Mol. Hum. Reprod. Advance Access originally published online on June 6, 2007
Molecular Human Reproduction 2007 13(8):577-585; doi:10.1093/molehr/gam040
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Retinoic acid pathway genes show significantly altered expression in uterine fibroids when compared with normal myometrium
1 Centre for Women's Health Research, Monash University Department of Obstetrics and Gynaecology, Monash Institute of Medical Research, Clayton, Victoria, Australia 2 Women's and Children's Program, Southern Health, Melbourne, Victoria, Australia
3 Correspondence address. Tel: +61 3 9594 5379; Fax: +61 3 9594 6389; E-mail: peter.rogers{at}med.monash.edu.au
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Abstract |
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Fibroids are benign neoplasms of myometrial smooth muscle cells (SMC). Despite being the most common tumor in humans, their etiology is poorly understood. Recent microarray studies have demonstrated that multiple members of the retinoid pathway are differentially expressed between myometrium and fibroids. The aim of this present study was to investigate gene expression of members of the retinoid pathway in matched myometrium and fibroids. We have demonstrated differential gene expression of two binding proteins [cellular retinol-binding proteins (CRBP) 1 and 2], three enzymes [alcohol dehydrogenase 1 (ADH1), aldehyde dehydrogenase (ALDH1) and retinol dehydrogenase (RODH)] and two receptors [retinoid X receptors (RXR)
and 
] involved in the retinoid pathway by real-time PCR. There were no differences in gene expression for retinoid receptors RAR, ß, and RXRß, and for the metabolizing enzyme cytochrome P450, family 26 subfamily A. We confirmed results for ADH1, ALDH1, CRBP1 and CRABP2 at the protein level by western blot. Using immunohistochemistry these proteins were mostly localized to myometrial and fibroid SMC. An exception to this was ALDH1 protein, which displayed strong staining localized to cells of the connective tissue, presumably fibroblasts, with a striking differential expression pattern between myometrium and fibroids. These results demonstrate that the retinoid pathway is altered in fibroids when compared with normal myometrium and specifically identify ALDH1 in fibroid fibroblasts. These alterations can lead to aberrant retinoic acid (RA) production and signaling, and alter the expression of RA target genes, which may be an important step in fibroid development. Key words: fibroids/retinoic acid pathway/ALDH1/CRBP1/CRABP2
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Introduction |
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Uterine leiomyomas (fibroids) are benign neoplasms of the smooth muscle cells (SMC) of the uterus and are the most common tumors in women, affecting at least half of US reproductive-age women (Payson et al., 2006). While these tumors are not malignant, for one third of these women fibroids are a significant cause of reproductive and gynecological problems, such as menorrhagia, dysmenorrhoea, chronic pelvic pain, as well as infertility, recurrent miscarriage, pre-term delivery and post-partum hemorrhage (Stewart, 2001; Flake et al., 2003). Uterine fibroids are a leading cause of hysterectomy in both Australia and the USA and account for over 200 000 hysterectomies in US women each year (Farquhar and Steiner, 2002). Recently, uterine artery embolization is emerging as an alternative treatment for patients with uterine fibroids (Marshburn et al., 2006).
Despite the fact that fibroids adversely affect many women, their etiology is poorly understood. These tumors grow during the reproductive years and regress after the menopause, implicating the sex hormones estrogen and progesterone as primary factors driving fibroid growth (Flake et al., 2003). However, there is little understanding of the processes of fibroid growth and regression at the cellular and molecular levels. In recent years, multiple published studies have used microarray platforms to try to elucidate genes important in fibroid growth (Tsibris et al., 2002; Ahn et al., 2003; Catherino et al., 2003; Weston et al., 2003; Hoffman et al., 2004; Arslan et al., 2005; Lee et al., 2005). Several genes and pathways appear to be consistently up-regulated between different microarray studies, including genes involved in the retinoic acid (RA) pathway, insulin growth factor 2 (IGF2) metabolism, transforming growth factor ß (TGF-ß) signaling and extracellular matrix formation. Some of these pathways and genes, such as IGF2 and TGF-ß, have been previously implicated in fibroid growth and investigated in multiple studies. Others, such as the RA pathway, warrant further investigation to confirm that the changes observed by microarray studies can be demonstrated by other approaches such as RNA and protein analysis, as well as to elucidate the possible role of these genes in fibroid growth and development.
