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Endocrinology Vol. 145, No. 2 976-982
Copyright © 2004 by The Endocrine Society

Estrogen Induces CCN5 Expression in the Rat Uterus in Vivo

Holly R. Mason, Danielle Grove-Strawser, Beverly S. Rubin, Romana A. Nowak and John J. Castellot, Jr.

Program in Cell, Molecular, and Developmental Biology (H.R.M., D.G.-S., B.S.R., J.J.C.), Sackler School of Biomedical Sciences, Tufts University, and Department of Anatomy and Cellular Biology (B.S.R., J.J.C.), Tufts University School of Medicine, Boston, Massachusetts 02111; and Department of Animal Sciences (R.A.N.), University of Illinois, Urbana, Illinois 61801

Address all correspondence and requests for reprints to: John J. Castellot, Jr., Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111. E-mail: john.castellot{at}tufts.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen plays an important role in the normal physiology as well as various pathologies of the uterus. Given the nature of uterine remodeling during the reproductive cycle and pregnancy, we sought to determine whether CCN5, a gene that we have shown to be important in smooth muscle cell proliferation and migration, is an estrogen-induced gene in the uterus. In the present study, we demonstrate that levels of CCN5 mRNA and protein expression were 5-fold higher in uteri from proestrous females relative to metestrous females, a finding consistent with estrogen induction of the CCN5 gene. Ovariectomized rats treated with exogenous estrogen or estrogen and progesterone exhibited 4- to 8-fold higher levels of CCN5 mRNA and protein than animals treated with either progesterone or vehicle alone. Analysis of rat uterine sections using immunohistochemistry demonstrates CCN5 localization throughout the uterus, including the endometrium and endometrial glands as well as the myometrium. Thus, our data indicate that CCN5 is positively regulated by estrogen in the rat uterus and suggests that this gene may play an important role in maintaining normal uterine physiology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN PLAYS AN important role in the normal physiology of the uterus (1, 2), and estrogen has also been implicated in various uterine pathologies including endometrial cancer, endometriosis, and leiomyomas (fibroids) (3, 4, 5, 6, 7, 8). There are many estrogen-induced genes in the uterus, including two members of the CCN family of genes, CCN1 (CYR61) and CCN2 (connective tissue growth factor, CTGF) (9, 10, 11, 12, 13).

The CCN family—named after the first three members discovered (CYR61, CTGF, and NOV)—is a small group of genes and proteins that has been implicated in the regulation of cell proliferation, migration, differentiation, apoptosis, angiogenesis, tumorigenesis, and fibrotic disease (14). To date, six members of the CCN family have been identified: CCN1 (CYR61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2, COP-1, CTGF-L, rCop-1), and CCN6 (WISP-3).

We originally identified CCN5 as a heparin-induced gene in vascular smooth muscle cells (SMC) (15). Further studies revealed that CCN5 was lost after transformation of rat embryo fibroblast cells (16), and that CCN5 was down-regulated in human colon adenocarcinoma compared with normal mucosa (17). Functional studies performed in our laboratory have indicated that CCN5 is expressed in both vascular and uterine smooth muscle cells and inhibits the proliferation and migration of both smooth muscle cell types (18 18A ). Moreover, recent data from our laboratory have revealed changes in CCN5 gene expression during the menstrual cycle in normal human myometrial tissues but not in leiomyoma tissue (18A ). These data are consistent with a role for estrogen in the regulation of CCN5 expression in normal myometrium but not in leiomyomas. Therefore, we wished to determine whether CCN5 is induced by estrogen in the uterus.

Although cell culture studies have demonstrated that CCN5 is an estrogen-responsive gene in human breast cancer cells (19), no studies have examined CCN5 expression in response to estrogen in vivo. Moreover, whether CCN5 is regulated by estrogen in the uterus is unknown. We therefore decided to examine the possibility that estrogen regulates CCN5 in vivo in a rat model.

