1 Department of Obstetrics & Gynecology, 2 Department of Environmental Medicine, 3 Department of Pathology and 4 Department of Medicine, New York University School of Medicine, New York, NY, USA
5 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, New York University School of Medicine, 550 First Avenue, NBV-9E2, New York, NY 10016, USA. Email: akhmea01{at}med.nyu.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: gene expression/leiomyoma/microarrays/myometrium/uterus
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Despite this major impact on gynaecological morbidity, the aetiology of uterine leiomyoma remains poorly understood. Epidemiological studies suggest that risk is inversely associated with parity, age at menarche, and age at menopause, whereas obesity, late reproductive age, and nulliparity are associated with increased risk (Flake et al., 2003).
A genetic basis of uterine leiomyoma is suggested by several lines of evidence. Compared to other ethnic groups, women of African-American descent are at higher risk of uterine fibroids, which develop at earlier ages, and tend to be larger, more numerous, and more symptomatic (Kjerulff et al., 1996; Marshall et al., 1997
; Baird et al., 2003
). Twin studies have shown that the concordance of hospitalization due to uterine fibroids or hysterectomy was significantly higher in monozygous twins compared to dizygous twins, providing further support for a genetic predisposition (Treloar et al., 1992
; Gross and Morton, 2001
).
Several reports of the familial clustering of uterine fibroids demonstrated that fibroids were 45-fold more common in first degree relatives of women with leiomyoma, compared to the general population (Winkler and Hoffmann, 1938; Kurbanova et al., 1989
; Vikhlyaeva et al., 1995
; Schwartz et al., 2000
). Reed's syndrome, a rare inherited disorder, is characterized by the appearance of multiple leiomyomas in the skin, uterus, or both (Fisher and Helwig, 1963
; Reed et al., 1973
). Recently, reports of several families in Finland and England with multiple uterine and cutaneous fibroids, and papillary renal cell carcinoma, were linked to mutations in the fumarate hydratase gene (Tomlinson et al., 2002
). In addition, mutations in the tuberous sclerosis 2 (TSC2) tumor suppressor gene leading to renal angiomyolipomas, cysts and carcinoma in humans are frequently associated with development of uterine leiomyomas in the Eker rat animal model (Walker et al., 2003
).
Cytogenetic studies have revealed that leiomyomas are monoclonal and 4050% of uterine fibroids are reported to have nonrandom chromosomal abnormalities (Ligon and Morton, 2001
). Common cytogenetic changes include translocation between chromosomes 12 and 14 (20%), deletions in chromosome 7 (17%), trisomy of chromosome 12 (12%), and aberrations of chromosome 6 (5%) (Nilbert and Heim, 1990
; Ligon and Morton, 2001
; Flake et al., 2003
). Based on the results of cytogenetic studies, several gene candidates have been proposed to be involved in the development of uterine leiomyoma. The potential candidates included high mobility group (HMG) genes on chromosomes 12q15 and 6p21, estrogen receptor beta (ESR2) on chromosome 14q22, and RAD51L1 gene on chromosome 14q23 (Nilbert and Heim, 1990
; Ligon and Morton, 2001
). HMG genes (HMGA1 and HMGA2) code for DNA-binding proteins that can induce conformational changes in DNA, thereby indirectly regulating transcription by influencing the access of other DNA-binding proteins to target genes. RAD51L1 is a member of the RAD51 recombination repair gene family and preferential fusion partner of HMGA2 in uterine leiomyoma (Takahashi et al., 2001
). In order to identify other critical genes and potential pathways involved in uterine leiomyoma, several studies have utilized the cDNA microarray screening methods.
![]() |
Microarray studies of uterine leiomyoma |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tsibris et al. (2002) have studied the leiomyoma samples and matched myometrium from nine patients (five African-American, two Caucasians, one Hispanic, one other ethnicity) in the proliferative and secretory phases of the menstrual cycle using the HUGeneFL6800 microarray chip (6800 genes per array) and the AffymetrixTM U95A GeneChip (12 000 genes per array). In the combined analyses, 67 genes were up-regulated and 78 genes were down-regulated by >2-fold in leiomyoma compared to matched myometrium.
Chegini et al. (2003b) used the ClontechTM Atlas microarray of 1176 cancer-related genes to study differential gene expression in the leiomyoma and matched normal myometrium from six patients: three untreated women and three women after GnRH analogue therapy. A total of 100 genes were differentially expressed, 18 up-regulated and 82 down-regulated in untreated leiomyoma compared with normal myometrium. After GnRH analogue treatment, the expression of 34 genes showed a significant decrease in leiomyoma, whereas 27 genes increased and 15 decreased in myometrium compared with their respective untreated tissues. The differentially expressed genes are involved in regulation of cell cycle and growth, signal transduction, transcription factors, and cell structure, although no fold changes were reported (Chegini et al., 2003b
).
Weston et al. used in-house prepared microarrays of 10 500 human cDNA sequences to compare gene expression between leiomyoma/myometrium pairs from 12 hysterectomy specimens. Twenty-five differentially expressed genes were identified (14 up-regulated, 11 down-regulated) in leiomyoma compared to the adjacent myometrium. Several genes were confirmed by RTPCR including overexpressed genes (IGF2, ETRA, COL4A2) and underexpressed angiogenesis-related genes (CTGF, CYR61) (Weston et al., 2003).
Wang et al. (2003) have analysed the leiomyoma and adjacent myometrium obtained from seven patients during the proliferative phase of the menstrual cycle using the HUGeneFL6800 microarrays. A comparison of expression patterns in each paired sample revealed 68 genes significantly different in each paired tissue sample (23 overexpressed and 45 underexpressed genes) (Wang et al., 2003
).