Multiple members of the RA pathway are among the genes that are identified most consistently as having altered expression in microarray studies (reviewed by Arslan et al., 2005). They include enzymes involved in RA synthesis, such as alcohol dehydrogenase 1 (ADH1), aldehyde dehydrogenase 1 (ALDH1) and cellular RA-binding protein 2 (CRABP2). Other genes include retinol dehydrogenase (RODH) (Tsibris et al., 2002; Skubitz and Skubitz, 2003) and cellular retinol-binding protein 1 (CRBP1) (Vanharanta et al., 2005; Zaitseva et al., 2006). Other than microarray studies, there are limited and sometimes contradictory data on the expression of retinoid pathway genes in normal myometrium and fibroids.
The RA pathway controls a number of biological processes including differentiation, proliferation and apoptosis (Napoli, 1996), and is implicated in the development of several cancers (Lin et al., 2001; Roberts et al., 2002; Farias et al., 2005). Given the evidence of aberrant expression of multiple genes involved in this pathway in fibroids when compared with normal myometrium, it is possible that alterations in RA synthesis and signaling play an important role in fibroid development. The aims of the present study were (i) to confirm and extend findings of microarray studies that expression of genes involved in RA pathway are altered in fibroids when compared with myometrium and (ii) to examine mRNA and protein expression and cellular localization of the members of the RA pathway in human myometrium and fibroids. We investigated mRNA expression for three enzymes involved in RA synthesis: ADH1, ALDH1 and RODH (Duester, 2000; Duester et al., 2003); cytochrome P450, family 26 subfamily A (CYP26A), which inactivates RA by selectively metabolizing it into polar metabolites (White et al., 1997; Haque and Anreola, 1998); two binding proteins: CRBP1 that presents retinol to the enzymes that are capable of RA synthesis, and CRABP2 that directs RA into the nucleus to the nuclear receptors (Dong et al., 1999; Delva et al., 1999; Noy, 2000) and six retinoid receptors: RA receptors (RAR, -ß, -) and retinoid X receptors (RXR, -ß, -). In addition, we investigated protein expression and cellular localization for four of these genes, ADH1, ALDH1, CRBP1 and CRABP2.
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Materials and Methods |
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Tissue collection
Human myometrial and fibroid tissue was obtained from 18 premenopausal women not taking exogenous hormones for at least 3 months prior to surgery (mean age 45 years, range 35–53), undergoing hysterectomy for fibroids. Informed consent was obtained from each patient and ethical approval was obtained from Southern Health Human Research and Ethics Committee B. Myometrium was sampled at least 2 cm away from the fibroid. For fibroids, the outside layer was removed and tissue was taken from the body of the tumor, trying to avoid the central region. When multiple fibroids were present in the same uterus, a sample was taken from the biggest tumor. Further information on samples used is provided in Table 1. Tissue was snap frozen on dry ice immediately after excision and stored at –80°C until RNA and protein extraction, or fixed with 10% formalin for immunohistochemistry. All samples were also routinely examined by a pathologist. Cycle stage was determined by endometrial dating. Fibroid samples were stored and processed separately from adjacent normal (host) myometrium.
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RNA and protein extraction
Total RNA and protein were extracted from cells and tissue samples using TrizolTM reagent (Invitrogen, Carlsbad, CA, USA) by following the manufacturer's protocol. RNA was further purified from the aqueous phase using RNeasy columns with on-column DNase I treatment (Qiagen, Doncaster, Victoria, Australia). The resulting RNA was ethanol precipitated and re-suspended in RNase-free water. The concentration and quality of resulting RNA was assessed using a UV spectrophotometer absorbance ratio of 260–280 nM (A260/280). RNA was stored at –80°C until further use. After removal of RNA in the aqueous phase and DNA in the interphase, protein was precipitated from the remaining organic phase. Resulting protein pellets were re-suspended in 1% sodium dodecyl sulfate (SDS) solution. Protein quantification was performed using the BCA protein assay (Pierce Biotech, Rockford, USA). Protein samples were stored at –80°C till further use.
