Leiomyoma or uterine fibroids are the most common benign tumor, occurring in approximately 60% of women during their lifetime1. In spite of its generally benign nature, uterine fibroids cause an array of substantial health problems in some women such as pressure or pain, excessive uterine bleeding and problems related to pregnancy 2. As a consequence, uterine fibroids account for approximately one-third of all hysterectomies in the United States or about 200,000 hysterectomies per year 3

Although the etiology of the disease is largely unknown, it is clear that growth of uterine fibroids depends on the ovarian hormones estrogen and progesterone 2. This hormonal dependency is supported by the following observations. Uterine fibroids are observed only after menarche, increase in size during pregnancy, and frequently regress after menopause (reviewed in 2). The tumors can be induced to regress by surgical ovariectomy or by treatment with GnRH agonists which induce a hypoestrogenic state4. Tissue estrogen concentrations are elevated in uterine fibroids compared to myometrium, which may result from increased aromatase activity 5. Estrogen produces diverse biological effects mediated by estrogen receptors (ER). When bound to estrogen, the ER modulates the transcriptional activity of target genes 67. Evidence shows that one effect of estrogen is to increase the levels of both estrogen receptor (ER) and progesterone receptor (PR) 2. It has been recently demonstrated that estrogen can stabilize ER mRNA, increasing the level of cellular ER protein 8.

While it is well established that growth of uterine fibroids depends on estrogen, molecular mechanisms of such estrogen dependency are largely unknown. Numerous studies have indicated that estrogen may mediate fibroid growth through the mitogenic effects of growth factors such as transforming growth factor-β and basic fibroblast growth factor (reviewed in 2). There have been a few recent studies addressing molecular mechanisms of functional interaction between estrogen signaling and growth factor-mediated signaling in the pathogenesis of uterine fibroids. Work by Hayashi et al 9 in estrogen-dependent cancers provides an example where the constitutively activated MAPK signaling pathway in endometrial cancer cells might enhance the transcriptional activity of ERα via phosphorylation of its AF-1 domain. Wnt signaling was recently implicated in the pathogenesis of uterine fibroids where the secreted frizzled related protein 1 (sFRP1) mRNA 10 was found to be significantly elevated in the tumor, and regulated by estrogen treatment. It was shown that sFRP1 contributes to fibroid growth through an anti-apoptotic effect.

A recent report has shown that PPARγ activation by its ligand (i.e., prostaglandin J2) in uterine fibroids is growth inhibitory and mediated at least in part by negative cross-talk between ER and PPARγ signaling pathways 11. However, the exact molecular mechanisms of how such interaction occurs between the two nuclear receptor signaling pathways remain to be answered. Elevated levels of PPARγ, retinoid × receptor alpha (RXRα), and all-trans retinoic acid were observed in human uterine fibroids, and retinoids in combination with estrogen was shown to induce development of uterine fibroids in a guinea pig model 12.

DNA microarray technology allows simultaneous interrogation of tens of thousands of genes 13. Several studies have applied the technology to identify genes that were differentially expressed in human uterine fibroids compared to adjacent normal myometrium 14. The technology has also been applied to identify estrogen-regulated genes in the adult rat uterus 15. To understand the molecular interactions involved in estrogenic support of uterine fibroid growth, we integrated results from the above DNA microarray studies using ortholog analysis. Orthologous genes are related by direct evolutionary descent and should play similar developmental or physiological roles 16. This study identified twelve human and rat orthologous genes that were differentially expressed in human uterine fibroids and estrogen-regulated in the adult rat uterus, and describes their possible cellular and physiological roles in estrogen-dependent tumor growth.

DNA microarray technology provides us with a great opportunity for looking at molecular mechanisms of disease development on a whole genome scale. While a few public databases have been built to facilitate data sharing 204142, it still remains a great challenge to integrate the data, particularly data generated from different organisms, in order to generate testable hypotheses. In the present work we integrated multiple microarray data sets generated by independent research groups from two different species using ortholog gene analysis to try to discover molecular clues to estrogen-dependent growth of human uterine fibroids. While this in silico analysis suggests pathways and individual gene product involvement in the regulation of fibroid tumor growth by estrogenic signaling, the authors recognize the need for experimental follow-up to prove these associations.