Skubitz and Skubitz (2003) have compared 20 uterine leiomyoma samples with 46 normal myometrial samples and 18 other tissues using the AffymetrixTM U95 GeneChip, containing
12 000 genes and
48 000 expressed sequence tags (EST). Ninety-six known gene fragments (comprising 78 independent genes) and 149 EST were overexpressed at least two-fold and 358 gene fragments/EST were underexpressed
2-fold in the leiomyoma compared to normal myometrium (Skubitz and Skubitz, 2003
).
Ahn et al. (2003) have screened up to 17 000 genes in leiomyoma and myometrium samples of six Korean patients using the GeneTrack Human cDNA HSVC 307 chip (Genomictree, Inc., Korea). A total of 71 differentially expressed genes (21 up-regulated and 50 down-regulated) were identified in this study (Ahn et al., 2003
).
Catherino et al. reported microarray analysis of up to 33 000 genes using the AffymetrixTM U133 platform (Catherino et al., 2003). Real time RTPCR, Western blot, and immunohistochemistry were used to confirm the concordant mRNA and protein overexpression of FZD2 and CD24 but did not confirm DLK overexpression in five patients with leiomyoma. Subsequently, three commonly overexpressed genes (IGF2, CRABP2, CD24) and five underexpressed genes (ADH1, DPT, ATF3, ABCA, PTGER3) were reported by this study (Catherino et al., 2004a
).
Quade et al. reported results of microarray analyses of about 7000 gene probes using Affymetrix HuFl GeneChips in four normal uterine myometria, seven uterine leiomyomas, and nine uterine leiomyosarcomas. A total of 153 probe sets representing 146 unique genes were selected by analysis of variance and included in the cluster analysis, which suggested that the molecular pathways in leiomyoma and leiomyosarcoma are distinct (Quade et al., 2004).
In a recent study, Hoffman et al. have compared tissues of normal myometrium and uterine leiomyoma from eight patients of varying age and race undergoing surgery for symptomatic fibroids. Using Affymetrix U133A GeneChip microarrays, 226 genes were found to be dysregulated by a 1.5-fold change between leiomyoma and normal myometrium. The authors identified several apoptosis-related genes, of particular interest were TRAIL and Ask1/MAP3K5, as well as several proliferation-related genes, including TGFB1, PDGFC, and two dual specificity phosphatases (Hoffman et al., 2004
).
We also have conducted a study to compare the expression of more than 22,000 genes between uterine leiomyoma and normal myometrium, as described below.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gene expression analysis
RNA was isolated from each sample using the Qiagen RNeasy Mini kit (Qiagen, Inc., USA). Sample preparation, hybridization and the array wash procedure were performed as described in the standard protocol provided by AffymetrixTM. In brief, 10 µg total RNA from each sample were used to synthesize double-stranded cDNA with the Superscript Choice System (Invitrogen). The cDNA was used as a template to generate biotinylated cRNA by an in vitro transcription reaction. RNA quality was checked by analysis of the 3':5' ratios for human actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) oligonucleotides on AffymetrixTM Test 3 chips. Fifteen micrograms of cRNA from each sample were added to the U133A array. The hybridization was carried out at 45 °C for 16 h in the AffymetrixTM Hybridization Oven. After sample hybridization, the microarrays were washed and stained with a steptavidin-conjugated fluorescent stain followed by antibody amplification on the AffymetrixTM Fluidics Station 400. Each microarray was scanned twice. The first scanning was performed after steptavidin-conjugated fluorescent staining and the data were used for analyzing expression levels of abundant RNA. The array was scanned again after the whole washing and staining procedure was complete. The expression levels of genes were measured using GeneChip software version 4.01.
Data analysis
The raw data from array scans were normalized by median-centering genes for each array, and log transformed. Additionally, genes with 50% or more of absent scores were filtered out. The remaining 9673 genes were analysed. To identify genes that are differentially expressed in leiomyoma samples compared to normal myometrium, the significance analysis of microarrays (SAM) method was used (Tusher et al., 2001). SAM uses a modified t-test statistic with sample-label permutations to estimate the false discovery rate (FDR). The FDR is defined as the expected proportion of false discoveries among the genes that are declared significant (Benjamini and Hochberg, 1995
; Storey, 2002
; Storey and Tibshirani, 2003
). The FDR is used to determine the significance threshold for genes and to limit the likelihood of type I error taking into account the fact that thousands of genes are simultaneously being tested.
In addition to the significance analysis of microarrays, fold-change analysis was performed, in which the ratios of geometric means of the expression intensities of the corresponding genes in leiomyoma samples relative to normal myometrium were calculated. The ratios were reported as the up- or down-fold change. To select the potentially important genes, we used threshold values of 2 and
2 in the fold change between leiomyoma and normal myometrium and the FDR significance level of <5%.
To compare the data on gene expression provided by different microarray studies of uterine leiomyoma, we have carefully examined the published lists of differentially expressed genes and identified the overlapping genes in two or more separate studies.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genes up-regulated in leiomyoma
Table I presents the 14 genes up-regulated, on average, by more than two-fold (FDR <5%) in leiomyoma relative to matched myometrium in the three patients. The up-regulated genes included growth factors (IGF2, MEST, NEGF2), factors involved in extracellular matrix formation (MMP11, CSPG2), factors involved in angiogenesis (TMSNB, SFRP1), and genes involved in cell differentiation and metabolism (CD24, HTR2B, QPRT, GAGEC1, PTK7, PEMT).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genes associated with retinoid metabolism
In agreement with our results, significant underexpression of ADH1 and ALDH1 in uterine leiomyoma compared to normal myometrium was one of the most common observations of gene array studies to date (Tsibris et al., 2002; Wang et al., 2003
; Skubitz and Skubitz, 2003
; Ahn et al., 2003
; Catherino et al., 2003
; Catherino et al., 2004a
; Quade et al., 2004
; Hoffman et al., 2004
). ADH1 and ALDH1 oxidize retinol to retinaldehyde and retinaldehyde to retinoic acid, respectively. Retinoic acid functions as a ligand controlling nuclear retinoic acid receptor (RAR) and retinoid X receptor (RXR) signaling pathways (Pfahl and Chytil, 1996
; Napoli, 1999
).