In the present study, we use rats exhibiting regular estrous cycles as well as ovariectomized (OVX) rats treated with exogenous estrogen and provide the first evidence that CCN5 is an estrogen-responsive gene in the uterus. We show that estrogen promotes CCN5 mRNA and protein expression using real-time PCR, Western blots, and immunohistochemistry.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Animal protocols were reviewed and approved by The Institutional Animal Care and Use Committee at Tufts University (Boston, MA). Albino Sprague Dawley rats (200–225 g) were obtained from Charles River Laboratories (Wilmington, MA). Animals were maintained on a 14-h light, 10-h dark cycle with lights on at 0400 h and off at 1800 h. Food and water were available ad libitum.

Estrous cycle studies
Vaginal smears were examined daily between 0800 and 1000 h; animals were chosen for further examination only if they had exhibited at least five consecutive 4-d estrous cycles. Intact animals were killed on the morning of proestrus to allow examination of animals with high levels of endogenous estradiol and the morning of metestrus to allow examination of animals with low levels of estradiol.

OVX animals
Ovaries were removed by bilateral abdominal incision under xylazine anesthesia (6 mg/kg, ip; Fermenta Animal Health Co., Kansas City, MO) supplemented with ketamine (90 mg/kg, ip; Fort Dodge Animal Health, Fort Dodge, IA) and atropine (0.05 mg/kg, im; Phoenix Scientific, Inc., St. Joseph, MO). Buprenorphine (0.05 mg/kg, im; Reckitt & Colman Products, Hull, UK) was administered after surgery to ease postoperative pain. OVX animals were allowed to recover for 10 d before receiving hormone injections. Animals were injected sc with hormones dissolved in sesame oil (Sigma, St. Louis, MO). Animals were treated with either ß-estradiol 3-benzoate (EB; 4 µg/100 g body weight; Sigma), progesterone (P; 4 mg/rat; Sigma), both EB + P, or sesame oil at 1000 h for 2 consecutive days. Animals were anesthetized with isofluorane and killed by decapitation on the third day. Half of the uterus was placed in crushed dry ice and stored at -80 C. The other half was immersed in a fixative containing 4% paraformaldehyde (Fisher Scientific, Fair Lawn, NJ) and 3% acrolein (Polysciences, Inc., Warrington, PA) in 0.1 M phosphate buffer (pH 7.2) for 1 h at room temperature. Tissues were then transferred to PBS and stored at 4 C until processing by immunohistochemistry.

Western blot
Rat uterine tissue was removed from -80 C storage, weighed, and homogenized using a Kontes Pellet Pestle mixer (Kimble/Kontes, Vineland, NJ) and a rotor/stator homogenizer (Fisher Scientific, Pittsburgh, PA). The homogenate was washed once in cold TBS [20 mmol/liter Tris (pH 7.6), 137 mmol/liter NaCl] and placed in 3 ml/g modified radioimmunoprecipitation buffer (RIPA) lysis buffer containing protease inhibitor (Sigma) for 1 h. Lysates were spun for 30 min at 16,100 x g in an Eppendorf centrifuge (Brinkmann, Westbury, NY), and supernatants were stored at -80 C. Protein estimations were performed using the bicinchoninic acid (BCA) method (Pierce Biotechnology, Rockford, IL). Extracts containing 25 µg protein were boiled in 1x sodium dodecyl sulfate sample loading buffer, resolved by SDS-PAGE (4–20% Tris-HCl gradient gels were used; Bio-Rad, Hercules, CA), and blotted onto 0.2 µm pore size Immuno-Blot polyvinylidene difluoride membranes (Bio-Rad) in Towbin buffer without methanol (25 mmol/liter Tris, 192 mmol glycine) at 200 mA for 2 h. The blots were dried for storage and then rewetted with methanol. Membranes were blocked for 1 h in TBS containing 5% milk, and Western blots were performed using the antirat CCN5 affinity-purified rabbit polyclonal antibody previously described [1:1000 dilution; (18)] or an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (1:2000 dilution; Research Diagnostics, Inc., Flanders, NJ). Blots were washed and incubated with horseradish peroxidase-conjugated donkey antirabbit antibody (1:5000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) or antimouse IgG (1:10000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in TBST (TBS + 0.2% Tween 20). Bands were visualized using the Bio-Rad ImmunStar HRP (Bio-Rad) enhanced chemiluminescence detection reagents and developed as described by the vendor. Prestained protein standard markers (Bio-Rad) were used as molecular weight markers. Densitometry analysis of films was performed using the Stratagene (La Jolla, CA) Eagle Eye II system. Care was taken to ensure that densitometry was only performed on films where the bands had not saturated the film. Blots were stained with amido black to confirm equal loading and proper transfer of proteins to the membrane.