Reverse transcription and real-time quantitative RT-PCR (RT-qPCR)
One microgram of DNase-treated RNA was mixed with 1 µl random primers (3 µg/µl, Invitrogen), 2 µl 10 mM dNTPs (Roche, Australia), 4 µl 5x RT buffer (Roche), 0.5 µl RNAsin (Promega, Madison, WI, USA), 2 µl 0.1 M DTT (Invitrogen) and 0.2 µl AMV-RT (Roche, Basel, Switzerland) and incubated at 42°C for 1 h.
A Corbett real-time PCR machine and Roche Light Cycler with LC fast-start DNA master SYBR green kit (Roche) were used to perform real-time QPCR according to the manufacturer's instructions. The primer sequences used are shown in Table 2. Relative mRNA levels for each of the genes were determined using specific cDNA standards. All results were normalized using 18S RNA as a housekeeping gene to correct for differences in concentration of the starting template.
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Western blotting
Twenty to thirty micrograms of protein from myometrial and fibroid tissues were subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Reduced protein samples were run on NuPage® Novex 10% Bis-Tris gels in MES SDS running buffer. The proteins were electrophoretically transferred to nitrocellulose membranes (Invitrogen). Membranes were washed twice with water and incubated for 1 h with blocking solution [this and all other reagents used for western blotting are from the WesternBreeze® chemiluminescent kit (Invitrogen)], followed by primary antibodies for CRBP1 (1:200), CRABP2 (1:200), ADH1 (1:200) and ALDH1 (1:200) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Membranes were washed, incubated for 30 min with secondary antibody and washed again. Signal was developed using CDP-Star® chemiluminescent substrate. Membranes were exposed to X-ray films (Amersham Biosciences, Buckinghamshire, UK), films were scanned and bands quantified by densitometry using Quantity One software (BioRad, CA, USA). An extra gel was run in parallel and probed with ß-actin antibody (1:4000) (Sigma-Aldrich, Castle Hill, NSW, Australia) for use as a loading control.
Immunohistochemistry
Formalin-fixed paraffin-embedded specimens were cut into 3 µ sections, mounted on slides, dewaxed and dehydrated. Endogenous peroxidase activity was blocked by incubation with 3% H2O2 in methanol for 10 min. Tissue sections were washed with phosphate-buffered saline (PBS), blocked with protein blocking agent (PBA) (Immunon Shandon, PA, USA) for 10 min and incubated with CRBP1, CRABP2, ADH1 or ALDH1 goat polyclonal antibody (all from Santa Cruz) 1:200 dilution in Tris-buffered saline/1% bovine serum albumin (TBS/BSA) overnight at 4°C. Goat IgG at equivalent concentration (1 µg/ml) was used as a negative control. Sections were washed and incubated with horse-radish peroxidase (HRP)-conjugated anti-goat secondary antibody (1:100) (Zymed, San Francisco, CA) for 30 min at room temperature, followed by ABC Vectastain kit (Vector laboratories, Burlingame, CA, USA) for 5 min. Chromogen was developed using SIGMAFASTTM 3,3'-Diaminobenzidine (Sigma) for 5 min. Sections were washed with water and counterstained with Harris hemotoxylin (Amber Scientific, Western Australia, Australia) to allow nuclei visualization.