Direct measurements of tissue levels of all-trans retinoic acid were shown to be increased in leiomyoma compared to normal myometrium, but only in the proliferative phase of the cycle (Tsibris et al., 1999). This may be a result of estrogen-dependent up-regulation of short-chain retinol dehydrogenase (RODH) (Napoli, 2001
; Rexer and Ong, 2002
), which is also overexpressed in uterine leiomyoma (Tsibris et al., 2002
; Skubitz and Skubitz, 2003
).
Down-regulation of class I ADH may result either in response to increased levels of all-trans RA in leiomyoma cells by negative feed-back mechanism or via overexpression of high-mobility group (HMGA1, HMGA2) genes (Figure 1). HMGA1 and HMGA2 are located respectively, on chromosomes 6p21 and 12q15, which are commonly rearranged in leiomyoma and in a variety of other mesenchymal tumours (Reeves, 2000). HMG proteins competitively bind to ADH class I HNF-1/HMGI promoter, affecting ADH1 gene transcription (Edenberg, 2000
).
|
Tsibris et al. had shown that human leiomyoma express higher levels of all-trans RA, RXR, and PPAR, and that sustained exposures to estrogen, PPAR
ligand (troglitazone) and all-trans RA were required for uterine leiomyoma growth in guinea pig in vivo model (Tsibris et al., 1999
). Based on available data, we conclude that in uterine leiomyoma there is a co-requirement of increased estrogens (via aromatase) and retinoic acid (via up-regulated retinol/retinaldehyde dehydrogenase) that sustains tumour growth.
Genes related to growth and proliferation
Up-regulation of IGF2 is one of the most consistent observations of gene array studies to date (Table III). IGF2 acts mainly through the IGF receptor type I (IGF1R), whereas IGF receptor type II (IGF2R) is involved with IGF2 degradation. Previously, IGF2 mRNA, but not its receptor levels, had been reported to be higher in the leiomyoma tissues compared to the myometrium (Hoppener et al., 1988; Boehm et al., 1990
; Vollenhoven et al., 1993
). Similar to IGF1, IGF2 is involved in proliferation, differentiation, and transformation in a wide variety of cell types. Effects of IGF2 are modulated by IGF-binding proteins (Stewart and Rotwein, 1996
). Six IGFBP with high affinity for IGFs have been identified and an additional four IGFBP-related proteins have been described (Kim et al., 1997
; Hwa et al., 1999
).
Interestingly, we and at least six other groups observed that genes encoding several IGF-binding proteins were significantly down-regulated in leiomyoma compared to normal myometrium, including IGFBP6 (Tsibris et al., 2002; Skubitz and Skubitz, 2003
; Hoffman et al., 2004
), CYR61/IGFBP10 (Sampath et al., 2001
; Tsibris et al., 2002
; Weston et al., 2003
; Ahn et al., 2003
; Hoffman et al., 2004
; Quade et al., 2004
), and CTGF (Ahn et al., 2003
; Weston et al., 2003
; Hoffman et al., 2004
).
IGFBP6 is of particular interest because its affinity for IGF2 is 20100-fold greater than that for IGF1 (Roghani et al., 1991; Bach, 1999
) indicating that it may serve as IGF2-specific binding protein, modulating IGF2 action. IGFBP6 expression is stimulated by all-trans RA in every cell model investigated thus far (Sheikh et al., 1993
; Martin et al., 1994
; Zhou et al., 1996
; Babajko and Binoux, 1996
; Gabbitas and Canalis, 1996
; Chambery et al., 1998
; Sueoka et al., 2000
; Kim et al., 2002
), while stromelysin 3 (MMP11) degrades IGFBPs (Fowlkes et al., 1999
). Therefore, substantial down-regulation of IGFBP6, as a result of decreased retinoid signaling or increased MMP11 expression, may lead to an increased bioavailability of IGF2 and further activation of the IGF2 autocrine loop, promoting growth of uterine leiomyoma.
In addition, some of the effects of RA may be independent of the action via the RAR and RXR nuclear receptors and involve direct binding to the IGF2R leading to an intracellular redistribution of the IGF2R and lysosomal enzymes (Kang et al., 1997, Kang et al., 1998
). Several studies have suggested that IGF2R is a tumor suppressor gene, which loss or mutation thereof had been implicated in various tumors (De Souza et al., 1995
; Hankins et al., 1996
; Ouyang et al., 1997
; Xu et al., 1997
). We hypothesize that reduction or loss of IGF2R and reduced expression of IGFBPs with high affinity to IGF2, may lead to overexpression of IGF2 and uterine leiomyoma growth.
Figure 1 illustrates a potential pathway of uterine leiomyoma development based on the data generated by the gene array studies. The most common findings suggest that interaction between the retinoid-related genes (RODH, ADH1, ALDH1, CRABP2) and IGF metabolism-related genes (IGF2, IGFBP6, CYR61/IGFBP10, MMP11) may play a significant role in uterine leiomyoma development.
Genes associated with differentiation
Leiomyoma cells are characterized by profound changes in cell differentiation. CD24 is commonly expressed by lymphoid cells, neuroblasts, certain cancer cells, as well as by regenerating muscle (Figarella-Branger et al., 1993) and had been correlated with a poor state of cell differentiation (Huang and Hsu, 1995
; Catherino et al., 2004a
). CD24 and its signal transducer (CD24ST) have been consistently reported to be up-regulated in uterine leiomyoma (Tsibris et al., 2002
; Catherino et al., 2003
; Skubitz and Skubitz, 2003
; Weston et al., 2003
). It has been proposed that CD24 overexpression is an important marker of a fundamental alteration in leiomyoma cell differentiation (Catherino et al., 2004a
).
