Mol. Hum. Reprod. Advance Access originally published online on May 6, 2005
Molecular Human Reproduction 2005 11(6):441-450; doi:10.1093/molehr/gah174
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Estrogen-induced changes in IGF-I, Myb family and MAP kinase pathway genes in human uterine leiomyoma and normal uterine smooth muscle cell lines
1Laboratory of Experimental Pathology and 2Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709
3 To whom correspondence should be addressed at: Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, P.O. Box 12233, MD C2-09, Research Triangle Park, NC 27709. Email: dixon{at}niehs.nih.gov
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Abstract |
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Many studies have implicated numerous hormones, growth factors, cytokines and other signal transduction molecules in the pathogenesis of uterine leiomyoma. Estrogen and estrogen-related genes are thought to play a key role in the growth of uterine leiomyomas, but the molecular mechanisms are unclear. In an attempt to investigate various pathways that might be involved in estrogen-regulated uterine leiomyoma growth as well as to identify any novel effector genes, microarray studies comparing estrogen-treated uterine leiomyoma cells (UtLM) and normal myometrial cells to untreated cells were performed. Several genes were differentially expressed in estrogen treated UtLM cells, including insulin-like growth factor-I (IGF-I) and others potentially involved in the IGF-I signalling pathway, specifically genes for A-myb, a transcription factor which promotes cell cycle progression and for MKP-1, a dual specificity phosphatase that dephosphorylates mitogen-activated protein kinase. IGF-I and A-myb were up-regulated in estrogen-treated cells while MKP-1 was down-regulated. Two other cell cycle promoting genes, c-fos and myc, were also down-regulated in estrogen treated UtLM cells. These genes are typically up-regulated in response to estrogen in some cells, notably breast epithelial cells, yet consistently have lower expression levels in uterine leiomyoma tissue when compared to autologous myometrium. Our results demonstrate some novel genes that may play a role in the growth of uterine leiomyoma, strengthen the case for involvement of the IGF-I pathway in the response of UtLM to estrogen and corroborate evidence that uterine smooth muscle cells respond to estrogen with a different gene expression pattern than that seen in epithelial cells.
Key words: A-myb/IGF-I/leiomyoma/microarray/MKP-1
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Introduction |
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Uterine leiomyomas (fibroids; myomas) are benign tumours that originate in the smooth muscle wall of the uterus (Buttram and Reiter, 1981
). These smooth muscle cell tumours are a leading cause of hysterectomies (Rein and Nowak, 1992; Wilcox et al., 1994) and are the most common type of pelvic neoplasm found in women (Buttram and Reiter, 1981). Even though they are benign, leiomyomas have a significant impact on the reproductive health of women, due to their high incidence (Cramer and Patel, 1990) and the lack of proven treatment options other than surgery (Olive, 2000; Stewart, 2001). It has become widely accepted that the growth of leiomyomas is regulated by steroid hormones, particularly estrogen and progesterone. Since these hormones are the key to regulating genes that direct the function and development of uterine tissues (Andersen, 1998), it follows that they may play a role in the growth and/or development of Leiomyomas. Clinical evidence that supports estrogen involvement includes the occurrence of these tumours primarily in premenopausal women and their regression under conditions of hypo-estrogenicity, as for example following menopause or administration of gonadotrophin releasing hormone agonists which decrease serum estrogen levels (Rein and Nowak, 1992; Friedman et al., 1993).
In addition, results of molecular studies also support a role for estrogen and progesterone in the pathogenesis of uterine leiomyoma. Many studies have demonstrated elevated levels of both estrogen (Andersen and Barbieri, 1995) and progesterone (Viville et al., 1997) receptors in leiomyoma compared to autologous myometrium. This would suggest that leiomyomas are capable of a heightened response to these hormones. Numerous studies have also implicated a host of other signalling molecules, such as growth factors and cytokines, as major drivers in the development and growth of these tumours and many of these factors are regulated by steroid hormones. For example, mRNA levels for epidermal growth factor (EGF), a progesterone-responsive molecule, are elevated in leiomyoma compared to autologous myometrium in women in the luteal (progesterone-dominated) phase of the menstrual cycle (Harrison-Woolrych et al., 1994), while addition of EGF to primary cultures of myometrial and leiomyoma cells causes a significant increase in DNA synthesis (Fayed et al., 1989). Other factors that have been suggested as having a major influence on uterine leiomyoma pathobiology include platelet-derived growth factor (PDGF) Fayed et al., 1989; Barbarisi et al., 2001), prolactin (Daly et al., 1984; Rein et al., 1990) and Transforming Growth Factor (TGF-ß) (Arici and Sozen, 2000; Lee and Nowak, 2001).
