a heparin-inducible gene in matched pairs of human uterine SMC as it is in vascular SMC.
We observed that CCN5 expression in leiomyomas was down-regulated compared to matched myometrium for all 10 of the matched pairs studied. While the differences between the amount of CCN5 expression in myometrium and leiomyoma tissue were statistically significant in all of the autologous pairs, this may not be a biologically significant difference in the menstrual phase as CCN5 levels were nearly equal (1.3 ratio of myometrium/fibroid CCN5). Interestingly, this effect was most pronounced in the proliferative phase, when estrogen levels are highest, and is lowest in the menstrual phase when estrogen levels are low. This suggests that expression of the CCN5 gene is positively regulated by estrogen in human myometrium and leiomyomas. This is consistent with our recent observation that CCN5 is strongly estrogen-regulated in the rat uterus (Mason et al., 2004). In addition, other studies have demonstrated that CCN5 is an estrogen-responsive gene in human breast cancer cells (Inadera et al., 2000).
The loss of CCN5 mRNA in tumours has also been observed in other tissues. A well-controlled study using autologous pairs of human colon adenocarcinoma and normal mucosa revealed that CCN5 was down-regulated in 79% of the tissue samples analysed (Pennica et al., 1998). In addition, another study has suggested that CCN5 is expressed in normal human osteoblasts but not osteosarcoma (Kumar et al., 1999).
Previously, we have demonstrated that CCN5 expression is induced and maintained by heparin in vascular SMC (Delmolino et al., 2001; Lake et al., 2003). In addition, heparin inhibits both vascular and uterine SMC proliferation (Clowes and Karnovsky, 1977; Guyton et al., 1980; Hoover et al., 1980; Castellot Jr et al., 1981; Horiuchi et al., 1999; Mason et al., 2003). To our surprise, we found that heparin does not induce CCN5 expression in matched pairs of myometrial and leiomyoma SMC. Thus, it appears that CCN5 expression is not heparin-regulated in uterine SMC and that the mechanism for inhibition of uterine SMC proliferation by heparin is not via the activation of CCN5. The reasons for the differences in CCN5 regulation in these two SMC types are currently under investigation and may include presence of an inhibitor/activator, differences in signalling pathways, absence of the heparin-regulated element from the promoter of the CCN5 gene in uterine SMC, and promoter cell-type specificity. It is interesting to note that CCN5 is still strongly correlated with the quiescent growth state in human uterine SMC, just as it is in vascular SMC (Delmolino et al., 2001; Lake et al., 2003).
Our data demonstrate that overexpression of CCN5 in human uterine SMC inhibits proliferation to a similar extent in both myometrial and leiomyoma SMC. Interestingly, the amount of inhibition observed is strikingly similar to that for vascular SMC (Lake et al., 2003). Thus, CCN5 has an anti-proliferative effect on both of these cell types but surprisingly has a very different pattern of regulation.
We have also found that ectopic expression of CCN5 infection inhibits leiomyoma SMC migration. Our data suggest that leiomyoma SMC may be more sensitive to the anti-motility effect than myometrial SMC. The reasons for this apparent difference are currently under investigation. We considered the possibility that cell proliferation may confound the motility results, but have ruled this out in our experiments for three reasons. First, we observed no difference in the number of cells migrating into the wound when the motility experiments were completed using 5 mmol/l hydroxyurea to inhibit cell proliferation (Mason et al., 2003). Second, because the wound is created from confluent cells in a quiescent growth state, the number of mitotic figures in all wounded cultures was <1%. Significant cell division is not observed until 36–40 h after wounding, at which time the uninfected control cultures tested had already filled in the wound. Finally, analysis of the wounds at earlier time-points also shows substantial inhibition of motility (data not shown).