Prominent among consistently up-regulated genes in the different studies were doublecortin (Tsibris et al., 2002; Ahn et al., 2003
; Skubitz and Skubitz, 2003
; Weston et al., 2003
; Hoffman et al., 2004
) and ionotropic glutamate receptor 2 (Tsibris et al., 2002
; Skubitz and Skubitz, 2003
; Wang et al., 2003
; Weston et al., 2003
; Hoffman et al., 2004
; Quade et al., 2004
). Doublecortin (DCX) was originally identified in studies of patients with defective cortical neuronal migration (lissencephaly) (des Portes et al., 1998
; Gleeson et al., 1998
). DCX functions as a microtubule-associated protein and contains two internal tandem repeats. While each repeat separately binds tubulin, both are required to bind concomitantly for microtubule polymerization (Taylor et al., 2000
).
Ionotropic glutamate receptor 2 (GRIA2) is a subunit of a ligand-gated cation channel and has been reported to be overexpressed in leiomyoma relative to myometrium up to 30-fold (Tsibris et al., 2002; Skubitz and Skubitz, 2003
; Wang et al., 2003
; Weston et al., 2003
; Hoffman et al., 2004
; Quade et al., 2004
). GRIA2 may play a role in local angiogenesis by increasing Ca2+ influx into leiomyoma and supporting vascularization (Tsibris et al., 2003
). In leiomyoma, a synergism between increased production of estradiol and all-trans retinoic acid with up-regulation of nuclear receptors PPAR
and RXR had been proposed (Tsibris et al., 1999
). GRIA2 might be coupled to this synergism directly or via interleukin 17 (IL17) and kinesin family 5 (KIF5C) genes (Tsibris et al., 2003
), both of which had been reported to be up-regulated in leiomyoma (Ahn et al., 2003
; Skubitz and Skubitz, 2003
; Tsibris et al., 2003
).
Among consistently down-regulated genes in leiomyoma were tryptase beta 2 (TPSB2) and carboxypeptidase A3 (CPA3), which are enzymes produced by mast cells. Decreased expression of these enzymes is consistent with a less differentiated state and may indicate a reduction in the number or function of mast cells in uterine leiomyoma, as previously suggested (Tsibris et al., 2002).
Other commonly down-regulated groups of genes include transcription factors (ATF3, GATA2, JUN, FOS), factors involved in lipid metabolism (APM2, ABCA, APOD), cell integrity proteins (KRT19, ABLIM, EFEMP1, FY, EMP1), nucleic acid metabolism (GBP2, RNASE4), and complement factors (CFH, C7) (Table III).
Genes related to the extracellular matrix
Extracellular matrix is thought to play an important role in the pathophysiology of uterine leiomyoma (Sozen and Arici, 2002). Overproduction of the ECM proteins contributes extensively to leiomyoma volume expansion. ECM is composed predominantly of collagens, proteoglycans, and matrix glycoproteins. In addition to its role in cell shape, proliferation, and differentiation, ECM serves as a repository for growth factors and cytokines and mediates their activation and turnover (Sozen and Arici, 2002
).
The proteoglycan, dermatopontin (DPT), was one of the most commonly down-regulated genes in our and previous gene array studies (Tsibris et al., 2002; Ahn et al., 2003
; Wang et al., 2003
; Catherino et al., 2004a
,b). DPT interacts with decorin for TGF-
binding and plays an important role in cell-matrix interactions and matrix assembly (Kuroda et al., 1999
; Okamoto et al., 1999
). It has been proposed that down-regulation of DPT would indirectly increase decorin function, leading to disruption of the processing of collagen fibrils (Ichii et al., 2001
; Catherino et al., 2004b
) and activation of TGF-
signaling pathway (Riquelme et al., 2001
). Factors decreasing the expression of DPT include collagen type I (Kuroda et al., 1999
), which had been shown to be overexpressed in leiomyoma (Stewart et al., 1994
). Reduction of DPT and increased TGF-
3 signalling have been proposed as a molecular link between uterine leiomyoma and keloid formation (Catherino et al., 2004b
).
The TGF-3-coding gene is located near the 14q2324 break-point, which is one of the most common translocation sites reported by cytogenetic studies of uterine fibroids (Andersen, 1998
). Compared to normal myometrium, TGF-
3 expression is 3.55-fold higher in leiomyoma (Arici and Sozen, 2000
; Lee and Nowak, 2001
; Tsibris et al., 2002
; Quade et al., 2004
). In addition, the expression of SMAD-3 and -4, intracellular proteins that transmit TGF-
receptor signals, is increased in leiomyoma compared to normal myometrium (Chegini et al., 2003a
). TGF-
proteins are potent inducers of collagens I, III and fibronectin (Ignotz and Massague, 1986
).
Extracellular matrix is regulated by the combined action of matrix metalloproteinases (MMP) and their inhibitors, tissue inhibitors of MMPs (TIMPs). It has been shown that both leiomyoma and myometrium express higher levels of MMP and TIMP mRNA during the secretory phase of the cycle compared to the proliferative phase, suggesting that MMP and TIMP expression in leiomyoma and myometrium are hormonally regulated (Dou et al., 1997). Palmer et al. have shown that the levels of MMP1 and MMP3 mRNA were similar in leiomyoma and myometrium, whereas stromelysin 3 (MMP11) was significantly elevated in uterine leiomyoma suggesting that stromelysin 3 may be involved in the formation of more fibrous extracellular matrix compared to unaffected myometrium (Palmer et al., 1998
).