Of these factors insulin-like growth factor-I (IGF-I) is one of the most widely studied. IGF-I is the product of an estrogen-regulated gene and is important in cell growth, proliferation and differentiation. IGF-I exerts its mitogenic action by increasing DNA synthesis, accelerating the progression of the cell cycle from G1 to S phase and by inhibiting apoptosis. It is a potent mitogen for a wide variety of neoplastic cell lines, including classically estrogen-responsive cells such as those from prostate, breast, ovary and uterus (Yu and Rohan, 2000).
Until recently, studies examining the role of growth factors and other signalling molecules were limited to comparing changes in expression levels between tumour and normal tissues for a few, and often for one, of these molecules at a time. The likelihood of having just one or two pathways involved, however, is low, and the above approach makes evaluation of any cross-talk among signalling pathways difficult. The development and growth of these tumours most likely involves the interaction of most, if not all, of the effectors studied thus far, and possibly a few others that have not yet been identified; however, the nature of the relationship of various signalling pathways to the pathogenesis of uterine leiomyoma is not yet known. Global analysis platforms such as cDNA microarray can be used to screen for the involvement of multiple pathways by examining the expression pattern of thousands of genes simultaneously. Studies examining differential gene expression between leiomyoma tissue and autologous myometrium by microarray have only begun to be published (Tsibris et al., 2002; Ahn et al., 2003; Chegini et al., 2003; Weston et al., 2003). Using UtLM (uterine leiomyoma) cells as a model for uterine leiomyoma tissue and uterine smooth muscle (UtSMC) cells as a model for normal uterine smooth muscle tissue, various hormonal growth factor and chemical treatments can be applied to these cell lines and gene expression patterns compared. Differences in expression patterns between the two cell lines may be used to tease out genes involved in leiomyoma development, while differences in expression patterns between treated and untreated cells will help in understanding which genes are important in development and subsequent growth of these tumours. In this study, we use microarray analysis of estrogen-treated uterine leiomyoma and normal uterine smooth muscle cells to examine the relationship of various factors so far implicated in the growth of these tumours and to identify novel factors that may also play a role.
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Materials and methods |
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Cells and culture conditions
GM10964 (UtLM) cells were obtained from Coriell Cell Repositories (Camden, NJ, USA) and the cells were grown in GM medium: Eagle's minimum essential medium [Invitrogen (Gibco), Carlsbad, CA, USA] with 19% fetal bovine serum (FBS) (Sigma St Louis, MO, USA) and supplemented with vitamins [Invitrogen (Gibco)], essential and non-essential amino acids [Invitrogen (Gibco)] and L-glutamine [Invitrogen (Gibco)]. Normal human UtSMC were purchased from Biowhittaker Laboratories (Walkersville, MD, USA) and maintained in smooth muscle growth medium-2 with SingleQuots (Biowhittaker). All culture maintenance and experiments were performed at 37°C and 5% CO2.