Thirty-eight human genes (Table 2) were identified in common from three independent studies showing differential expression between uterine fibroids and normal myometrium. Of these, twelve human and rat orthologous genes (Table 3) were shown to be estrogen-regulated in the rat uterus. Since they are human and rat orthologs, we inferred that they should share similar expression regulation and biological functions in both species. These genes provide important clues to understand estrogen-dependent growth of human uterine fibroids.

Prostaglandin E2 receptor subtype 3 (PTGER3 or EP3) is a G-protein-coupled receptor activated by prostaglandin E2 that was down regulated in uterine fibroids. Alternative splicing generates three isoforms: EP3 alpha, EP3 beta and EP3 gamma, which differ in the putative cytoplasmic carboxy-terminal tail. It was demonstrated that while EP3 gamma is coupled to both stimulation and inhibition of adenylate cyclase, EP3 alpha and beta are exclusively coupled to inhibition of adenylate cyclase 43. Shoji et al. 44 demonstrated that EP3 plays an important role in suppression of cell growth and its down-regulation enhances colon carcinogenesis at a later stage. Transforming growth factor-beta (TGF-β) is a potent inhibitor of normal epithelial cell proliferation and is increasingly implicated in fibroid growth. Many tumor cell lines do not respond to the inhibitory effects of TGF-β due to a reduction or absence of the type II receptor (TGF-β R2) 45. The down-regulation of TGF-β R2 in uterine fibroids is consistent with this finding. The PCP4 gene encoding PEP-19 is a calmodulin-regulatory protein found abundantly within neurons that was found to be increased in uterine fibroids. A study in PC12 cells 46 demonstrated that PEP-19 could inhibit apoptosis in the cells, suggesting that its up-regulation in human uterine fibroids may be similar. Thus, estrogen may promote cell survival and tumor growth by increasing expression of the anti-apoptotic PCP4 gene and by suppressing the expression of growth inhibitory receptors PTGER3 and TGF-β R2.

The calcitonin receptor-like receptor (CALCRL or CRLR), a G-protein coupled receptor, acts as a receptor for adrenomedullin (ADM) or calcitonin gene related peptide (CGRP) depending on which receptor activity modifying protein (RAMP) it partners with 47. Using a rat uterine fibroid-derived cell line (ELT3), Thota and Yallampalli 34 demonstrated that expression of CALCRL and RAMP1 increased with progesterone and decreased with estrogen, consistent with what Helvering et al found in the array work (Table 3). Down-regulation of CALCRL expression in the tumor and in response to estrogen may implicate two important aspects of the tumor growth, that is, cell proliferation and angiogenesis.

L-PGDS has dual molecular functions. It catalyzes the conversion of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2) inside the cell and binds to small non-substrate lipophilic molecules including retinal, retinoic acid and thyroid hormone and serve as a transporter for these molecules once secreted 48. ANXA1 is a calcium-dependent phospholipid binding protein and belongs to the annexin family. It was originally identified as a protein that apparently modulated the release of arachidonic acid from cells 49. Recent data have shown that ANXA1 may specifically target cytosolic phospholipase A2 (PLA2) by both direct enzyme inhibition and suppression of cytokine-induced activation of the enzyme 50. CRABP2 is a member of a large family of small proteins that specifically bind lipophilic compounds such as fatty acids and retinoids 28. Recent work has suggested that CRABP2 may have a role in the movement of retinoic acid (RA) to the RA receptors (RARs), thereby enhancing the action of RA in the cells in which it is expressed. ALDH1A1 belongs to the aldehyde dehydrogenase family and is a terminal enzyme of the pathway catalyzing conversion of retinal to RA 51. NR4A1 (encoded by Nur77 in mouse, NGFI-B in rat, and TR3 in human) is an immediate-early responsive orphan nuclear receptor whose expression is rapidly induced by a variety of extracellular stimuli including estrogen as evidenced here (Table 3). Using Northern blot RNA analysis Cicatiello et al 37 reported that NR4A1 was transiently activated in the uterus (up to 20-fold) within 30–120 min after treatment of adult ovariectomized rats with estrogen.