Figure 2 shows the potential relationships between TGF-3, induction of collagens types I, III, and IV and down-regulation of dermatopontin (DPT). In this model, TGF-
overexpression may induce tissue inhibitor of metalloproteinase (TIMP1) (Ma and Chegini, 1999
), thus shifting MMP/TIMP balance and further promoting matrix accumulation.
|
TGF-3 and MMP11 may represent the common molecules shared by the pathways described in Figures 1 and 2. The proposed pathways point to an important role of the extracellular matrix expansion in uterine leiomyoma. We hypothesize that the ECM accumulation and shift in MMP/TIMP balance may in part explain such key features of leiomyoma as local expansion and lack of invasion.
In summary, despite the substantial differences in patients characteristics, laboratory methods and analytical approaches, the results from gene expression studies point to several themes essential for uterine leiomyoma development. The consistently observed changes in genes regulating retinoid synthesis, IGF-II metabolism, TGF- signaling and ECM formation indicate the potential importance of these factors in the development of leiomyoma. Ongoing and future research should clarify whether the commonly observed changes in gene expression correspond to protein alterations in uterine leiomyoma. Understanding the common molecular pathways of uterine leiomyoma initiation and progression is critical for development of novel strategies for treatment and prevention of this common condition.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andersen J (1998) Factors in fibroid growth. Baillieres Clin Obstet Gynaecol 12, 225243.[ISI][Medline]
Arici A and Sozen I (2000) Transforming growth factor-beta3 is expressed at high levels in leiomyoma where it stimulates fibronectin expression and cell proliferation. Fertil Steril 73, 10061011.[CrossRef][ISI][Medline]
Arici A and Sozen I (2003) Expression, menstrual cycle-dependent activation, and bimodal mitogenic effect of transforming growth factor-beta1 in human myometrium and leiomyoma. Am J Obstet Gynecol 188, 7683.[CrossRef][ISI][Medline]
Babajko S and Binoux M (1996) Modulation by retinoic acid of insulin-like growth factor (IGF) and IGF binding protein expression in human SK-N-SH neuroblastoma cells. Eur J Endocrinol 134, 474480.[Abstract]
Bach LA (1999) Insulin-like growth factor binding protein-6: the "forgotten" binding protein? Horm Metab Res 31, 226234.[ISI][Medline]
Baird DD, Dunson DB, Hill MC, Cousins D and Schectman JM (2003) High cumulative incidence of uterine leiomyoma in black and white women: ultrasound evidence. Am J Obstet Gynecol 188, 100107.[CrossRef][ISI][Medline]
Benjamini Y and Hochberg Y (1995) Controlling the false discovery rate a practical and powerful approach to multiple testing. J R Stat Soc Ser B Stat Methodol 57, 289300.
Boehm KD, Daimon M, Gorodeski IG, Sheean LA, Utian WH and Ilan J (1990) Expression of the insulin-like and platelet-derived growth factor genes in human uterine tissues. Mol Reprod Dev 27, 93101.[ISI][Medline]
Budhu A, Gillilan R and Noy N (2001) Localization of the RAR interaction domain of cellular retinoic acid binding protein-II. J Mol Biol 305, 939949.[CrossRef][ISI][Medline]
Buttram VC and Reiter RC (1981) Uterine leiomyomata: etiology, symptomatology, and management. Fertil Steril 36, 433445.[ISI][Medline]
Catherino WH, Prupas C, Tsibris JC, Leppert PC, Payson M, Nieman LK and Segars JH (2003) Strategy for elucidating differentially expressed genes in leiomyomata identified by microarray technology. Fertil Steril 80, 282290.[ISI][Medline]
Catherino W, Salama A, Potlog-Nahari C, Leppert P, Tsibris J and Segars J (2004a) Gene expression studies in leiomyomata: new directions for research. Semin Reprod Med 22, 8390.[CrossRef][ISI][Medline]
Catherino WH, Leppert PC, Stenmark MH, Payson M, Potlog-Nahari C, Nieman LK and Segars JH (2004b) Reduced dermatopontin expression is a molecular link between uterine leiomyomas and keloids. Genes Chromosomes Cancer 40, 204217.[CrossRef][ISI][Medline]
Chambery D, de Galle B and Babajko S (1998) Retinoic acid stimulates IGF binding protein (IGFBP)-6 and depresses IGFBP-2 and IGFBP-4 in SK-N-SH human neuroblastoma cells. J Endocrinol 159, 227232.
Chegini N, Luo X, Ding L and Ripley D (2003a) The expression of Smads and transforming growth factor beta receptors in leiomyoma and myometrium and the effect of gonadotropin releasing hormone analogue therapy. Mol Cell Endocrinol 209, 916.[CrossRef][ISI][Medline]
Chegini N, Verala J, Luo X, Xu J and Williams RS (2003b) Gene expression profile of leiomyoma and myometrium and the effect of gonadotropin releasing hormone analogue therapy. J Soc Gynecol Investig 10, 161171.[CrossRef][ISI][Medline]
Coronado GD, Marshall LM and Schwartz SM (2000) Complications in pregnancy, labor, and delivery with uterine leiomyomas: a population-based study. Obstet Gynecol 95, 764769.
Cramer SF and Patel A (1990) The frequency of uterine leiomyomas. Am J Clin Pathol 94, 435438.[ISI][Medline]
De Souza AT, Hankins GR, Washington MK, Orton TC and Jirtle RL (1995) M6P/IGF2R gene is mutated in human hepatocellular carcinomas with loss of heterozygosity. Nat Genet 11, 447449.[ISI][Medline]
Delva L, Bastie JN, Rochette-Egly C, Kraiba R, Balitrand N, Despouy G, Chambon P and Chomienne C (1999) Physical and functional interactions between cellular retinoic acid binding protein II and the retinoic acid-dependent nuclear complex. Mol Cell Biol 19, 71587167.
des Portes V, Pinard JM, Billuart P, Vinet MC, Koulakoff A, Carrie A, Gelot A, Dupuis E, Motte J, Berwald-Netter Y et al. (1998) A novel CNS gene required for neuronal migration and involved in X-linked subcortical laminar heterotopia and lissencephaly syndrome. Cell 92, 5161.[ISI][Medline]
Dong D, Ruuska SE, Levinthal DJ and Noy N (1999) Distinct roles for cellular retinoic acid-binding proteins I and II in regulating signaling by retinoic acid. J Biol Chem 274, 2369523698.