Microarray study
UtLM and UtSMC cells in T175 flasks (at passage 8–14 and 80–95% confluency) were serum starved by incubating in phenol red-free, stripped-serum GM medium [GM medium as above with phenol-red free MEM and charcoal/dextran-treated FBS (Hyclone, Logan, UT, USA)] for 4 days. Cells were then treated with either 10–8 M 17ß-estradiol (E2) (Sigma) or with 0.01% ethanol (vehicle control) in phenol red-free, stripped-serum GM medium for 24 h. For both cell lines, cells from flasks that received the same treatment (4-5 T175 flasks per cell line per treatment) were pooled and total RNA extracted using an RNeasy MidiKit (QIAGEN, Valencia, CA, USA). Due to the expense of chip printing and the large amount of RNA needed for each hybridization, cells from multiple flasks representing biological replicates were pooled. Since only those genes that were consistently under- or over-expressed on three or four of 4 chip replicates were reported, results from these pooled samples can be considered average changes from the biological replicates. Additional biological replicates are provided in the expression analysis at the protein level (See Verification of microarray results section below). The samples were concentrated to >9 µg/µl using Microcon 30 columns (Amicon:Millipore, Bedford, MA, USA). RNA concentration was determined spectrophotometrically and the quality of the RNA was checked on a formaldehyde agarose gel. Microarray experiments were performed at the National Institute of Environmental Health Sciences Microarray Center (NIEHS; Research Triangle Park, NC). Detailed protocols for the microarray procedures used are available at the website, http://www.dir.niehs.nih.gov/microarray or found in Lobenhofer et al., 2002. Validation of chip clones by resequencing is an ongoing activity at NIEHS and updated chip annotations are also available at the website, http://www.dir.niehs.nih.gov/microarray/clones. Briefly, each RNA pair was independently labelled two times with Cy3 and with Cy5 in order to conduct duplicate dye reversal hybridizations to the NIEHS 2K ToxChip Version 1.0 (for a total of four hybridizations per sample pair). ToxChip Version 1.0 contains 1901 known human genes and ESTs and includes genes from the following functional categories: apoptosis, cell cycle control, DNA replication and repair, heat shock proteins, oncogenes and tumour suppressor genes, kinases, phosphatases and transcription factors. The database of genes present on this chip can be searched at the website, http://www.dir.niehs.nih.gov/microarray/chips.htm. Pixel intensity values from microarray digital image files were acquired using IPLabs image processing software (Scanalytics, Fairfax, VA) and ArraySuite v1.3 extensions and adjusted by subtracting the local background surrounding each gene feature. The ratio of the pixel intensity values for each gene feature was normalized to a ratio of approximately 1.0 using a set of control genes which, on average, were not differentially expressed (Chen et al., 1997). Differentially expressed genes were identified using a probability-based distribution method to calculate a 95% confidence interval for the ratio data. Gene features with a normalized ratio intensity value outside of the confidence interval were considered significantly altered. Expression profiles from the differentially expressed genes were managed in the MicroArray Project System database for statistical validation of differential expression (Bushel et al., 2001, 2002). Briefly, a modified Z-score for each gene feature expression ratio value was computed using the median and the median of the absolute deviates about the median (MAD) in order to detect and remove outlying ratio values from the calculation of the mean ratio fold change. In addition, a binomial distribution (Casella and Berger, 1990) was used to determine the probability of detecting genes altered by chance. For example, scoring genes as differentially expressed at the 95% confidence level (P=0.005) three or more times (k
3) out of four replicate microarray experiments (n=4) has a probability of 0.00048 being detected by chance. Finally, gene features with a ratio value coefficient of variation >0.4 were not used for determining the mean fold change. Differentially expressed genes in at least three of the four replicate experiments were further considered using pathway annotation and mechanistic consideration (Bushel et al., 2001).
Verification of microarray results
Northern blot analysis
Equal amounts of total RNA from estrogen-treated and untreated UtLM and UtSMC cells were separated on a 1.2% formaldehyde agarose gel and transferred to a charged nylon membrane (Immobilon Ny+: Millipore) using the Vacugene XL vacuum blotting system (Amersham/Pharmacia Biotech, Piscataway, NJ, USA). RNA was crosslinked to the membrane using a Stratagene (La Jolla, CA, USA) UV Crosslinker on the Auto setting. Probes for A-myb and MKP-1 were generated from cDNA clones originally obtained from Invitrogen/Research Genetics [I.M.A.G.E. clone numbers 136609 (A-myb) and 417357 (MKP-1)]. A probe for IGF-I was generated from a plasmid containing a human transgene kindly provided by Dr Ping Ye, University of North Carolina-Chapel Hill. Briefly, overnight clone cultures were lysed and plasmids recovered using the Qiaprep Spin Miniprep Kit (QIAGEN) according to manufacturer's protocol. The cDNA inserts were removed with the appropriate restriction enzymes (EcoR1 and Not1 for A-myb and EcoR1 and Pac1 for MKP-1), separated on a 1% agarose gel, then purified using a Qiaquick Gel Extraction Kit (QIAGEN) to obtain probes of approximately 1300 bp for A-myb and 850 bp for MKP-1. Probes were labelled with [
-32P] dCTP by random priming to a specific activity of >1x106 cpm. Hybridization was performed overnight using the QuickHyb Hybridization Solution (Stratagene) according to manufacturer's directions. Films were exposed at –70°C for 72 h. Band intensities were quantified using a Molecular Dynamics Personal Densitometer SI and the ImageQuant software package.