Double immunostaining for ALDH1–smooth muscle actin (SMA), ALDH1–vimentin and vimentin–SMA was also performed on formalin-fixed sections. For ALDH1–SMA, slides were incubated with 3% H2O2 in methanol followed by block with PBA, and then incubated with SMA antibody (Dako, Carpentaria, CA, USA) 1:400 for 1 h at room temperature. Sections were further incubated with biotinylated secondary antibody for 15 min, followed by streptavidin-alkaline phosphatase (AP) for 15 min, using reagents from LSAB + AP kit (Dako). Vector blue (Vector laboratories) was used as a chromogen. Slides were then washed, blocked with PBA and stained for ALDH1 as described above with the exception of nuclear counterstain, which was not performed for all double stains. For ALDH1–vimentin double stain, slides were stained for ALDH1 as described above, followed by antigen retrieval by boiling slides in 0.01 M citrate buffer for 15 min. Further staining was performed using EnVisionTM G/2 double stain system (Dako) steps 5–8 and anti-vimentin antibody (1:50) (Dako) for 1 h at room temperature. Vector blue was used as a chromogen. For vimentin–SMA staining, antigen retrieval was performed by boiling slides in citrate buffer, followed by 3% H2O2 and PBA, and then incubated with SMA antibody 1:400 for 1 h. Sections were further incubated with biotinylated secondary antibody for 15 min, followed by streptavidin-HRP for 15 min, using reagents from LSAB + HRP kit (Dako) and visualized using AEC chromogen (Zymed). Slides were then stained with vimentin as described above.
Staining intensity was assessed visually by a single observer with limited rigor since protein expression was quantified by western blotting. However, differences for staining intensity were recorded as observed.
Statistical analysis
RT-qPCR and western blot data were analysed using Excel and Graph Pad PrizmTM software (version 4.03, Graph Pad software, CA, USA). All data were normalized using loading control (18S RNA for RT-qPCR and ß-actin for western blot). mRNA and protein expression between myometrium and fibroids were compared using Wilcoxon matched pairs test.
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mRNA expression of CRBP1, CRABP2, ADH1, ALDH1, RODH and CYP26A1
Gene expression profiles of myometrium and fibroid were analysed by real-time PCR. Figure 1 shows mRNA expression levels for six genes involved in the RA pathway: ADH1 (Fig. 1A), ALDH1 (Fig. 1B), RODH (Fig. 1C), CYP26A1 (Fig. 1D), CRBP1 (Fig. 1E) and CRABP2 (Fig. 1F). Apart from CYP26A, all these genes showed differential expression between myometrium and fibroids. Two genes, CRABP2 (P = 0.0098) and RODH (P = 0.027), were up-regulated in fibroids, whereas, three genes, CRBP1 (P = 0.007), ADH1 (P = 0.001) and ALDH1 (P = 0.001), were down-regulated. There were no differences in expression levels between proliferative and secretory stages of the menstrual cycle for any of the genes examined.
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Retinoid receptor expression in myometrium and fibroids
Data showing mRNA expression levels for RA receptors (RAR
, -ß and -; Fig. 2A–C) and RX receptors (RXR , -ß and -; Fig. 2D–F) were generated using real-time PCR. RXR and levels were down-regulated in fibroids when compared with paired myometrium (RXR: P = 0.014; RXR P = 0.001). No differences were detected in expression levels between myometrium and fibroids for RAR , ß and and for RXRß. There were no significant differences between proliferative and secretory stages of the menstrual cycle.
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Protein expression and localization
Alcohol dehydrogenase 1
Western blotting with ADH1 antibody detected a 46 kDa band in all myometrial and fibroid samples (Fig. 3A). Significantly higher levels of ADH1 protein were observed in myometrial than fibroid samples (P = 0.0098), confirming mRNA data. ADH1 protein cytoplasmic imunolocalization occurred in myometrial and fibroid SMC, vascular SMC (VSMC) and endothelial cells (EC) (Fig. 4A and B). Myometrial SMC displayed stronger immunostaining when compared with paired fibroid samples, but variable staining was observed between different samples. Variable staining intensity was observed between different muscle fibers present in the same sample. Strongest staining was observed in vascular cells, and this staining was similar between myometrial and fibroid tissue. Connective tissue was negative for ADH1.