Dou Q, Tarnuzzer RW, Williams RS, Schultz GS and Chegini N (1997) Differential expression of matrix metalloproteinases and their tissue inhibitors in leiomyomata: a mechanism for gonadotrophin releasing hormone agonist-induced tumour regression. Mol Hum Reprod 3, 10051014.[Abstract]
Edenberg HJ (2000) Regulation of the mammalian alcohol dehydrogenase genes. Prog Nucleic Acid Res Mol Biol 64, 295341.[ISI][Medline]
Evanko SP, Johnson PY, Braun KR, Underhill CB, Dudhia J and Wight TN (2001) Platelet-derived growth factor stimulates the formation of versican-hyaluronan aggregates and pericellular matrix expansion in arterial smooth muscle cells. Arch Biochem Biophys 394, 2938.[CrossRef][ISI][Medline]
Farquhar CM and Steiner CA (2002) Hysterectomy rates in the United States 19901997. Obstet Gynecol 99, 229234.
Figarella-Branger D, Moreau H, Pellissier JF, Bianco N and Rougon G (1993) CD24, a signal-transducing molecule expressed on human B lymphocytes, is a marker for human regenerating muscle. Acta Neuropathol (Berl) 86, 275284.[CrossRef][ISI][Medline]
Fisher WC and Helwig EB (1963) Leiomyomas of the skin. Arch Dermatol 88, 510520.[ISI][Medline]
Flake GP, Andersen J and Dixon D (2003) Etiology and pathogenesis of uterine leiomyomas: a review. Environ Health Perspect 111, 10371054.[ISI][Medline]
Fowlkes JL, Serra DM, Nagase H and Thrailkill KM (1999) MMPs are IGFBP-degrading proteinases: implications for cell proliferation and tissue growth. Ann N Y Acad Sci 878, 696699.
Gabbitas B and Canalis E (1996) Retinoic acid stimulates the transcription of insulin-like growth factor binding protein-6 in skeletal cells. J Cell Physiol 169, 1522.[CrossRef][ISI][Medline]
Gleeson JG, Allen KM, Fox JW, Lamperti ED, Berkovic S, Scheffer I, Cooper EC, Dobyns WB, Minnerath SR, Ross ME et al. (1998) Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein. Cell 92, 6372.[ISI][Medline]
Gross KL and Morton CC (2001) Genetics and the development of fibroids. Clin Obstet Gynecol 44, 335349.[CrossRef][ISI][Medline]
Hankins GR, de Souza AT, Bentley RC, Patel MR, Marks JR, Iglehart JD and Jirtle RL (1996) M6P/IGF2 receptor: a candidate breast tumor suppressor gene. Oncogene 12, 20032009.[ISI][Medline]
Hoffman PJ, Milliken DB, Gregg LC, Davis RR and Gregg JP (2004) Molecular characterization of uterine fibroids and its implication for underlying mechanisms of pathogenesis. Fertil Steril 82, 639649.[CrossRef][ISI][Medline]
Hoppener JW, Mosselman S, Roholl PJ, Lambrechts C, Slebos RJ, Pagter-Holthuizen P, Lips CJ, Jansz HS and Sussenbach JS (1988) Expression of insulin-like growth factor-I and -II genes in human smooth muscle tumours. EMBO J 7, 13791385.[Abstract]
Huang LR and Hsu HC (1995) Cloning and expression of CD24 gene in human hepatocellular carcinoma: a potential early tumor marker gene correlates with p53 mutation and tumor differentiation. Cancer Res 55, 47174721.[Abstract]
Hwa V, Oh Y and Rosenfeld RG (1999) The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocr Rev 20, 761787.
Ichii T, Koyama H, Tanaka S, Kim S, Shioi A, Okuno Y, Raines EW, Iwao H, Otani S and Nishizawa Y (2001) Fibrillar collagen specifically regulates human vascular smooth muscle cell genes involved in cellular responses and the pericellular matrix environment. Circ Res 88, 460467.
Ignotz RA and Massague J (1986) Transforming growth factor-beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J Biol Chem 261, 43374345.
Janat MF and Liau G (1992) Transforming growth factor beta 1 is a powerful modulator of platelet-derived growth factor action in vascular smooth muscle cells. J Cell Physiol 150, 232242.[CrossRef][ISI][Medline]
Kang JX, Li Y and Leaf A (1997) Mannose-6-phosphate/insulin-like growth factor-II receptor is a receptor for retinoic acid. Proc Natl Acad Sci USA 94, 1367113676.
Kang JX, Bell J, Leaf A, Beard RL and Chandraratna RA (1998) Retinoic acid alters the intracellular trafficking of the mannose-6-phosphate/insulin-like growth factor II receptor and lysosomal enzymes. Proc Natl Acad Sci USA 95, 1368713691.
Kim EJ, Kang YH, Schaffer BS, Bach LA, MacDonald RG and Park JH (2002) Inhibition of Caco-2 cell proliferation by all-trans retinoic acid: role of insulin-like growth factor binding protein-6. J Cell Physiol 190, 92100.[CrossRef][ISI][Medline]
Kim HS, Nagalla SR, Oh Y, Wilson E, Roberts CT, Jr and Rosenfeld RG (1997) Identification of a family of low-affinity insulin-like growth factor binding proteins (IGFBPs): characterization of connective tissue growth factor as a member of the IGFBP superfamily. Proc Natl Acad Sci USA 94, 1298112986.