Western blot analysis
UtLM and UtSMC cells were treated with estrogen and ethanol as described above and total protein was extracted as previously reported (Carney et al., 2002). Equal amounts of total protein were separated on a 4–12% NuPage (Invitrogen) Bis-Tris gel and transferred to a 0.45 µm PVDF membrane (Immobilon-P; Millipore). The membrane was blocked, then incubated with primary antibody (rabbit polyclonal anti-MKP-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted 1:500, overnight at 4°C. The membrane was incubated with horseradish peroxidase-conjugated secondary antibody (donkey anti-rabbit; Amersham), diluted 1:5000. Signal was developed using the enhanced chemiluminescence (ECL) Western Blotting Analysis System (Amersham Pharmacia) according to manufacturer's protocol. Films were exposed to Hyperfilm ECL (Amersham) for 40 min. Images were quantified as described for the northern analysis. Membranes were stripped and reprobed with HPRT antibody for loading control and normalization.
Immunocytochemistry
UtLM cells were seeded onto 1-well chamber slides (Labtek:Nalge Nunc International, Naperville, IL, USA) at 100 000 cells per slide and allowed to attach overnight. The culture medium was then changed to stripped serum GM for 4 days following which the cells were treated with 10–8 M E2 or 0.1% ethanol (vehicle control) in stripped-serum GM medium for 24 or 72 h. Following treatment, the cells were fixed onto the slide with 4% paraformaldehyde and stained for A-myb or IGF-I expression. Briefly, endogenous peroxide activity was blocked with 0.3% H2O2 for 30 min, then primary antibody (goat polyclonal anti-A-myb or rabbit polyclonal anti-IGF-I; Santa Cruz) or normal goat or rabbit serum (negative control) was applied at 1:100 for 1 h (A-myb) or at 1:75 for 2 h (IGF-I). Secondary antibodies (Vector Laboratories, Burlingame, CA, USA) were applied at 1:500 (A-myb) or 1:200 (IGF-I) for 30 min. The colour was visualized using 3,3'-diaminobenzidine tetrahydrachloride (DAB, DAKO, Carpinteria, CA, USA) and the cells were counterstained with Harris hematoxylin (Richard-Allan Scientific, Kalamazoo, MI, USA). The amount of staining on each slide was evaluated using a modification of a previously developed semiquantative ‘quickscore’ method (Detre et al., 1995). The modification involves broadening the intensity scale to accommodate staining in our monocultures, all of which were assigned a proportion score of six since 100% of the cells stained positively, relegating any treatment differences to the intensity score, and still allow for differential staining among cells on a single slide. The modified intensity scale is 0=negative, 1=weak, 1.5=minimal, 2=moderate, 2.5=strong, 3=very intense. Scores were assigned independently by two scorers and averaged for each slide scored.
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Results |
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Microarray analysis
Duplicate pairs of cDNA microarrays, with dye labelling reversal, were used to assess gene expression differences in UtLM and UtSMC cells after estrogen exposure. Nineteen genes were differentially and significantly regulated in estrogen treated UTLM cells; 6 were up-regulated and 13 were down-regulated. Twelve genes were differentially regulated in UtSMC cells; 1 gene was up-regulated and 11 genes were down-regulated. All up-regulated genes were uniquely up-regulated in the respective cell lines. Seven of the down-regulated genes were common to both cell lines (Table I). Up-regulated genes in UtLM included IGF-I and A-myb while down-regulated genes included c-fos and myc. MKP-1 was down-regulated in both cell lines (Figures 1 and 2). It is interesting to note that, in UtLM cells, twice as many genes were down-regulated as were up-regulated and in UtSMC cells all differentially expressed genes, expect one, were down-regulated (Table I).