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Aldehyde dehydrogenase 1
Western blotting with ALDH1 detected a 55 kDa band in both myometrial and fibroid samples (Fig. 3B). Densiometry analysis of data showed that there were higher levels of ALDH1 protein present in myometrium as compared to fibroids (P = 0.002). ALDH1 protein displayed cytoplasmic staining and was localized to various cell types in both tissues (Fig. 5A and B). Diffuse cytoplasmic staining with weak to moderate intensity was observed in myometrial and fibroid SMC. Similar to ADH1, stronger staining was observed around some, but not all blood vessels. Very strong staining was observed in cells that were associated with connective tissue. Even though this intense staining was present in both tissue types, the morphology of immunostained cells was markedly different between these tissues. In myometrium, the staining was concentrated around blood vessels and in between muscle fibers in cells with the appearance of fibroblasts. In fibroids, this pattern was not present, and stained cells appeared round or ovoid with staining often concentrated around the nucleus. To further investigate these intensely stained cells, we co-stained ALDH1 with
-SMA as a SMC marker and (Fig. 5C and D), vimentin as a fibroblast marker (Fig. 5E), as well as vimentin with -SMA (Fig. 5F). The results demonstrate that intense ALDH1 staining in both tissues was associated with some but not all vimentin-positive fibroblasts. Our results also demonstrate that -SMA and vimentin have different cellular distribution, with -SMA being expressed in uterine and vascular SMC, and vimentin in fibroblasts and EC.
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Cellular retinol-binding protein 1
Western blotting with CRBP1 antibody detected a 15 kDa band in all myometrial and fibroid samples (Fig. 3C). Significantly higher levels of CRBP1 protein were observed in myometrial than in fibroid samples (P = 0.049), confirming mRNA data. CRBP1 protein displayed cytoplasmic staining and was localized to myometrial and fibroid SMC, VSMC and EC of some, but not all blood vessels (Fig. 4C and D). Myometrial samples mostly showed stronger staining when compared with paired fibroids, but variable staining was observed between different samples. Variable intensity of staining was observed between different muscle fibers present in the same sample. Strong staining was observed in VSMC. Connective tissue was negative.
Cellular retinoic acid-binding protein 2
Western blotting with CRABP2 antibody detected a 15 kDa band in all myometrial and fibroid samples (Fig. 3D). Significantly lower levels of CRABP2 protein were observed in myometrial than in fibroid samples (P = 0.0195), confirming mRNA data. CRABP2 protein displayed mostly cytoplasmic staining, but some nuclear staining was also observed (Fig. 4E and F). CRABP2 was localized to myometrial and fibroid SMC, VSMC and EC. Fibroid samples showed stronger staining when compared with paired myometrium. Strong staining was observed in the vasculature.
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Discussion |
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The current study is a comprehensive investigation of the major RA pathway genes in both myometrium and fibroids. This study systematically evaluated mRNA and protein expression, as well as cellular localization, of multiple genes involved in the RA pathway in these tissues, and investigate gene expression of RA and RX receptors in the same tissues. Here we report altered gene expression of ADH1, ALDH1, RODH, CRBP1 and CRABP2 in uterine fibroids when compared with matching myometrium. We also confirm that these differences are maintained at the protein level for ADH1, ALDH1, CRBP1 and CRABP2 and investigate cellular localizations of these proteins. A major finding from this study is the discovery of ALDH1 expressing cells, which are not SMC but vimentin-positive fibroblasts that have a markedly different morphological appearance in myometrium when compared with fibroids. We were able to detect mRNA for RA receptors
, ß, , and RX receptors , ß, , as well as CYP26A in all samples, with significantly higher levels of RXR and in myometrium when compared with fibroids (Fig. 6). The only studies that have reported differential levels of expression for ADH1, ALDH1 and RODH in fibroids compared with myometrium have used gene expression microarrays (Tsibris et al., 2002; Ahn et al., 2003; Skubitz and Skubitz, 2003; Wang et al., 2003; Hoffman et al., 2004). In the present study, we confirm that these genes have altered expression by real-time qPCR, and that these differences are maintained at the protein level for ADH1 and ALDH1. Both ADH1 and ALDH1, which are down-regulated in fibroids, are major players in the conversion of retinol to RA (Molotkov et al., 2002; Molotkov and Duester, 2003). Immunohistochemical studies revealed that both enzymes are expressed in the cytoplasm of myometrial and fibroid SMC, and in both SMC and EC of some blood vessels, indicating that RA synthesis occurs in these cell types. Decreased expression of these enzymes may indicate that RA synthesis and signaling is reduced in fibroids, as has been observed in invasive breast cancer (Triano et al., 2003) and prostate cancer (Kim et al., 2005). The third enzyme that we have investigated in the present study, RODH, was the only enzyme that was up-regulated in fibroids when compared with myometrium. The role of this enzyme in retinol metabolism is still unclear, and it is possible that RODH facilitates conversion of retinal back to retinol to promote retinol storage (Duester, 1996). The fourth enzyme that we have investigated in this study, CYP26A1, belongs to the family of cytochrome P450 enzymes and is responsible for generation of hydroxylated forms of RA (White et al., 1997), which are considered to be elimination forms of RA. We did not detect significant differences in gene expression for CYP26A between myometrium and fibroids, which suggests that the degradation component of RA metabolism is not altered in fibroids when compared with normal myometrium.