Our group has shown that CCN5 can inhibit cell proliferation and migration in two different SMC types. This finding is in contrast to the functions of the other members of the CCN family as both CCN1 and CCN2 have mitogenic and motogenic activity for many different cell types (Bradham et al., 1991; Frazier et al., 1996; Kireeva et al., 1996; Brigstock et al., 1997; Shimo et al., 1998; Babic et al., 1999; Shimo et al., 1999; Fan et al., 2000; Nakanishi et al., 2000; Nishida et al., 2000; Blom et al., 2001; Crean et al., 2002; Grzeszkiewicz et al., 2002; Leu et al., 2002; Paradis et al., 2002). It is tempting to speculate that this difference in cell function is the lack of the C-terminal domain in CCN5 that is present in CCN1 and CCN2. CCN proteins are cysteine rich and contain four modular domains (Bork, 1993). The first domain has sequence similarity to the insulin-like growth factor binding proteins (IGFBP) and is thought to be responsible for binding IGF. The second domain is a von Willebrand factor type C repeat and is thought to be responsible for oligomerization. The third domain is a thrombospondin-1 domain that may be involved in cell attachment. The fourth domain, the C-terminal domain, contains a cysteine knot motif and is thought to be important for dimerization and receptor binding. Earlier work suggests that the C-terminal domain may be important for the proliferative activity of CCN2 (Brigstock et al., 1997). Further experiments to examine the role of the C-terminal domain in the proliferative activity of CCN2 are currently underway.
CCN1 has also been found to be down-regulated in human leiomyomas compared to matched normal myometrium (Sampath et al., 2001). Furthermore, treatment with 17ß-estradiol enhanced CCN1 mRNA expression in myometrial but not leiomyoma explants (Sampath et al., 2001). Taken together with the CCN5 data, this suggests a potential role of CCN family members in leiomyoma pathogenesis.
In summary, we have demonstrated that CCN5 has an anti-proliferative and anti-motility effect on both myometrial and leiomyoma SMC. We have also shown that CCN5 expression is strongly down-regulated in leiomyomas. Loss of this anti-proliferative molecule in leiomyomas could account, at least in part, for the aberrant proliferation and tumorigenesis, and experiments to test this hypothesis are underway.
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Acknowledgements |
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We are grateful to Jerold Harmatz (Tufts University School of Medicine) for his biostatistical expertise and consultation on the experimental design and analysis of data. This work was supported by NIH Grants HD 046251, HD23681 and HL49973 to J.J.C.; and HD35148 and HD046227 to R.A.N.
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REFERENCES |
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Babic AM, Chen CC and Lau LF (1999) Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 19,2958–2966.
Blom IE, van Dijk AJ, Wieten L, Duran K, Ito Y, Kleij L, Denichilo M, Rabelink TJ, Weening JJ, Aten J et al (2001) In vitro evidence for differential involvement of CTGF, TGFbeta, and PDGF-BB in mesangial response to injury. Nephrol Dial Transplant 16,1139–1148.
Bork P (1993) The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett 327,125–130.[CrossRef][ISI][Medline]
Bradham DM, Igarashi A, Potter RL and Grotendorst GR (1991) Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J Cell Biol 14,1285–1294.
Brigstock DR (1999) The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr Rev 20,189–206.
Brigstock DR, Steffen CL, Kim GY, Vegunta RK, Diehl JR and Harding PA (1997) Purification and characterization of novel heparin-binding growth factors in uterine secretory fluids Identification as heparin- regulated Mr 10,000 forms of connective tissue growth factor. J Biol Chem 272,20275–20282.
Castellot JJ, Jr, Addonizio ML, Rosenberg R and Karnovsky MJ (1981) Cultured endothelial cells produce a heparinlike inhibitor of smooth muscle cell growth. J Cell Biol 90,372–379.
Chirgwin JM, Przybyla AE, MacDonald RJ and Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonucleases. Biochemistry 18,5294–5299.[CrossRef][Medline]
Clowes AW and Karnovsky MJ (1977) Suppression by heparin of smooth muscle cell proliferation in injured arteries. Nature 265,625–626.[CrossRef][Medline]
Cramer DW (1992) Epidemiology of myomas. Semin Reprod Endocrinol 10,320–324.
Crean JK, Finlay D, Murphy M, Moss C, Godson C, Martin F and Brady HR (2002) The role of p42/44 MAPK and protein kinase B in connective tissue growth factor induced extracellular matrix protein production, cell migration, and actin cytoskeletal rearrangement in human mesangial cells. J Biol Chem 277,44187–44194.