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Northern analysis
Total RNA extracted from treated UtLM and UtSMC cells was examined for expression levels of IGF-I, A-myb and MKP-1 genes. Bands were detected at 7.2 kb for IGF-I, 5 kb for A-myb and 2.5 kb for MKP-1, consistent with previous reports in the literature (Nomura et al., 1988
; Gloudemans et al., 1990; Keyse and Emslie, 1992). Results of the northern analyses confirmed the changes seen on microarray. Expression of IGF-I and A-myb were higher in E2 treated UtLM cells than in the ethanol treated controls and expression of MKP-1 was lower in E2 treated UtLM and UtSMC cells than in the ethanol treated cells. IGF-I and A-myb were expressed at much lower levels overall in UtSMC cells than in UtLM cells and there were no apparent treatment differences in UtSMC cells (Figure 3).
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Western analysis
We were able to analyse expression of MKP-1 at the protein level by western blot. Expression of MKP-1 protein corroborated the microarray results and mimicked the pattern seen at the mRNA level for UtLM cells. However, while protein expression was higher overall in UtSMC cells than in UtLM cells, as was seen with expression of MKP-1 mRNA, the down-regulation in E2 treated UtSMC cells seen at the mRNA level was not reflected at the protein level (Figure 4).
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Immunocytochemistry
A-myb staining in UtLM cells was predominantly nuclear while IGF-I staining was localized to the cytoplasm. No differences in A-myb or IGF-I protein expression between 17ß-E2- and ethanol-treated cells were apparent by immunocytochemistry after 24 h. However, 72 h following treatment, A-myb and IGF-I expression were elevated in E2-treated cells compared to ethanol-treated cells (Figure 5).
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Discussion |
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Although uterine leiomyoma are benign, they can grow to very large sizes and have a high incidence of occurrence in women. These tumours have a low mitotic rate, however, an indication that their growth does not involve the typical loss of control mechanisms seen in malignant cancers. While many potential mitogens, oncogenes, tumour suppressors and other molecules that may be involved in the pathogenesis of uterine leiomyoma have been studied individually, no one factor or system can be pinpointed as being causal. Clinical and molecular evidence overwhelmingly points to involvement of steroid hormones in the growth of uterine leiomyoma; the exact mechanism of this involvement, however, is unknown. Estrogen may be a mediator of mitogenic effects on leiomyoma cells by triggering the production of autocrine factors which stimulate mitogenesis (Barbarisi et al., 2001
). Some genes that are potential effectors of this estrogen response appear to be differentially expressed in estrogen treated leiomyoma cells. IGF-I, PDGF, EGF, and TGF-ß, among others, have been individually implicated as the major factor influencing the growth of these tumours. An interaction among several signalling systems is likely to be involved, however, requiring a global approach to investigate these connections. Moreover, since proteins are the ultimate effectors of changes in gene expression it is also important to demonstrate that the gene changes are reflected by changes in protein levels. In this study we employed the cDNA microarray technique to attempt to discover novel players that may provide links among the various pathways that appear to influence the growth of uterine leiomyoma. We found changes in the levels of both mRNA and protein for IGF-I, and for possible effectors of the IGF-I signalling pathway, MAP kinase phosphatase-1 and A-myb, in UtLM cells following estrogen treatment.
The typical endpoint of estrogen response in classical estrogen-responsive cells, such as MCF-7 cells, is cellular proliferation. Proliferation of cultured UtLM in response to estrogen, on the other hand, has proven to be quite variable. It is the opinion of some that estrogen alone is insufficient to elicit a proliferative response in these cells and that other factors, growth factors and/or cytokines, are necessary in order to induce proliferation (Rauk et al., 1995, Salmi and Rutanen, 1996). Other markers of estrogen responsiveness, such as changes in expression of estrogen-regulated genes, can be used as endpoints in estrogen response studies, however; part of the purpose of this paper is to define such markers in the UtLM and UtSMC cell lines. For example, studies have shown that the expression of the estrogen-regulated gene c-fos is lower in leiomyoma and normal myometrium (smooth muscle) compared to endometrium (epithelium) demonstrating a difference in the regulation of this gene in the different tissue types (Salmi and Rutanen, 1996). Fos is often up-regulated in epithelial cells (e.g. MCF-7 and endometrium), but not in leiomyoma or myometrium, in response to estrogen. The fact that this gene is down-regulated in the UtLM cells in this study provides evidence that these cells are indeed responding appropriately to estrogen.