Five of the ortholog genes (ANXA1, L-PGDS, ALDH1A1, CRABP2 and NR4A1) were found to be linked via their physiological functions following pathway analysis as summarized in Fig. 4. Estrogen may antagonize PPARγ mediated signaling first by up-regulating ANXA1 gene expression, thereby inhibiting phospholipase A2 activity and reducing arachinoic acid production, a common substrate for prostaglandin synthesis. Secondly, estrogen may antagonize PPARγ mediated signaling by down-regulating L-PGDS gene expression, which reduces conversion of PGH2 to PGD2 thus decreasing synthesis of PGJ2, an endogenous ligand for PPARγ activation. Recent work may provide clues to understand the physiological significance of these molecular events in the context of estrogen-dependent fibroid growth. Houston et al. 11 demonstrated that PPARγ upon its activation by its endogenous ligand PGJ2, suppressed estrogen-dependent fibroid growth. The authors hypothesized that PPARγ achieved its antagonistic effect by competitively binding to estrogen-responsive elements on promoters of genes which may be required for estrogen-dependent tumor growth. Yuan et al 52 showed that PPARγ activation by pioglitazone greatly retarded the progression of rat hepatic fibrosis. Shimada et al 53 reported that PPARγ ligands, including PGJ2, induced apoptosis in colon cancer cells and the same group 54 demonstrated that PPARγ plays an important role in the regulation of cell growth and cell death in gastric epithelial cells. It was reported 12 that human uterine fibroids expressed significantly higher levels of PPARγ, retinoid × receptor α and all-trans retinoic acid than myometrium. Thus we hypothesize that estrogen helps tumor cell survival and growth by interfering with PPARγ-mediated apoptosis at multiple steps in the biosynthesis of its ligand.

Retinoic acid (RA) has been established as a biologically active form of vitamin A (retinol). Fig 4 shows that biosynthesis of RA occurs by two sequential irreversible oxidations, first producing retinal from retinol by retinal dehydrogenases (RolDHs). The retinal is then oxidized to RA by retinal dehydrogenases (RalDHs) of the alcohol dehydrogenase (ADH) family 1 (also known as ALDH1A1-3). Tsibris et al 12 demonstrated that estrogen induced formation of abdominal fibroids in the guinea pig, and RA in combination with estrogen was required to specifically induce formation of fibroids in the uterus. Estrogen-induced guinea pig uterine fibroids are similar to the human fibroids, in that they also expressed increased levels of PPARγ, RXR-α and all-trans retinoic acid. Our integrated analysis of expression data collected by independent studies revealed that expression of two genes, ALDH1A1 and CRABP2 for RA synthesis or mobilization was differentially expressed in human uterine fibroids and regulated by estrogen in the rat uterus. Previous studies in rodents have suggested that estrogen directly induces uterine RA synthesis by increasing expression of epithelial retinal dehydrogenase (eRolDH) and ALDH1A2 (reviewed in 29). Reduction of ALDH1A1 expression (Table 3) may not directly result from estrogen regulation but rather be due to feedback inhibition by RA 55. RA acts on tumor growth by binding and regulating the tumor cell heterodimeric receptors RAR and RXR. RXR may also exert its effects on tumor cell survival and growth by partnering with PPARγ and/or NR4A1. NR4A1 was originally recognized for its role in cell proliferation and differentiation. It may confer its growth effects by trans-activating target genes required for cell proliferation in nucleus 56. On the other hand, it also acts on mitochondria (non-trans-activation activity) to induce apoptosis. It was demonstrated that its nuclear export and initiation of NR4A1-dependent apoptosis depends on the nuclear exporting signal (NES) residing on its binding partner RXR and is suppressed by RA 36. Thus, increased expression of NR4A1 and elevated levels of RA in response to estrogen could promote cell proliferation and suppress pro-apoptosis activity. Proteolipid and DM20 encoded by the PLP1 gene through alternative splicing are major structural components of central nervous system myelin. It has been well established that steroid hormones such as estrogen and progesterone regulate the expression of myelin proteins such as proteolipid protein (reviewed in 57). However, what roles PLP1 may play in the development of human uterine fibroids needs further study. Similarly we have little knowledge of what functions that RNase A family, 4 (RNASE4) plays in the tumor growth. It is worthwhile to note that directions of expression changes of ANXA1, NR4A1 and PLP1 were opposite from that of the estrogen treated rat uterus.