Delmolino LM, Stearns NA and Castellot JJ Jr (2001) COP-1, a member of the CCN family, is a heparin-induced growth arrest specific gene in vascular smooth muscle cells. J Cell Physiol 188,45–55.[CrossRef][ISI][Medline]
Fan WH, Pech M and Karnovsky MJ (2000) Connective tissue growth factor (CTGF) stimulates vascular smooth muscle cell growth and migration in vitro. Eur J Cell Biol 79,915–923.[CrossRef][ISI][Medline]
Frazier K, Williams S, Kothapalli D, Klapper H and Grotendorst GR (1996) Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor. J Invest Dermatol 107,404–411.[CrossRef][ISI][Medline]
Grzeszkiewicz TM, Lindner V, Chen N, Lam SC and Lau LF (2002) The angiogenic factor cysteine-rich 61 (CYR61, CCN1) supports vascular smooth muscle cell adhesion and stimulates chemotaxis through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans. Endocrinology 143,1441–1450.
Guyton JR, Rosenberg RD, Clowes AW and Karnovsky MJ (1980) Inhibition of rat arterial smooth muscle cell proliferation by heparin In vivo studies with anticoagulant and nonanticoagulant heparin. Circ Res 46,625–634.
Hoover RL, Rosenberg R, Haering W and Karnovsky MJ (1980) Inhibition of rat arterial smooth muscle cell proliferation by heparin II In vitro studies. Circ Res 47,578–583.
Horiuchi A, Nikaido T, Ya-Li Z, Ito K, Orii A and Fujii S (1999) Heparin inhibits proliferation of myometrial and leiomyomal smooth muscle cells through the induction of alpha-smooth muscle actin, calponin h1 and p27. Mol Hum Reprod 5,139–145.
Inadera H, Hashimoto S, Dong HY, Suzuki T, Nagai S, Yamashita T, Toyoda N and Matsushima K (2000) WISP-2 as a novel estrogen-responsive gene in human breast cancer cells. Biochem Biophys Res Commun 275,108–114.[CrossRef][ISI][Medline]
Kireeva ML, Mo F-E, Yang GP and Lau LF (1996) Cyr61, a product of a growth factor-inducible immediate-early gene, promotes cell proliferation, migration, and adhesion. Mol Cell Biol 16,1326–1334.[Abstract]
Kumar S, Hand AT, Connor JR, Dodds RA, Ryan PJ, Trill JJ, Fisher SM, Nuttall ME, Lipshutz DB, Zou C et al (1999) Identification and cloning of a connective tissue growth factor-like cDNA from human osteoblasts encoding a novel regulator of osteoblast functions. J Biol Chem 274,17123–17131.
Lake AC, Bialik A, Walsh K and Castellot JJ, Jr (2003) CCN5 is a growth arrest-specific gene that regulates smooth muscle cell proliferation and motility. Am J Pathol 162,219–231.
Lau LF and Lam SC (1999) The CCN family of angiogenic regulators: the integrin connection. Exp Cell Res 248,44–57.[CrossRef][ISI][Medline]
Leu SJ, Lam SC and Lau LF (2002) Pro-angiogenic activities of CYR61 (CCN1) mediated through integrins alphavbeta3 and alpha6beta1 in human umbilical vein endothelial cells. J Biol Chem 277,46248–46255.
Marshall LM, Spiegelman D, Barbieri RL, Goldman MB, Manson JE, Colditz GA, Willett WC and Hunter DJ (1997) Variation in the incidence of uterine leiomyoma among premenopausal women by age and race. Obstet Gynecol, 90, 967–973.[Abstract]
Mason HR, Nowak RA, Morton CC and Castellot JJ, Jr (2003) Heparin inhibits the motility and proliferation of human myometrial and leiomyoma smooth muscle cells. Am J Pathol 162,1895–1904.
Mason HR, Grove-Strawser D, Rubin BS, Nowak RA and Castellot JJ, Jr (2004) Estrogen induces CCN5 expression in the rat uterus in vivo. Endocrinology, published online Nov. 6, 2003.
Mishra-Gorur K, Delmolino LM and Castellot JJ, Jr (1998) Biological functions of heparan sulfate and heparan sulfate proteoglycans. Trends Glycosci Glycotechnol 10,193–210.