As has been previously described by our lab and others, IGF-I protein levels are elevated in leiomyoma compared to matched myometrium (Dixon et al., 2000; Englund et al., 2000). The increase appears to be a combination of increased uptake (van der Ven et al., 1994; Dixon et al., 2000) and increased expression of IGF-I in the cells (Giudice et al., 1993; Englund et al., 2000). Some discrepancy exists as to the influence of the menstrual cycle, and hence the hormonal state of the uterus, on the increase in IGF-I levels (Giuduce et al., 1993; Englund et al., 2000), but it is widely accepted that IGF-I is an estrogen-regulated gene, in part because of studies that have seen cycle stage-dependent increases in IGF-I levels in leiomyoma (Andersen, 1996). This study provides additional evidence that an increase in IGF-I in human leiomyoma cells is at least in part a result of increased expression and that this increased expression is related to exposure to estrogen.
The myb gene family has three members: A-, B-, and c-myb and there is high homology within their DNA binding domains (Oh and Reddy, 1999). C-myb is a positive regulator of proliferation and A-myb may serve as a similar mediator. A-myb is a potent transactivator of Myb-binding site promoter constructs (e.g. c-myc promoter) in smooth muscle cells and its expression induces progression into the S-phase of the cell cycle (Marhamati et al., 1997). It seems to functionally cooperate with c-myc to induce DNA synthesis in quiescent smooth muscle cells. Increased A-myb mRNA levels have been detected in proliferating cells and increased mRNA levels in smooth muscle cells during the cell cycle appear to be due to increased rate of gene transcription (Marhamati et al., 1997). In this study, we found A-myb expression and protein levels to be elevated in UtLM cells following estrogen stimulation. To our knowledge, this is the first report of expression of this gene in uterine cells or tissue or of its estrogen dependence.
MKP-1 is believed to target phosphorylated MAPK in the nucleus and inactivate it (Volmat et al., 2001) and it is thought that these phosphatases act as a negative control on MAPK signalling (Sun et al., 1993). MAPK activation plays a prominent role in the regulation of cell cycle progression and cellular proliferation. When activated (phosphorylated), MAPK accumulates in the nucleus; indeed, nuclear translocation is necessary for progression into the S-phase of the cell cycle. MKP-1 and MKP-2 are specifically induced by the p42/44 MAP kinase pathway and are believed to constitute an autoregulatory loop regulating the activity of this pathway (Volmat et al., 2001). MAPK is one of the downstream effectors of the IGF-I signalling pathway and previous studies in our lab have shown an increase in the level of phosphorylated MAPK in leiomyoma compared to matched myometrium (He et al., 2002). We did not see an increase in the expression of MAPK as a result of estrogen exposure in the UtLM cells in this study. However, we postulate that functional (phosphorylated) MAPK may still be increased in leiomyomas even without increased MAPK expression because of the decreased inhibition by MKP-1.
As noted in the results section, the down-regulation of MKP-1 in E2-treated UtSMC cells seen at the mRNA level was not reflected at the protein level as it was in E2-treated UtLM cells. One possible explanation for this could be differences in the post-translational regulation of MKP-1 between UtLM and UtSMC cells. Brondello et al. (1999) showed that p42/44MAPK-dependent phosphorylation of MKP-1 stabilizes the protein by reducing its ubiquitin-mediated degradation. This phosphorylation may be prevented or a phosphatase that dephosphorylates the stable form of MKP-1 may be differentially up-regulated in E2-treated UtLM cells. As a result, MKP-1 would be quickly degraded following translation and the level of MKP-1 protein reduced, thus mirroring the E2-induced reduction of MKP-1 mRNA levels in the UtLM (tumour), but not the UtSMC (normal smooth muscle) cells. In addition, the expression of MKP-1 protein in UtSMC cells at higher doses of estrogen (10–6 M) actually does mimic the mRNA expression changes (i.e. is down-regulated; data not shown) indicating that reduction in MKP-1 protein levels in UtLM and UtSMC cells is indeed an estrogen-mediated event and that UtLM cells are more sensitive to this estrogen-regulated effect than UtSMC cells.