Real-time PCR
Rat uterine tissue was removed from -80 C storage, weighed, and homogenized as above. RNA isolation was performed using the RNeasy Mini kit (QIAGEN, Valencia, CA). DNA was removed using the DNA-free kit (Ambion, Austin, TX), 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) and examination of products on a 1.8% agarose gel confirmed the absence of genomic DNA. Primers were purchased from Integrated DNA Technologies (IDT, Coralville, IA). The sense CCN5 (GenBank accession no. gi 7739780) primer was 5'-CACCAACTTTCTGCCCTTGT-3' (position 796–815), and the antisense CCN5 primer was 5'-ATCTCCAGTTGGCAGAATCG-3' (position 922–941). These primers produced a product size of 146 base pairs. The sense GAPDH (GenBank accession number gi 8393417) primer was 5'-GAAGGGCTCATGACCACAGT-3' (position 538–557), and the antisense GAPDH primer was 5'-GGATGCAGGGATGATGTTCT-3' (position 635–654), producing a product size of 117 bp. Real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). The reactions were performed on the GeneAmp 5700 Sequence Detection System (PE Applied Biosystems). Controls included reactions with no template and no reverse transcriptase. The cycling conditions were: 95 C 10 min, and 40 cycles of 95 C 15 sec/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 vs. log C0) was constructed using a dilution series of rat lung total RNA (Ambion) transcribed to cDNA using the same protocol outlined above, and relative amounts of CCN5 and GAPDH were determined.

Plasma hormone levels
Blood was collected into heparinized tubes at time the rats were killed and centrifuged for 30 min at 4 C at 1000 x g. Plasma was aliquotted and stored at -30 C. Estradiol and P levels were determined using the Coat-A-Count Estradiol and Progesterone kits (Diagnostic Products Corp., Los Angeles, CA). The assays were performed according to the manufacturer’s protocol. A LKB-Wallac CliniGamma 1272 {gamma} counter was used to quantitate samples (Wallac, Turku, Finland).

Immunohistochemistry
Rat tissue was fixed as noted above and placed in 30% sucrose overnight at 4 C. Seven-micrometer sections were cut on a Leica CM3050 S cryostat (Leica Microsystems, Inc., Bannockburn, IL), mounted on Superfrost Plus microscope slides (Fisher Scientific), and stored at -20 C. Sections were placed in.05 M TBS overnight at room temperature. On the following day, slides were pretreated with 0.01 M sodium metaperiodate for 15 min, washed, and treated with 1% sodium borohydride for 10 min to remove residual aldehydes. After washing, the sections were placed in 5% dimethylsulfoxide for 10 min. Endogenous peroxide activity was quenched by treatment with 0.3% H2O2 in methanol for 30 min. After appropriate washes, the slides were incubated with blocking serum and then incubated in primary antibody overnight at 4 C. CCN5 protein was detected using an antirat CCN5 affinity-purified rabbit polyclonal antibody previously described [1:100 dilution; (18)] and EMMPRIN (used as a positive control) was detected using an antimouse EMMPRIN goat polyclonal antibody (1:50 dilution; R & D Systems, Minneapolis, MN). Slides were developed using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and the diaminobenzidine (DAB) substrate kit (Vector Laboratories). All sections from all animals were run in a single chemistry.