A major novel finding from this study was the discovery of strong ALDH1 immunostaining in some SMA negative, vimentin-positive cells, present in the connective tissue between muscle fibers and in between muscle cells inside the muscle fibers. These ALDH1-positive cells had a distinctly different morphological appearance in fibroids when compared with myometrium. The major cell type of the connective tissue is the fibroblast, which typically appear as elongated cells with extended cell processes that show a fusiform or spindle-like shape (Kalluri and Zeisberg, 2006). In contrast to this typical fibroblast morphology in myometrium, ALDH1 immunopositive cells in fibroid were round or ovoid, without cytoplasmic processes. Very little work has been undertaken investigating the cellular composition of fibroids and myometrium. Using human myometrium, it has been possible to successfully isolate and culture human fibroblasts and identify two distinct fibroblastic phenotypes, Thy1+ and Thy1– (Koumas et al., 2001, 2003; Koumas and Phipps, 2002). These distinct subsets vary in their ability to respond to and produce inflammatory cytokines (Koumas et al., 2001); COX-1 and -2 expression and prostaglandin synthesis (Koumas and Phipps, 2002); and their ability to differentiate into myofibroblastic or lipofibroblastic phenotypes (Koumas et al., 2003). Based on the evidence that not all vimentin-positive fibroblasts co-expressed ALDH1, as well as the different morphology for these cells in myometrium versus fibroids, it is possible that different fibroblast subsets are present in different proportions in fibroids when compared with myometrium. In recent years, there has been an increased understanding of the importance of fibroblasts in tumor development (Baglole et al., 2006; Kalluri and Zeisberg, 2006). Fibroblasts are now recognized as active players in creating a microenvironment that supports tumor development. Nothing is known about the role of fibroblasts in fibroid growth.
The findings from the current study confirm for both mRNA and protein the results of microarray and other studies that reported reduced levels of CRBP1 (Vanharanta et al., 2005; Zaitseva et al., 2006) and increased levels of CRABP2 (Tsibris et al., 2002; Skubitz and Skubitz, 2003; Wang et al., 2003; Catherino et al., 2003; Hoffman et al., 2004; Lattuada et al., 2006) in uterine fibroids. Retinol and RA-binding proteins play an important role in regulating retinol storage, RA synthesis and inactivation, as well as facilitating RA signaling (Dong et al., 1999; Delva et al., 1999; Noy, 2000). Aberrant expression of both of these proteins has been reported in a variety of cancers, including endometrial carcinoma (Orlandi et al., 2006), ovarian cancer cells (Roberts et al., 2002) and renal cell carcinoma (Goelden et al., 2005). Down-regulation of CRBP1 in breast cancer cells compromises RAR signaling, which in turn leads to loss of cell differentiation and tumor progression (Farias et al., 2005). It is possible that down-regulation of CRBP1 in fibroids may have similar effect.