Human genetics studies have shown that 40–50% of human uterine fibroids display karyotypically detectable chromosomal abnormalities 2. Twenty percent of the abnormality is the characteristic translocation t(12:14) 5859. The twelve estrogen-regulated genes identified in the present study did not map to this translocation. The most prominent predisposition genes involved in human uterine fibroids, HGMI(c) and HMGI (Y), account for nearly 50% of the genetic variation in human uterine fibroids 59. Dysregulation of these genes through chromosomal translation is a major event in uterine fibroid formation. Rearrangement of HMGI(C) and HMGI(Y) is also a very frequent event in many mesenchymal tumors, suggesting a critical role of the HMGI complex in tumorigenesis. The two key estrogen regulated pathways (e.g. RA and PPARγ) identified in the present study were both reported to be interacting with the HMG complex. For example, orchestrated action from PPARγ, HMGI(C), and other transcription factors is required in directing adipocyte differentiation 60. Altered activity of these transcription factors could lead to biased differentiation of adipocytes or even adipocyte hyperplasia. Moreover, the retinoic acid pathway could regulate or be regulated by HMGI(C) during neuroblastoma tumorigenesis. Both HMGI(C), and HMGI(Y) are expressed in neuroblastoma cell lines and tumors and they are regulated by RA both at the RNA and protein levels, and can affect the responsiveness of these cells to RA 6162. A causal link has been proposed between HMGI(C) expression and RA induced growth arrest during tumorigenesis. Given the pivotal roles of the HMG complex in the fibroids and their interaction with PPARγ and RA pathways in controlling adipocyte growth and tumorigenesis of neuroblastoma, we speculate that estrogen may regulate fibroid growth through the PPARγ and RA pathways and their interaction with the HMGI(C) and HGMI(Y) complex. However, the direct link between HMG complex with RA and/or PPARγ in human uterine fibroids needs to be established experimentally, and is an area for future investigation.

In conclusion, integration of multiple DNA microarray studies through ortholog gene analysis identified twelve uterine fibroid disease genes that may respond to estrogen in the fibroid. Functional and pathway analyses suggested multiple molecular mechanisms for estrogen-dependent growth of human uterine fibroids: enhanced tumor cell survival by increased expression of PCP4 and decreased expression of TGF-β R2 and PTGER3 and the complex interplay among five distinct nuclear receptors (ER, RAR, RXR, NR4A1 and PPARγ) that may enhance tumor cell survival and growth. Fully understanding the exact molecular interactions among these genes requires further study to validate their role in uterine fibroids. This work provides direction for studies which could influence the future direction of therapeutic intervention for the disease.

The author(s) declare that they have no competing interests.

TW, AGG, CS, JS and MNC participated in collecting, analyzing and interpreting array data. TW drafted the manuscript. HQ performed statistical analysis of the array data published by Hoffman et al (2003). LMH, NHK, HUB and JEO designed the rat uterus array study. JEO supervised the overall work. All authors read and approved the final manuscript.