Statistical analysis
Data are presented as the mean ± SEM. Significance of difference was assessed using a Student’s t test or one-way ANOVA when appropriate. Differences were considered significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intact females
Proestrous animals have increased CCN5 mRNA and protein levels when compared with metestrous animals.
RNA was harvested from both proestrous and metestrous animals, and RT was performed. Real-time PCR was completed using both rat CCN5- and rat GAPDH-specific primers to determine relative CCN5 mRNA levels. As shown in Fig. 1Go, the relative level of CCN5 to GAPDH in proestrous animals is 5.0-fold higher than that of metestrous animals (P < 0.05).



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  		FIG. 1. CCN5 mRNA levels are higher during proestrus than during metestrus. RNA was harvested from whole uterus and real-time PCR performed using rat CCN5- and GAPDH-specific primers. Expression of CCN5 mRNA was determined relative to the GAPDH control. n = 4 for each condition. *, P < 0.05 when compared with metestrus.

 
To determine whether the protein expression closely followed the mRNA expression pattern, we also performed Western blots to examine the CCN5 protein levels of both proestrous and metestrous animals. As shown in Fig. 2Go, proestrous animals have higher levels of CCN5 relative to GAPDH than do metestrous animals. The relative levels of CCN5 are 4.7-fold higher in proestrous animals compared with metestrous animals (P < 0.05; Fig. 2CGo).



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  		FIG. 2. Proestrous animals have increased CCN5 protein expression compared with metestrous animals. Protein was harvested from whole uterus, and Western blot analysis was performed using an antirat CCN5 affinity-purified rabbit polyclonal antibody. Relative CCN5 expression was determined by densitometry relative to a GAPDH control. Four animals were used for each condition. A, Western blot using whole cell lysates from metestrous and proestrous animals probed with both anti-CCN5 and anti-GAPDH antibodies. B, Densitometry (CCN5 expression relative to GAPDH) for each of the lanes shown in A. C, Average of CCN5 expression for metestrous vs. proestrous animals shown in B. *, P < 0.05 when compared with metestrus.

 
To determine the tissue distribution of CCN5 in the uterus from the cycling rat, we performed immunohistochemistry. Proestrous animals have a higher level of CCN5 throughout the uterus (Fig. 3AGo). Interestingly, proestrous animals appear to have a high level of CCN5 expression in the endometrium, including the luminal epithelium (Fig. 3EGo) and the luminal surface of the uterine glands (Fig. 3IGo). By comparison, the levels of CCN5 are considerably lower in both of these locations for the metestrous animals (Fig. 3Go, F and J). Negative controls incubated with either secondary antibody alone or primary antibody in the presence of competing peptide exhibit no CCN5 expression throughout the uterus (data not shown). EMMPRIN was used here as a positive control as it has previously been shown to be up-regulated in the presence of estrogen in the uterus (20). As expected, the levels of EMMPRIN are higher in uterine sections from proestrous animals relative to sections from metestrous animals (Fig. 3Go, C and D, G and H, and K and L).



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  		FIG. 3. CCN5 is strongly expressed in the uterus of proestrous animals when compared with metestrous animals. IHC was performed using an anti-CCN5 antibody. Anti-EMMPRIN antibodies were used as a positive control for estrogen responsiveness. CCN5 was visualized using DAB. Negative controls incubated with secondary antibody alone or with primary antibody in the presence of competing peptide display no CCN5 expression anywhere in the uterus (data not shown). Endometrium is marked E, myometrium M, and serosa S. Expression in whole uterus is shown in A–D (magnification, x200), endometrium in E–H (magnification, x630), and uterine glands in I–L (magnification, x630). A, E, and I, Proestrus, CCN5 antibody. B, F, and J, Metestrus, CCN5 antibody. C, G, and K, Proestrus, EMMPRIN antibody. D, H, and L. Metestrus, EMMPRIN antibody.