Ultimately, the goal of mitogenesis is progression through the cell cycle and cell division. One of the mitogenic actions of IGF-I is progression through G1 to S-phase (Yu and Rohan, 2000). A-myb, also, appears to promote progression from G1- to S-phase and A-myb mRNA levels peak during late G1- and early S- phases (Marhamati et al., 1997; Ziebold and Klempnauer, 1997). These findings along with results from this study suggest that A-myb and IGF-I may act together to push the leiomyoma cell through the cell cycle at the G1-to-S transition point. It is possible that the association between these two factors is through cell-cycle active cyclins. The C-terminus of A-myb appears to function in an auto-regulatory fashion, inhibiting the trans-activation function of A-myb in the unphosphorylated state. Ziebold and Klempnauer have shown that phosphorylation of A-myb induced by cyclins A and E relieves this inhibitory effect, thus allowing A-myb to act to influence gene transcription (Ziebold and Klempnauer, 1997). Moreover, IGF-I has been shown to induce expression of cyclins A and E. Therefore, a part of the mitogenic signalling pathway for UtLM may involve IGF-I activation of A-myb via cyclins A and/or E. Further research into this possible pathway is planned.
The gene expression changes seen in this study suggest a potential model for an estrogen-induced signalling cascade in UtLM cells (Figure 6). In this model, the UtLM cell's heightened response to E2 results in increased expression of IGF-I. IGF-I may also act on UtLM cells in a paracrine fashion from increased expression in surrounding stromal cells. IGF-I then acts to increase levels of phosphorylated MAPK (MAPK-P) (He et al., 2002). MAPK-P translocates to the nucleus where, because of the down-regulation of MKP-1, its effect on transcription is prolonged. IGF-I may also act, possibly through interactions with cyclins A or E, to increase activity of the A-myb transcription factor. These two pathways may act independently or in concert to increase transcription of factors necessary to move the cell through the G1-to-S phase transition of the cell cycle.
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Over the past 2 years, several studies have reported gene expression differences in leiomyoma versus normal myometrium using global platforms such as microarray and differential display (Tsibris et al., 2002
; Wu et al., 2002; See Table II). Depending on the platform used, the numbers of genes or gene fragments assayed, and the bioinformatics analysis methods employed, the number of differentially expressed sequences identified ranged from a few to several hundred. Many of these were unique to the study in which they were identified and because of the inherent nature of gene nomenclature at present, it is often difficult to determine what role, if any, they may play in the growth and development of uterine leiomyoma. A few, however, have consistently appeared in several of these studies, including the current study (Table II). For example, -cardiac actin has been shown to be up-regulated in several studies, while early growth response-1 (EGR-1) and fos genes are consistently down-regulated. The CCN family members connective tissue growth factor (CTGF) and cyr61 are commonly down-regulated genes. Curiously, IGF-II, but not IGF-I, has been shown to be up-regulated in several of the tissue studies using the microarray platform. Some of these changes are consistent with previous reports from the literature (e.g. down-regulation of fos) while others are novel changes that were first reported from global expression analysis. Genes that are reproducibly up- or down-regulated provide a solid starting point for further investigations into the roles they may play in the pathogenesis of uterine leiomyoma.
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Despite the benign nature of uterine leiomyoma, the molecular basis of their development and growth appears to be quite complex. Numerous factors from seemingly unrelated pathways have been shown to be differentially expressed in uterine leiomyoma compared to myometrium. Interactions between these factors and pathways, however, undoubtedly occur. As more studies of global expression changes in uterine leiomyoma are reported, factors and pathways that are important to the pathogenesis of this disease will become more prominent and new factors that may provide links among these pathways will be found. In this study, we have used just such a global expression platform, microarray, to explore gene expression changes in estrogen-treated UtLM. In summary, we found expression changes in genes of the IGF-I and MAP kinase pathways in agreement with previous studies that have implicated these pathways in the pathogenesis of uterine leiomyoma. In addition, we show expression changes in genes, such as A-myb, that were not previously known to play a role in this disease. The results obtained here suggest potential clarifications of the IGF-I signalling pathway in uterine leiomyomas.
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Acknowledgements |
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We would like to thank the staff of the NIEHS microarray center for their support of this work including Jennifer Collins, C. Jeff Tucker, Sherry Grissom, Danica Ducharme and Rick Paules and thanks to Pierre Bushel for his help with the microarray analysis. We would also like to thank Lysandra Castro for her help with the immunocytochemistry and Norris Flagler for his help with imaging. We thank Drs Gary Boorman and Rick Irwin for their critical review of the manuscript.
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References |
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Submitted on February 22, 2005; resubmitted on March 24, 2005; accepted on March 31, 2005.
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