RA signals through six retinoid receptors (RAR, -ß, - and RXR, -ß, -). These receptors form RAR–RXR heterodimers upon stimulation by the ligand, and act as inducible nuclear transcription factors to stimulate transcription of numerous genes (Bastien and Rochette-Egly, 2004). Here we report that myometrium and fibroids express decreased levels of RXR and RXR mRNA, with no differences for other receptor subtypes. There is conflicting evidence on retinoid receptor expression in fibroids when compared with myometrium. There has been large variability in results for retinoid receptor expression reported by different studies, some in agreement and some in disagreement with the current study (Tsibris et al., 1999; Lattuada et al., 2006). Different criteria for patient selection and ethnic differences between patient groups may contribute to the different gene expression profiles observed. It has been demonstrated that women of black ethnicity have different expression profiles of several genes, including RAR and RXR, when compared with white, hispanic or asian women (Wei et al., 2006). In the study by Catherino and Malik (2007), out of eight subjects in the study, three were Caucasian and five African American, two were on oral contraceptives and two had undefined stage of the menstrual cycle. Other studies do not provide information with regards to ethnicity (Tsibris et al., 1999; Lattuada et al., 2006), and one study does not state if patients were receiving any exogenous hormones (Tsibris et al., 1999). In the current study, we did not include any African American patients, and none of our patients received exogenous hormones for at least 3 months prior to surgery.
Results from the current study demonstrate alterations in multiple genes that control RA synthesis and transport, as well as in some of the retinoid receptors, in uterine fibroids when compared with myometrium. Most of these genes, including major enzymes, are down-regulated in fibroids when compared with myometrium. It is possible that down-regulation of CRBP1, ADH1 and ALDH1 results in decreased RA synthesis in uterine fibroids, which results in decreased signaling through the retinoid receptors. This, together with down-regulation of RXR and receptor expression, causes alterations in transcription of RA response genes, as well as possible alterations in signaling through other pathways that interact with the retinoid receptors. If this thesis is correct, it could result in decreased cellular differentiation and apoptosis, as well as increased proliferation, which all contribute to fibroid development (Fig. 5). A very recent paper has reported similar but not identical findings to our data for gene expression, as well as decreased levels of all-trans and 9-cis RA in fibroids when compared with myometrium, providing additional evidence that the RA pathway is down-regulated in fibroids when compared with myometrium (Catherino and Malik, 2007). In contrast, others have reported increased levels of atRA in uterine fibroids in the follicular stage of the menstrual cycle, and could not detect any 9-cis RA (Tsibris et al., 1999).
Functional studies are needed to determine if the differences observed here are translated into differential regulation of RA response genes. Limited data is available to suggest that both myometrial and fibroid SMC can respond to retinoids in vitro, and that some of these responses may be different between the two cell types (Boettger-Tong et al., 1997; Broaddus et al., 2004; Mangioni et al., 2005).
In conclusion, in the present study we report that expression of multiple genes that belong to RA pathway, including enzymes, binding proteins and receptors, is altered in fibroids when compared with myometrium. We also demonstrate for the first time that some SMA-negative fibroblast-like cells of the connective tissue strongly express ALDH1, and that these cells have different morphology in myometrium when compared with fibroids. Further studies are warranted to investigate the importance of alterations of genes involved in RA synthesis in fibroid development, and the possible involvement of fibroblasts in this process.
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Acknowledgements |
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Thanks are due to nurses Nancy Taylor and Nicky Sam for the collection of tissue samples and to various gynecological surgeons affiliated with Monash Medical Centre who provided subjects for the study. Thanks are also due to Anna Ponnampalam, Leonie Cann and Fiona Lederman for expert technical advice and Gareth Weston for critical review of the manuscript. This work was supported by a Royal Australian and New Zealand College of Obstetricians and Gynaecologists Members Research Foundation Scholarship to B.V. P.A.W.R. is a Principal Research Fellow of the National Health and Medical Research Council of Australia (NHMRC Fellowship grant # 134063). M.Z. is a holder of an Australian postgraduate award.
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Submitted on February 20, 2007; resubmitted on April 18, 2007; accepted on April 23, 2007.
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