 
OVX rats
Exogenous estrogen induces a high level of CCN5 mRNA expression in OVX females.
To more directly establish the role of estrogen in the regulation of CCN5 mRNA expression, we performed real-time PCR using the uteri from OVX animals treated with exogenous steroids. There was a statistically significant difference among the different treatment groups (one-way ANOVA, F = 6.9, P < 0.01). Animals treated with EB had 5.5-fold higher CCN5 levels compared with animals treated with oil (P < 0.05; Fig. 4Go). Animals treated with both EB and P had a 4.2-fold higher CCN5 mRNA expression when compared with oil-treated animals (P < 0.05). Interestingly, the difference in CCN5 mRNA levels in animals treated with EB + P vs. EB alone was statistically significant (P < 0.05). Animals treated with P alone had levels of CCN5 similar to animals treated with oil. Thus, OVX animals treated with exogenous estradiol have higher levels of CCN5 mRNA than do animals treated with oil alone or P alone.



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  		FIG. 4. Exogenous estrogen induces a high level of CCN5 mRNA expression in OVX females. RNA was harvested from uterine tissue and real-time PCR was performed using rat CCN5- and GAPDH-specific primers. CCN5 expression was determined relative to GAPDH. n = 4 for each condition. *, P < 0.05 when compared with oil. **, P < 0.05 when compared with EB.

 
High CCN5 protein levels are present in OVX animals treated with exogenous estrogen.
Because we had determined that estrogen was capable of up-regulating CCN5 mRNA expression in OVX rats, we wished to determine whether estrogen had a similar effect on CCN5 protein expression. Animals treated with EB in the presence or absence of P had the highest levels of CCN5 relative to GAPDH (Fig. 5AGo). Animals treated with P alone or oil had considerably lower relative levels of CCN5. The relative expression level of CCN5 was 6.9-fold and 7.6-fold higher in animals treated with EB and EB + P, respectively (Fig. 5BGo). Thus, estradiol or estradiol and P administration promote CCN5 protein expression in OVX rats.



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  		FIG. 5. High CCN5 protein levels are present in OVX animals treated with exogenous estrogen. Protein was harvested from uterine tissue, and Western blot analysis performed using anti-CCN5 and anti-GAPDH antibodies. Relative CCN5 expression was determined by densitometry relative to a GAPDH control. A representative experiment is shown. A, Western blot of whole cell lysates isolated from animals in each treatment group. Oil treatment lane was overloaded with protein, as a low level of CCN5 expression was expected for these animals. B, Densitometry of Western blot shown in A.

 
To examine the tissue distribution of CCN5 in the uterus, we performed immunohistochemistry. Animals treated with EB in the presence or absence of P have a high level of CCN5 expression throughout the uterus (Fig. 6Go, C and D). This is most prominent in the apical surface of the uterine epithelium (Fig. 6Go, I and J). By comparison, animals treated with P alone or oil have noticeably lower CCN5 levels (Fig. 6Go, A and B, and G and H). Examination of CCN5 expression in the uterine glands revealed low CCN5 expression in animals treated with oil or P (Fig. 6Go, M and N). Interestingly, animals treated with EB had a prominent luminal surface staining (Fig. 6OGo) that was not present in animals treated with EB + P (Fig. 6PGo). Negative controls consisting of uterine sections incubated with either secondary antibody alone or primary antibody in the presence of competing peptide have no detectable CCN5 expression (data not shown). EMMPRIN levels are significantly higher in animals treated with EB than animals treated with oil (Fig. 6Go, E and F, K and L, and Q and R). Thus, it appears that when examining CCN5 regulation by estrogen, a strong up-regulation is observed in the uterus. Whereas P alone did not affect CCN5 protein expression, animals treated with P in combination with estrogen exhibit a different pattern of CCN5 expression on the luminal epithelium of the uterine glands relative to those treated with estradiol alone.