Kjerulff KH, Langenberg P, Seidman JD, Stolley PD and Guzinski GM (1996) Uterine leiomyomas. Racial differences in severity, symptoms and age at diagnosis. J Reprod Med 41, 483490.[ISI][Medline]
Kurbanova MK, Koroleva AG and Sergeev AS (1989) Genetic-epidemiologic analysis of uterine myoma: assessment of repeated risk. Genetika 25, 18961898.[ISI][Medline]
Kuroda K, Okamoto O and Shinkai H (1999) Dermatopontin expression is decreased in hypertrophic scar and systemic sclerosis skin fibroblasts and is regulated by transforming growth factor-beta1, interleukin-4, and matrix collagen. J Invest Dermatol 112, 706710.[CrossRef][ISI][Medline]
Lee BS and Nowak RA (2001) Human leiomyoma smooth muscle cells show increased expression of transforming growth factor-beta 3 (TGF beta 3) and altered responses to the antiproliferative effects of TGF beta. J Clin Endocrinol Metab 86, 913920.
Li XH and Ong DE (2003) Cellular retinoic acid-binding protein II gene expression is directly induced by estrogen, but not retinoic acid, in rat uterus. J Biol Chem 278, 3581935825.
Ligon AH and Morton CC (2001) Leiomyomata: heritability and cytogenetic studies. Hum Reprod Update 7, 814.
Ma C and Chegini N (1999) Regulation of matrix metalloproteinases (MMPs) and their tissue inhibitors in human myometrial smooth muscle cells by TGF-beta1. Mol Hum Reprod 5, 950954.
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, 967973.
Martin JL, Coverley JA and Baxter RC (1994) Regulation of immunoreactive insulin-like growth factor binding protein-6 in normal and transformed human fibroblasts. J Biol Chem 269, 1147011477.
Napoli JL (1999) Retinoic acid: its biosynthesis and metabolism. Prog Nucleic Acid Res Mol Biol 63, 139188.[ISI][Medline]
Napoli JL (2001) 17beta-Hydroxysteroid dehydrogenase type 9 and other short-chain dehydrogenases/reductases that catalyze retinoid, 17beta- and 3alpha-hydroxysteroid metabolism. Mol Cell Endocrinol 171, 103109.[CrossRef][ISI][Medline]
Nilbert M and Heim S (1990) Uterine Leiomyoma Cytogenetics. Genes Chromosomes Cancer 2, 313.[ISI][Medline]
Nishio E and Watanabe Y (1997) Transforming growth factor beta is a modulator of platelet-derived growth factor action in vascular smooth muscle cells: a possible role for catalase activity and glutathione peroxidase activity. Biochem Biophys Res Commun 232, 14.[CrossRef][ISI][Medline]
Nowak RA (1999) Fibroids: pathophysiology and current medical treatment. Baillieres Best Pract Res Clin Obstet Gynaecol 13, 223238.[CrossRef][ISI][Medline]
Okamoto O, Fujiwara S, Abe M and Sato Y (1999) Dermatopontin interacts with transforming growth factor beta and enhances its biological activity. Biochem J 337, 537541.[CrossRef][ISI][Medline]
Ouyang H, Shiwaku HO, Hagiwara H, Miura K, Abe T, Kato Y, Ohtani H, Shiiba K, Souza RF, Meltzer SJ et al. (1997) The insulin-like growth factor II receptor gene is mutated in genetically unstable cancers of the endometrium, stomach, and colorectum. Cancer Res 57, 18511854.[Abstract]
Palmer SS, Haynes-Johnson D, Diehl T and Nowak RA (1998) Increased expression of stromelysin 3 mRNA in leiomyomas (uterine fibroids) compared with myometrium. J Soc Gynecol Invest 5, 203209.[ISI][Medline]
Pfahl M and Chytil F (1996) Regulation of metabolism by retinoic acid and its nuclear receptors. Annu Rev Nutr 16, 257283.[CrossRef][ISI][Medline]
Quade BJ, Wang TY, Sornberger K, Dal Cin P, Mutter GL and Morton CC (2004) Molecular pathogenesis of uterine smooth muscle tumors from transcriptional profiling. Genes Chromosomes Cancer 40, 97108.[CrossRef][ISI][Medline]
Reed WB, Walker R and Horowitz R (1973) Cutaneous leiomyomata with uterine leiomyomata. Acta Derm Venereol 53, 409416.[ISI][Medline]
Reeves R (2000) Structure and function of the HMGI(Y) family of architectural transcription factors. Environ Health Perspect 108 (Suppl 5), 803809.[ISI][Medline]
Rexer BN and Ong DE (2002) A novel short-chain alcohol dehydrogenase from rats with retinol dehydrogenase activity, cyclically expressed in uterine epithelium. Biol Reprod 67, 15551564.
Riquelme C, Larrain J, Schonherr E, Henriquez JP, Kresse H and Brandan E (2001) Antisense inhibition of decorin expression in myoblasts decreases cell responsiveness to transforming growth factor beta and accelerates skeletal muscle differentiation. J Biol Chem 276, 35893596.
Roghani M, Lassarre C, Zapf J, Povoa G and Binoux M (1991) Two insulin-like growth factor (IGF)-binding proteins are responsible for the selective affinity for IGF-II of cerebrospinal fluid binding proteins. J Clin Endocrinol Metab 73, 658666.[Abstract]
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, 17071715.