Moorehead ME and Conard CJ (2001) Uterine leiomyoma: a treatable condition. Ann NY Acad Sci 948,121–129.
Nakanishi T, Nishida T, Shimo T, Kobayashi K, Kubo T, Tamatani T, Tezuka K and Takigawa M (2000) Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture. Endocrinology 141,264–273.
Nishida T, Nakanishi T, Asano M, Shimo T and Takigawa M (2000) Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro. J Cell Physiol 184,197–206.[CrossRef][ISI][Medline]
Nowak RA (1999) Fibroids: pathophysiology and current medical treatment. Baillières Best Pract Res Clin Obstet Gynecol 13,223–238.[CrossRef][Medline]
Nowak RA (2001) Identification of new therapies for leiomyomas: what in vitro studies can tell us. Clin Obstet Gynecol 44,327–334.[CrossRef][ISI][Medline]
Nowak RA, Rein MS, Heffner LJ, Friedman AJ and Tashjian AH, Jr (1993) Production of prolactin by smooth muscle cells cultured from human uterine fibroid tumors. J Clin Endocrinol Metab 76,1308–1313.[Abstract]
Noyes RA, Hertig AT and Rock J (1950) Dating the endometrial biopsy. Fertil Steril 1,3–25.[Medline]
Paradis V, Dargere D, Bonvoust F, Vidaud M, Segarini P and Bedossa P (2002) Effects and regulation of connective tissue growth factor on hepatic stellate cells. Lab Invest 82,767–774.[ISI][Medline]
Pennica D, Swanson TA, Welsh JW, Roy MA, Lawrence DA, Lee J, Brush J, Taneyhill LA, Deuel B, Lew M et al (1998) WISP genes are members of the connective tissue growth factor family that are up-regulated in wnt-1-transformed cells and aberrantly expressed in human colon tumors. Proc Natl Acad Sci USA 95,14717–14722.
Perbal B (2001) NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues. Mol Pathol 54,57–79.
Sampath D, Zhu Y, Winneker RC and Zhang Z (2001) Aberrant expression of Cyr61, a member of the CCN (CTGF/Cyr61/Cef10/NOVH) family, and dysregulation by 17 beta-estradiol and basic fibroblast growth factor in human uterine leiomyomas. J Clin Endocrinol Metab 86,1707–1715.
Saxena N, Banerjee S, Sengupta K, Zoubine MN and Banerjee SK (2001) Differential expression of WISP-1 and WISP-2 genes in normal and transformed human breast cell lines. Mol Cell Biochem, 228, 99–104.[CrossRef][ISI][Medline]
Shimo T, Nakanishi T, Kimura Y, Nishida T, Ishizeki K, Matsumura T and Takigawa M (1998) Inhibition of endogenous expression of connective tissue growth factor by its antisense oligonucleotide and antisense RNA suppresses proliferation and migration of vascular endothelial cells. J Biochem (Tokyo) 124,130–140.
Shimo T, Nakanishi T, Nishida T, Asano M, Kanyama M, Kuboki T, Tamatani T, Tezuka K, Takemura M, Matsumura T et al (1999) Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo. J Biochem (Tokyo) 126,137–145.
Stewart EA (2001) Uterine fibroids. Lancet 357,293–298.[CrossRef][ISI][Medline]
Wilcox LS, Koonin LM, Pokras R, Strauss LT, Xia Z and Peterson HB (1994) Hysterectomy in the United States, 1988–1990. Obstet Gynecol 83,549–555.[ISI][Medline]
Zhang R, Averboukh L, Zhu W, Zhang H, Jo H, Dempsey PJ, Coffey RJ, Pardee AB and Liang P (1998) Identification of rCop-1, a new member of the CCN protein family, as a negative regulator for cell transformation. Mol Cell Biol 18,6131–6141.
Zoubine MN, Banerjee S, Saxena NK, Campbell DR and Banerjee SK (2001) WISP-2: a serum-inducible gene differentially expressed in human normal breast epithelial cells and in MCF-7 breast tumor cells. Biochem Biophys Res Commun 282,421–425.[CrossRef][ISI][Medline]
Submitted on June 27, 2003; resubmitted on November 17, 2003; accepted on November 18, 2003.
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