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  		FIG. 6. Exogenous estrogen induces a high level of CCN5 protein expression in the uterus of OVX females. IHC was performed using the anti-CCN5 antibody described above. Anti-EMMPRIN antibodies were used as a positive control. CCN5 expression was visualized using DAB. Negative controls incubated with either secondary antibody alone or primary antibody in the presence of competing peptide exhibit no CCN5 expression anywhere in the uterus (data not shown). Endometrium is marked E, myometrium M, and serosa S. Expression in whole uterus is shown in A–F (magnification, x200), endometrium in G–L (magnification, x630), and uterine glands in M–R (magnification, x630). A, G, and M, Oil, CCN5 antibody. B, H, and N, P, CCN5 antibody. C, I, and O, EB, CCN5 antibody. D, J, and P, EB + P, CCN5 antibody. E, K, and Q, Oil, EMMPRIN antibody. F, L, and R, EB, EMMPRIN antibody.

 
Plasma estradiol and P levels in both cycling and OVX animals.
As expected, RIA measurements confirmed elevated levels of estradiol on the morning of proestrus (59.4 ± 9.4 pg/ml) and lower levels of estradiol on the morning of metestrus (24.3 ± 2.7 pg/ml) (P < 0.05; Table 1Go). Circulating P levels were elevated on metestrus (17.3 ± 1.7 ng/ml) relative to proestrus (2.0 ± 0.2 ng/ml) (P < 0.005; see Table 1Go for details). The levels of estradiol and P differed in the different treatment groups for the OVX animals, as was expected (one-way ANOVA, F = 24.9, P < 0.0001 for estradiol; F = 7.8, P < 0.01 for P; see Table 1Go for details).


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  		TABLE 1. Plasma hormone levels in both normal cycling and OVX animals

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this communication, we provide the first in vivo evidence that estrogen induces CCN5 expression in the uterus. Using intact regularly cycling females, we have determined that CCN5 levels are significantly increased in the uterus of proestrous females relative to metestrous females. Analysis of both the mRNA and protein data show that proestrous animals express 5-fold higher CCN5 levels than metestrous animals. Immunohistochemistry reveals that this increase in CCN5 expression is seen throughout the uterus. These data are consistent with up-regulation of CCN5 expression by estrogen.

More direct evidence for the up-regulation of CCN5 expression by estradiol is provided by the OVX, steroid-treated females. Estradiol administration alone or in combination with P enhanced CCN5 expression over that seen in females treated with P alone or with oil. Analysis of mRNA levels shows that P may have antagonized the effect of estrogen, as animals treated with both hormones had a modest but statistically significant reduction in CCN5 mRNA levels than animals treated with estrogen alone. This potential antagonistic effect of P was also seen in immunohistochemistry. On the luminal surface of the uterine glands, animals treated with estrogen alone had higher levels of CCN5 expression than did animals treated with both hormones. In both intact and OVX females, CCN5 expression was noted throughout the uterus in the presence of high circulating levels of estradiol and was not restricted to the myometrium.

It is interesting that CCN5 appears to be estrogen-regulated throughout the uterus, not just in the myometrium, as we initially posited from our observations in cultured uterine smooth muscle cells. This is analogous to the situation in the artery wall, in which both endothelial cells and smooth muscle cells express CCN5 (18). One plausible explanation for this observation is based on the hypothesis that CCN5 may be an important proliferation- and motility-regulating gene. In the myometrium, the predominant cell type is the smooth muscle cell, and CCN5 may act to limit the proliferation of this cell as it does in the artery wall. In the endometrium, there is active proliferation, migration, and remodeling of vascular structures, glands, and the supporting endometrial cells throughout the menstrual cycle. CCN5 might play an important role in regulating these processes, under the control of estrogen as noted above.