Schwartz SM, Marshall LM and Baird DD (2000) Epidemiologic contributions to understanding the etiology of uterine leiomyomata. Environ Health Perspect 108(Suppl 5), 821827.[ISI][Medline]
Sheikh MS, Shao ZM, Hussain A, Clemmons DR, Chen JC, Roberts CT, Jr, LeRoith D and Fontana JA (1993) Regulation of insulin-like growth factor-binding-protein-1, 2, 3, 4, 5, and 6: synthesis, secretion, and gene expression in estrogen receptor-negative human breast carcinoma cells. J Cell Physiol 155, 556567.[ISI][Medline]
Skubitz KM and Skubitz AP (2003) Differential gene expression in uterine leiomyoma. J Lab Clin Med 141, 297308.[CrossRef][ISI][Medline]
Sozen I and Arici A (2002) Interactions of cytokines, growth factors, and the extracellular matrix in the cellular biology of uterine leiomyomata. Fertil Steril 78, 112.[ISI][Medline]
Stewart CE and Rotwein P (1996) Insulin-like growth factor-II is an autocrine survival factor for differentiating myoblasts. J Biol Chem 271, 1133011338.
Stewart EA, Friedman AJ, Peck K and Nowak RA (1994) Relative overexpression of collagen type I and collagen type III messenger ribonucleic acids by uterine leiomyomas during the proliferative phase of the menstrual cycle. J Clin Endocrinol Metab 79, 900906.[Abstract]
Storey JD (2002) A direct approach to false discovery rates. J Roy Stat Soc B 64, 479498.[CrossRef][ISI]
Storey JD and Tibshirani R (2003) Statistical significance for genomewide studies. Proc Natl Acad Sci U S A 100, 94409445.
Sueoka N, Lee HY, Walsh GL, Fang B, Ji L, Roth JA, LaPushin R, Hong WK, Cohen P and Kurie JM (2000) Insulin-like growth factor binding protein-6 inhibits the growth of human bronchial epithelial cells and increases in abundance with all-trans-retinoic acid treatment. Am J Respir Cell Mol Biol 23, 297303.
Takahashi T, Nagai N, Oda H, Ohama K, Kamada N and Miyagawa K (2001) Evidence for RAD51L1/HMGIC fusion in the pathogenesis of uterine leiomyoma. Genes Chromosomes Cancer 30, 196201.[CrossRef][ISI][Medline]
Taylor KR, Holzer AK, Bazan JF, Walsh CA and Gleeson JG (2000) Patient mutations in doublecortin define a repeated tubulin-binding domain. J Biol Chem 275, 3444234450.
Tomlinson IP, Alam NA, Rowan AJ, Barclay E, Jaeger EE, Kelsell D, Leigh I, Gorman P, Lamlum H, Rahman S et al. (2002) Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet 30, 406410.[CrossRef][ISI][Medline]
Treloar SA, Martin NG, Dennerstein L, Raphael B and Heath AC (1992) Pathways to hysterectomy: insights from longitudinal twin research. Am J Obstet Gynecol 167, 8288.[ISI][Medline]
Tsibris JC, Porter KB, Jazayeri A, Tzimas G, Nau H, Huang H, Kuparadze K, Porter GW, O'Brien WF and Spellacy WN (1999) Human uterine leiomyomata express higher levels of peroxisome proliferator-activated receptor gamma, retinoid X receptor alpha, and all-trans retinoic acid than myometrium. Cancer Res 59, 57375744.
Tsibris JC, Segars J, Coppola D, Mane S, Wilbanks GD, O'Brien WF and Spellacy WN (2002) Insights from gene arrays on the development and growth regulation of uterine leiomyomata. Fertil Steril 78, 114121.[CrossRef][ISI][Medline]
Tsibris JC, Maas S, Segars JH, Nicosia SV, Enkemann SA, O'Brien WF and Spellacy WN (2003) New potential regulators of uterine leiomyomata from DNA arrays: the ionotropic glutamate receptor GluR2. Biochem Biophys Res Commun 312, 249254.[CrossRef][ISI][Medline]
Tusher VG, Tibshirani R and Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98, 51165121.
Vikhlyaeva EM, Khodzhaeva ZS and Fantschenko ND (1995) Familial predisposition to uterine leiomyomas. Int J Gynaecol Obstet 51, 127131.[CrossRef][ISI][Medline]
Vollenhoven BJ, Herington AC and Healy DL (1993) Messenger ribonucleic acid expression of the insulin-like growth factors and their binding proteins in uterine fibroids and myometrium. J Clin Endocrinol Metab 76, 11061110.[Abstract]
Walker CL (2002) Role of hormonal and reproductive factors in the etiology and treatment of uterine leiomyoma. Recent Prog Horm Res 57, 277294.
Walker CL, Hunter D and Everitt JI (2003) Uterine leiomyoma in the Eker rat: a unique model for important diseases of women. Genes Chromosomes Cancer 38, 349356.[CrossRef][ISI][Medline]
Wang H, Mahadevappa M, Yamamoto K, Wen Y, Chen B, Warrington JA and Polan ML (2003) Distinctive proliferative phase differences in gene expression in human myometrium and leiomyomata. Fertil Steril 80, 266276.[ISI][Medline]
Weston G, Trajstman AC, Gargett CE, Manuelpillai U, Vollenhoven BJ and Rogers PA (2003) Fibroids display an anti-angiogenic gene expression profile when compared with adjacent myometrium. Mol Hum Reprod 9, 541549.
Winkler VDH and Hoffmann W (1938) Regarding the question of inheritance of uterine myoma. Deutsche-Med Wochenschr 68, 235257.
Wu X, Blanck A, Norstedt G, Sahlin L and Flores-Morales A (2002) Identification of genes with higher expression in human uterine leiomyomas than in the corresponding myometrium. Mol Hum Reprod 8, 246254.
Xu YQ, Grundy P and Polychronakos C (1997) Aberrant imprinting of the insulin-like growth factor II receptor gene in Wilms' tumor. Oncogene 14, 10411046.[CrossRef][ISI][Medline]
Zhou Y, Mohan S, Linkhart TA, Baylink DJ and Strong DD (1996) Retinoic acid regulates insulin-like growth factor-binding protein expression in human osteoblast cells. Endocrinology 137, 975983.[Abstract]
Submitted on August 6, 2004; resubmitted on October 11, 2004; accepted on November 30, 2004.