CCN5 is a member of the CCN family of genes that have diverse cell functions. The CCN family includes CCN1 (CYR61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2, COP-1, CTGF-L, rCop-1), and CCN6 (WISP-3) (14). To our knowledge, only three of the six CCN family members (CCN1, CCN2, and CCN5) have now been identified as estrogen-responsive genes. CCN1 (CYR61) was the first of the CCN family members demonstrated to be estrogen-induced using a subtractive hybridization approach in the rat uterus (9). More recently, two different groups have determined that CCN1 mRNA and protein levels are up-regulated by treatment with 17ß-estradiol in cultured human breast cancer cells (10, 11). CCN2 (CTGF) was also found to be estrogen regulated in the mouse uterus (13). OVX mice treated with 17ß-estradiol had a greater level of CCN2 expression in the uterine epithelium than animals with no steroid treatment (13). Interestingly, similar to our results with OVX animals, P appeared to have an antagonistic effect as animals treated with both 17ß-estradiol and P had moderately lower levels of CCN2 than animals treated with 17ß-estradiol alone. The physiological significance of this putative antagonistic effect of P is unknown.

As CCN1, CCN2, and CCN5 appear to be regulated by estrogen, one might expect that an estrogen-response element exists in the promoter of these genes. An estrogen response element has been identified in the promoter of CCN1 (12, 21). Whereas an estrogen response element has not yet been identified in the 5' flanking sequence of the CCN2 or CCN5 genes, estrogen responsiveness can also be conferred in other ways, including AP-1 or SP-1 sites (22, 23). SP-1 sites have been identified in the promoter of all three genes (24, 25), whereas AP-1 sites have been identified in the promoter of CCN2 and CCN5 (25).

In addition to estrogen responsiveness of CCN5, our group has recently shown that CCN5 inhibits vascular SMC motility and proliferation (18). We are currently investigating the role of CCN5 in human uterine SMC proliferation and motility (18A ). When CCN5 expression is examined during the menstrual cycle, the highest levels of CCN5 mRNA are observed in normal myometrial tissue during the proliferative phase of the menstrual cycle when estradiol levels are high, thus supporting the observations reported in this communication that CCN5 is an estrogen-regulated gene. Interestingly, CCN5 mRNA levels are very low in matched leiomyoma tissues in all phases of the menstrual cycle. Whereas estrogen is thought to promote myometrial proliferation (26, 27), our recent observations suggest that CCN5 may inhibit myometrial proliferation (18A ). Taken together, these observations suggest that CCN5 may be a component of a negative feedback loop controlling myometrial proliferation induced by estrogen.

Other studies have compared the expression of CCN5 in both normal and tumor cells (17, 28, 29, 30); however, the role of CCN5 in tumor development and progression remains unclear. Interestingly, CCN1 has also been found to be down-regulated in human leiomyomas compared with matched normal myometrium (12). Furthermore, treatment with 17ß-estradiol enhanced CCN1 mRNA expression in myometrial but not leiomyoma explants (12), even though leiomyomas have elevated levels of ER mRNA (31). These data also support a potential role for CCN family members in leiomyoma pathogenesis.

In summary, our data indicate that estrogen promotes CCN5 expression in the rat uterus in vivo. This may have important implications for uterine pathologies including endometrial cancer and endometriosis as well as uterine leiomyomas, and we are currently examining the role of estrogen and CCN5 in human leiomyomas. We postulate that, although estrogen may promote CCN5 expression in the normal myometrium, dysregulation of the CCN5 gene or altered estrogen responsiveness may account for some of the increased proliferation associated with leiomyomas.


   Acknowledgments
 
The authors thank Dr. Andrew Lake for valuable advice.


   Footnotes
 
This work was supported by NIH Grants HD046251, HD23681, and HL49973 (to J.J.C.); HD35148 (to R.A.N.); and AG14974 (to B.S.R.).

Abbreviations: CCN, Named after CYR61, CTGF, and NOV; CTGF, connective tissue growth factor; DAB, diaminobenzidine; EB, ß-estradiol 3-benzoate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; P, progesterone; SMC, smooth muscle cells.

Received July 7, 2003.

Accepted for publication October 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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